Antitumor, cytotoxic, and antioxidant evaluation of six heterocyclic compounds containing different heterocycle moieties

Article abstract of DOI:10.1002/jhet.3847

Heterocyclic compounds with different heterocycle moieties, namely benzoxazinone, benzimidazole, quinazolinone, and benzofuranone heterocyclic rings, were synthesized, characterized, and evaluated for their anticancer activity against human hepatocellular carcinoma cell line (HepG2) using sulforhodamine B (SRB) and dimethylthiazol-diphenyltetrazolium bromide (MTT) assays. Also, their cytotoxic activities were tested against human epithelioid carcinoma (Hela) cell line in comparison with normal cell, amniotic epithelial (WISH) cell line, as an in vitro toxicity estimation model. The results showed clearly that 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide 4 is the most potent antioxidant and anticancer agents. Although, 3-amino-2-benzylquinazolin-4(3H)-one 5 is less potent anticancer agent against Hela but it is more safe against normal cell (WISH).

Full text of DOI:10.1002/jhet.3847

Received: 2 October 2019
DOI: 10.1002/jhet.3847
Revised: 8 November 2019
Accepted: 19 November 2019
A R T I C L E
Antitumor, cytotoxic, and antioxidant evaluation of six
heterocyclic compounds containing different heterocycle
moieties
Tasneem Mokhtar Radwan1
| Maher Abdel-Aziz El-Hashash2
|
Ashraf Abdel-Hamid Farouk Wasfy1 | Suzan Abdelhalim Abdallah3
1Chemistry Department, Faculty of
Abstract
Science, Benha University, Benha, Egypt
2Chemistry Department, Faculty of
Science, Ain-Shams University, Cairo,
Egypt
3Botany and Microbiology Department,
Faculty of Science, Benha University,
Benha, Egypt
Heterocyclic compounds with different heterocycle moieties, namely ben-
zoxazinone, benzimidazole, quinazolinone, and benzofuranone heterocyclic
rings, were synthesized, characterized, and evaluated for their anticancer activ-
ity against human hepatocellular carcinoma cell line (HepG2) using sul-
forhodamine B (SRB) and dimethylthiazol-diphenyltetrazolium bromide
(MTT) assays. Also, their cytotoxic activities were tested against human epithe-
lioid carcinoma (Hela) cell line in comparison with normal cell, amniotic epi-
thelial (WISH) cell line, as an in vitro toxicity estimation model. The results
showed clearly that 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide 4 is
the most potent antioxidant and anticancer agents. Although, 3-amino-2-ben-
zylquinazolin-4(3H)-one 5 is less potent anticancer agent against Hela but it is
more safe against normal cell (WISH).
Correspondence
Tasneem Mokhtar Radwan, Chemistry
Department, Faculty of Science, Benha
University, Benha, Qalyubia, Egypt.
Email: tasneemmokhatar@gmail.com
1 | INTRODUCTION
to an insoluble blue MTT formazan product.[7] In con-
trast, colorimetric assay for the sulforhodamine B (SRB)
Cancer is moving to be a significant health problem in
the world.[1–3] According to a World Health Organization
(WHO) report, cancer is the second leading cause of
death globally and is responsible for an estimated 9.6 mil-
lion deaths in 2018.[4] Globally, about one in six deaths is
due to cancer.[4] The mortality rate of patients is 782 000
deaths for liver cancer.[4] Cervical cancer is the fourth
most frequent cancer in women with an estimated
570 000 new cases in 2018 representing 6.6% of all female
cancers. Approximately 90% of deaths from cervical can-
cer occurred in low- and middle-income countries.[5]
For the in vitro chemosensitivity testing of tumor cell
lines, several rapid calorimetric assays have been
described. The tetrazolium (MTT) test was the most com-
monly used in latest years.[6] The colorimetric test for
MTT (dimethylthiazol-diphenyltetrazolium bromide) is
based on mitochondrial uptake and succinate dehydroge-
nase reduction of soluble, yellow, MTT tetrazolium salt
is based on the ability of the SRB dye to bind basic amino
acid residues on proteins.[7]
Several of the present treatments have problems with
toxicity and drug-resistance, these encouraged researchers
to explore effective new treatments for cancer disease.[8]
Consequently, synthesis of new bioactive compounds such
as heterocyclic compounds is an important aim for many
researchers. Heterocyclic compounds have an active role as
antibacterial, antiviral, antifungal, anti-inflammatory,
and antitumor drugs.[9] For example, benzoxazinone deriv-
atives are biologically interesting and they are now receiv-
ing considerable attention due to their applications in
medicine use for antifungal, antiviral, antibacterial, antima-
larial, antipyretic, anticancer, and anti-HIV (human immu-
nodeficiency virus) activities.[10] Moreover, 4H-
3,1-benzoxazin-4-one derivatives are shown to be inhibitors
of α-chymotrypsin[11] and rhomboid proteases.[12] They
consist of six-membered ring containing oxygen and
J Heterocycl Chem. 2020;1–12.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

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RADWAN ET AL.
nitrogen atoms.[10] Benzoxazinone derivatives showed a
variety of pharmacological activities, such as antifungal,
antiviral, antibacterial, antimalarial, antipyretic, antican-
cer, and anti-HIV activities.[13–19] On the other hand, a
benzimidazole compound consists of the fusion of benzene
and imidazole. They have been reported to have potential
antitumor activity.[20] While quinazolinone derivatives are
containing two nitrogen atoms in a six-membered ring
fused with a benzene ring. They reveal a variety of biologi-
cal effects, such as antibacterial and antitumor activities.[21]
Moreover, benzofuranone derivatives exhibited anticancer
activity.[22] It composed of a benzene ring fused to
furanone.
The search for a bioactive compound for treating can-
cer stimulates us to turn our interest in the synthesis of
different heterocyclic compounds to select the best hetero-
cyclic nucleus for treating different types of cancer.
Depending upon their biological activity, different six het-
erocyclic compounds, represented four heterocyclic classes
(2-benzyl-3,1-benzoxazin-4-one (1) represented ben-
zoxazinone class, N-(2-(1H-benzo[d]imidazol-2-yl)phenyl)-
2-phenyl acetamide (2) represented benzimidazole class,
ethyl 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acetate (3),
It has been noted that compound (1) which has been
synthesized in our study was synthesized before by
M.E. Diener et al. during conduction of this study. In
addition, they did not evaluate its potential activity as
anticancer activity against any cancerogenic cell lines.[23]
Here, this study is the first to evaluate the potential impli-
cations of this compound in several cancerogenic cell
lines.
Heterocyclic compounds with different heterocycles
moieties, namely benzoxazinone, benzimidazole,
quinazolinone, and benzofuranone heterocyclic rings,
were synthesized, characterized, and evaluated for their
anticancer activity against human HepG2 using two dif-
ferent methods, SRB and tetrazolium (MTT) assays.
Additionally, DNA binding affinity of these compounds
was investigated as a potential mechanism for the anti-
cancer activity against
a
human HepG2 using
DNA/methyl green assay. Moreover, the obtained com-
pounds were evaluated for their in vitro cytotoxic activi-
ties against human epithelioid carcinoma (Hela) cell
line in comparison with normal cell, amniotic epithelial
(WISH) cell line, as an in vitro model for toxicity assess-
ment. Furthermore, synthesized compounds were
screened for their antioxidant activities.
2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide
(4),
3-amino-2-benzylquinazolin-4(3H)-one (5) represented
quinazolinone class and 2-benzyl-3-((3-oxoisobenzofuran-1
(3H)-ylidene)amino)quinazolin-4(3H)-one (6) referred to
benzofuranone and quinazolinone classes as shown in
Figure 1), have been designed, synthesized, and character-
ized. Hence, we selected those compounds to be evaluated
for their anticancer effect against human hepatocellular
carcinoma cell line (HepG2) using SRB and tetrazolium
(MTT) assays.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
In this paper, we used 2-benzyl-3,1-benzoxazin-4-one (1)
to synthesize novel benzimidazole, quinazolinone, and
benzofuranone compounds. In the present study,
FIGURE 1 This design illustrates the
different heterocyclic compounds with different
moieties, namely benzoxazinone,
benzimidazole, quinazolinone, and
benzofuranone heterocyclic rings. (i) o-
Phenylenediamine, ethanol. (ii) Ethyl glycinate
hydrochloride, ethanol. (iii) Hydrazine hydrate,
ethanol. (iv) Hydrazine hydrate, ethanol.
(v) Phthalic anhydride, ethanol

RADWAN ET AL.
3
2-benzyl-4H-3,1-benzoxazin-4-one (so-called metastable
benzoxazinone) was obtained via interaction of
phenylacetyl chloride with anthranilic acid in pyridine
and yielded 2-phenylacetylamino benzoic acid followed
by ring closure in acetic anhydride to afford the desired
product benzoxazinone (1) in high yield (Scheme 1).
The structure of compound (1) was confirmed by its
1H NMR spectrum when run DMSO-d6 shows the follow-
ing signals ppm at δ 4 (2H, s, CH2Ph), 7.3 to 8.5 (9H, m,
ArH) and devoid any band for a hydroxyl group and its
IR spectrum exhibits absorption at 1760 cm−1 for υC=O of
δ-lactone and lacked any band for υNH conforming the
formation of oxazinone ring. Its structure confirmed by
its 13C NMR analysis in DMSO-d6 shows the following
signals ppm: 169.536 (2C), 161.085, 159.149, 145.890,
136.839, 134.702 (2C), 131.049, 129.523 (2C), 128.607
(2C), 127.892 (2C), 126.976 (2C), 119.765, 116.503, 44.698,
and 40.425.
While, δ C signals of 40 169 were indicated for open
structure (S) of compound (1) as indicated in Scheme 2.
It is well known that benzoxazinone derivatives are
opened by time, so we prepare benzoxazinone compound
(1) and divided it into different lots for physical analysis
(IR, mass, 1H NMR, and 13C NMR) and biological assays.
These lots were stored under control condition (desicca-
tor) until use. Compound (1) was not immediately ana-
lyzed in NMR instrument because it was analyzed in
other university (at NMR unit, Faculty of Science, Man-
soura University). However, it not affected their potential
as indicated because we confirmed chemical structure of
compound (1) using IR spectra (IR spectrum exhibits
absorption at 1760 cm−1 for υC=O of δ-lactone and lacked
any band for υNH conforming the formation of oxazinone
ring) before performing anticancer assays.
The enzyme will not be functioning if the oxazinone
ring opens. Thus, the acid has no effect on the resulting
activity. Moreover, oxazine derivatives are shown to be
antioxidant and anticancer agents.[24]
The reaction mechanism for synthesis compound
(1) possibly takes place via the following mechanism
(Figure 2):
Synthesis of benzimidazole, quinazolinone, and
benzofuranone compounds (N-(2-(1H-benzo[d]imidazol-
2-yl)phenyl)-2-phenyl acetamide (2) represented benzimid-
azole class, ethyl 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)
acetate (3), 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acet-
ohydrazide (4), 3-amino-2-benzylquinazolin-4(3H)-one (5)
represented
quinazolinone
class
and
2-benzyl-
3-((3-oxoisobenzofuran-1(3H)-ylidene)amino) quinazolin-4
(3H)-one (6) represented to benzofuranone and
quinazolinone classes), has been achieved following an
individual steps procedure starting from 2-benzyl-3,-
1-benzoxazin-4-one (1). When a benzoxazinone compound
(1) was subjected to react with nitrogen nucleophile
namely o-phenylenediamine in boiling ethanol afforded
benzimidazole compound N-(2-(1H-benzo[d]imidazol-
2-yl)phenyl)-2-phenyl acetamide (2) (Scheme 5).
The structure of compound (2) was inferred by its
1H NMR spectrum when run DMSO-d6 shows the fol-
lowing signals ppm at δ 3.4 (2H, s, CH2Ph), 7.1 to 8.5
(9H, m, ArH), and 11.1 (1H, s, NH). In IR spectra,
strong absorption peaks at 1674, 3283, 3208 cm−1
attributable to υC=O and υNH nonbonded and bonded
respectively indicates the formation of the imidazole
ring. Its structure confirmed by its 13C NMR analysis in
DMSO-d6 shows the following signals ppm: 169.612
(2C), 140.797, 134.855 (2C), 131.096, 129.551, 128.626,
127.033, 122.760, 119.860, and 44.698. EIMS shows m/e
at 327 (M•+).
The mechanism of inactivation of enzyme, as one
way to treat cancer disease by benzoxazinone is depended
upon the nucleophilic reaction of benzoxazinone with
enzyme hydroxyl group at the active site of the enzyme,
which leads to ring opening and formation of an acylated
enzyme (inactive form). The inhibitory activity of ben-
zoxazinone may be based on the electrophilicity of the
lactone carbonyl group which may form a hydrogen bond
with enzyme as indicated in Scheme 3.
While, the open structure (S) of compound (1) cannot
react with enzyme and it cannot form a hydrogen bond
with enzyme as indicated in Scheme 4.
SCHEME 2 Hydrolysis of benzoxazinone compound (1)
SCHEME 3 Mechanism of the inactivation of the enzyme by
SCHEME 1 Synthesis of benzoxazinone compound (1)
benzoxazinone

4
RADWAN ET AL.
SCHEME 4 The impossibility of a chemical reaction between
the acid and the enzyme
SCHEME 7 Synthesis of hydrazide derivative (4)
On the other hand, when the ester (3) was allowed to
react with hydrazine hydrate in boiling ethanol yielded
the hydrazide derivative (2-(2-benzyl-4-oxoquinazolin-3
(4H)-yl)acetohydrazide) (4) (Scheme 7).
The structure of compound (4) was confirmed by its
1H NMR spectrum when run DMSO-d6 shows the follow-
ing signals ppm at δ 3.4 (2H, s, CH2Ph), 5 (2H, s, CH2),
7.2 to 8.8 (10H, m, ArH), and 9.3 (1H, s, NH). Its IR spec-
trum revealed strong absorption bands at 1669 cm−1
(υC=O), 3170, 3323 cm−1 due to υNH indicating the con-
version of ester into hydrazide. Its structure confirmed by
its 13C NMR analysis in DMSO-d6 shows the following
signals ppm: 180.934, 166.131, 161.409, 155.992, 146.901,
135.541, 134.654, 128.779 (2C), 127.005, 126.833 (2C),
119.870, 44.403, 41.017 and disappearance of two signals
at 14.014. This confirmed the formation of hydrazide.
On other hand, the interaction of benzoxazinone
compound (1) with hydrazine hydrate in boiling ethanol
FIGURE 2 Mechanism of synthesis of benzoxazinone
SCHEME 5 Synthesis of benzimidazole compound (2)
afforded
3-amino-2-benzylquinazolin-4(3H)-one
(5)
(Scheme 8).
The structure of compound (5) was confirmed by its
1H NMR spectrum when run DMSO-d6 shows the fol-
lowing signals ppm at δ 3.7 (2H, s, CH2Ph), 7.1 to 8.5
(9H, m, ArH), and 11.2 (2H, t, NH2) indicating the for-
mation of quinazolinone and its IR spectrum exhibits
strong absorption bands at 1672, 3198, and 3315 cm−1
attributable to υC=O and υNH bonding and nonbonding
respectively. Its structure confirmed by its 13C NMR
analysis in DMSO-d6 shows the following signals ppm:
169.603 (2C), 140.797, 134.845 (2C), 131.087, 129.551,
128.617 (2C), 127.024, 122.760, 119.851, 116.407, and
44.689.
SCHEME 6 Synthesis of ester (3)
Quinazolinone
derivative
ethyl
2-(2-benzyl-
4-oxoquinazolin-3(4H)-yl)acetate (3) was obtained via
unambiguous route via the reaction of benzoxazinone (1)
with ethyl glycinate hydrochloride in refluxing ethanol
(Scheme 6).
The structure of compound (3) was confirmed by its
1H NMR spectrum when run DMSO-d6 shows the follow-
ing signals ppm at δ 3.4 (2H, s, CH2Ph), 3.9 (3H, t, CH3
(ethyl)), 4.1 (2H, q, CH2 (ethyl)), 4.8 (2H, s, CH2), 7.1 to
8.5 (9H, m, ArH) and absence of NH proton peak. More-
over, its IR spectrum revealed strong absorption bands at
1672, 1752 cm−1 attributable to υC=O of two carbonyl
groups and devoid any band for υNH conforming the for-
mation of ester. Its structure confirmed by its 13C NMR
analysis in DMSO-d6 shows the following signals ppm:
169.593 (2C), 167.685 (2C), 161.343, 155.524, 140.787,
134.902 (3C), 131.087, 129.551, 129.551, 128.769 (3C),
127.1 (2C), 126.213, 122.741, 119.832 (2C), 61.600 (2C),
45.519, 44.708, 40.988, and 14.014 (2C, these two signals
confirm the formation of ester).
EIMS of compound (5) showed peaks at m/e 251(M•+),
146 (M-PhCH2, NH2)•+, 119 (M-PhCH2, NH2, HCN)•+,
91(M-PhCH2, NH2, HCN, CO)•+, and 65.
The interaction of compound (5) with phthalic
anhydride gave the lactone derivative (2-benzyl-
3-((3-oxoisobenzofuran-1(3H)-ylidene)amino)quinazolin-
4(3H)-one)(6) (Scheme 9).
IR spectrum of compound (6) revealed strong absorp-
tion bands at 1660, 1747 cm−1 due to υmax of two car-
bonyl groups confirming the formation of the lactone
ring. EIMS of compound (6) showed m/e at 381 (M•+).
1
When spectrum of compound (6) run in DMSO, the H
NMR exhibits signals at δ 3.7 (s, 2H, benzylic proton) and
7.1 to 8.5 (m, 13H, ArH).

RADWAN ET AL.
5
Benzoxazinone (1) > Quinazolinone (hydrazide) (4) > Quinazolinone
(ester) (3) > Quinazolinone fused with Benzofuranone (6) >
Benzoimidazole (2) > Quinazolinone (5)
FIGURE 4 Illustrated the order of investigated compounds,
represented heterocyclic classes, according to their activity against
human hepatocellular carcinoma cell line (HepG2)
SCHEME 8 Synthesis of quinazolinone derivative (5)
against HepG2 cell line at 100 μm can be summarized in
Figure 4.
At 100 μm, the arrangement of tested compound
toward the same cell line was different than at 10 μm
(Figure 5). This different may be due to the increasing
the concentration of compounds. Hence, the highest anti-
cancer activity was displayed by benzoxazinone com-
pound (1) which showed cancer cell viability (76.24%).
Moreover, all remaining compounds exhibited weak
inhibitory activity. By comparing the activity of com-
pounds reported in this study to their structures, the
structure-activity relationship can be predicted. Ben-
zoxazinone compound (1), possess oxygen atom in addi-
tion to a nitrogen atom, is the most potent compound
toward HepG2 cell line. This activity may be related to
the electronegativity of the oxygen in comparison to the
nitrogen atom and the ability to be only a hydrogen bond
acceptor. Dependence on all previous data, the mecha-
nism of kill cancer cells can be predicted.
SCHEME 9 Synthesis of benzofuranone derivative (6)
Quinazolinone fused with Benzofuranone (6) > Benzoxazinone (1)
> Benzoimidazole (2) > Quinazolinone (ester) (3)> Quinazolinone
(5)> Quinazolinone (hydrazide) (4).
FIGURE 3 Illustrated the order of investigated
compounds, represented heterocyclic classes, according to their
activity against human hepatocellular carcinoma cell line
(HepG2)
2.2 | Anticancer activity
2.2.1 | Anticancer activity against
human HepG2
The mechanism of inactivation of enzyme, as one
way to treat cancer disease, for example, seine protein-
ase[27] (such as α-chymotrypsin enzyme,[11] neutrophil
elastase,[28] or rhomboid proteases[12]), by benzoxazinone
is depended upon the nucleophilic reaction of ben-
zoxazinone with enzyme hydroxyl group at the active site
of the enzyme,[11] which leads to ring opening and forma-
tion of an acylated enzyme (inactive form).[29] The inhibi-
tory activity of benzoxazinone may be based on the
electrophilicity of the lactone carbonyl group which may
form a hydrogen bond with enzyme[11] (Scheme 3).
When oxygen atom of benzoxazinone moiety is rep-
laced with the nitrogen atom to yield quinazolinone, the
activity decreased as shown in Scheme 10, because the
electronegativity of oxygen is greater than nitrogen.
We conclude from the above that benzoxazinone
compound (1) was the best compound among tested com-
pounds having slight effect toward human HepG2.
This fact stimulates us to focus on the benzoxazinone
compound. So, we tested various concentrations of
2-benzyl-3,1-benzoxazin-4-one ranging from 0.01, 0.1,
1, 10, 100 μg/mL against HepG2 using SRB to evaluate
IC50 (the inhibition concentration where the response is
reduced by half) (Table 1).
The synthesized compounds were screened for their anti-
cancer activity against human HepG2 using two different
methods, SRB and tetrazolium (MTT) assays. In the SRB
assay, the dye binds to basic amino acids of cellular pro-
teins and cell density determination refers to total protein
mass.[25] While, tetrazolium (MTT) technique depended
upon mitochondrial dehydrogenase enzyme in which
these enzymes can reduce the tetrazolium dye to its insol-
uble formazan with a purple color.[26]
First, we are screening six compounds against human
HepG2 using SRB assay at two concentrations 10 μm and
100 μm. The obtained results revealed that six investi-
gated compounds exhibited different degrees of activity.
At 10 μm, all compounds had slight effects on HepG2.
However, the activities of different tested heterocyclic
compounds against HepG2 cell line can be summarized
in Figure 3.
From these results, we can conclude that compound
(6) with benzofuranone moiety has the best activity
among other tested compounds. This may be due to
furanone ring which contain carbonyl group besides
oxygen atom.
Also, the same behavior, slight activities of tested
compounds against HepG2 cell line, has been indicated
at 100 μm. So, the activities of the tested compounds
Using sigmablot program version 6, the half maximal
inhibitory concentration (IC50) value was calculated and
the standard curve was drawn as shown in Figure 6.

6
RADWAN ET AL.
120
100
80
60
40
20
0
10 µm
100 µm
1
2
3
4
5
6
Tested compounds
FIGURE 5 Comparison between in vitro activities of
investigated compounds against HepG2 at two concentrations
10 μm and 100 μm
FIGURE 6 Spectrum of the antiproliferative activity of the
compound (1) against HepG2 via sulforhodamine B (SRB) assay
TABLE 2 In vitro cytotoxic activity of tested compounds
against hepatocellular carcinoma cell line (HepG2) using MTT
assay
Compound
IC50 (μM)a
64.02 3.6
51.94 3.2
17.08 1.4
42.33 3.0
10.71 0.9
26.83 2.1
34.52 2.5
4.50 0.2
SCHEME 10 Synthesis of quinazolinone moiety
Acidb
1
2
TABLE 1 In vitro anticancer assay for benzoxazinone
compound (1) against HepG2 at different concentrations
3
4
% Viabilitya
5
Compound
Concentration
Mean
100.00
94.89
86.76
81.85
75.77
62.36
SDb
6
1
0
0
DOXc
0.01
0.1
1
2.880709
1.501332
0.66099
0.880606
0.27677
aIC50 (μM): (mean SD), 1 to 10 (very strong), 11 to 20 (strong), 21 to
50 (moderate), 51 to 100 (weak), and above 100 (noncytotoxic).
bAcid: 2-(2-phenylacetamido)benzoic acid.
cDOX: doxorubicin.
10
100
very strong to weak. On the contrast to SRB assay, com-
pound (4), quinazolinone hydrazide derivative, was
found to be the most potent compound against HepG2
cell line. This activity may be due to the presence of
hydrazide moiety. On the other hand, the lowest potent
compound was benzoxazinone compound (1). However,
compound (1), possess oxazinone ring, was more potent
than acid (2-(2-phenylacetamido)benzoic acid) owing
opened ring. It proved that the activity of compound (1)
was arising from the presence of oxazinone ring.
In tetrazolium (MTT) assay, the order of investigated
compounds according to their activity against human
HepG2 has been different from these results have been
obtained in SRB assay. These differences can be due to
the differences between mechanisms of the two methods,
SRB and tetrazolium (MTT) assays. In the SRB assay, the
dye binds to basic amino acids of cellular proteins.[25]
aThe % viability: percentage of cancer cells is alive.
bSD is standard deviation.
We found that compound (1) has slight antip-
roliferative activity against hepatocellular carcinoma cell
line. From this value, we conclude that this compound
had not anticancer activity against HepG2 using SRB
assay.
This stimulated us to evaluate the cytotoxicity effect
for these tested compounds against HepG2 using tetrazo-
lium (MTT) assay (Table 2). Doxorubicin was used as a
positive control.
These results can be summarized in Figure 7.
The obtained results (Table 2, Figure 7) revealed that
six investigated compounds exhibited different degrees of
activity against HepG2 using MTT assay beginning from

RADWAN ET AL.
7
Quinazolinone (hydrazide) (4)> Benzoimidazole (2)> Quinazolinone (5) >
Quinazolinone fused with Benzofuranone (6)> Quinazolinone (ester) (3) >
Benzoxazinone (1)> Acid.
TABLE 3 DNA/methyl green colorimetric assay of the DNA-
binding compounds using MTT assay
Compound
IC50 (μM)a
74.89 3.2
81.47 3.5
33.96 1.8
49.42 2.5
28.35 1.5
40.06 2.1
42.81 2.3
31.27 1.8
FIGURE 7 Illustrated the order of investigated compounds,
represented heterocyclic classes, according to their activity
against human hepatocellular carcinoma cell line (HepG2) using
MTT assay
Acidb
1
2
3
While, tetrazolium (MTT) technique depended upon
mitochondrial dehydrogenase enzyme in which these
enzymes can reduce the tetrazolium dye to its insoluble
formazan with a purple color.[26] This can be appearing
clarify when G. Zheng et al. found that isonicotinic
acid hydrazide, owing hydrazide moiety, cause human
hepatoblastoma cell line (HepG2), healthy cell, injury
by mitochondrial DNA damage.[30] In addition to, MTT
is based on mitochondrial uptake.[7] From all the previ-
ous reasoning, it can be easily explained the difference
between the obtained results from two assays. In tetra-
zolium (MTT) assay, compound (4), owing hydrazide
moiety, could be damaging mitochondrial DNA of
human HepG2, cancerogenic cell line. To elicit this
prediction, DNA/methyl green assay was performed.
Methyl green reversibly binds to DNA, and the colored
complex is stable at neutral pH, whereas free methyl
green fades at this pH value. DNA-binding active com-
pounds displace DNA from its methyl green complex.
The displacement was determined by a spectrophoto-
metric assay as a decrease in the absorbance at 630 nm.
The obtained results were shown in Table 3.
The results of the binding DNA test were in line
with cytotoxicity studies, using tetrazolium (MTT)
assay, where the most potent compound, compound
(4), quinazolinone (hydrazide), showed the best DNA
binding. Also, the lowest potent compound, compound
(1), benzoxazinone compound, showed the worst DNA
binding and the same arrangement of tested com-
pounds, in tetrazolium (MTT) cytotoxicity assay, was
noticed. There is only one exception that acid showed
DNA binding better than benzoxazinone compound
(1). So, other studies will be performed to elicit the
underlying mechanism of the activity benzoxazinone
compound (1) toward HepG2 cell line. These results
can be summarized in Figure 8.
4
5
6
DOXc
aIC50 values represent the concentration (mean SD, n = 3-5 separate
determinations) required for a 50% decrease in the initial absorbance of the
DNA/methyl green solution.
bAcid: 2-(2-phenylacetamido)benzoic acid.
cDOX: doxorubicin.
Quinazolinone (hydrazide) (4) > Benzoimidazole (2)>
Quinazolinone (5)> Quinazolinone fused with Benzofuranone
(6)> Quinazolinone (ester) (3) > Acid > Benzoxazinone (1).
FIGURE 8 Illustrated the order of investigated compounds,
represented heterocyclic classes, according to their DNA binding
affinity
From the obtained results, compound (4), hydrazide
derivative, has been showed the most potent compound
against Hela cell line. Furthermore, compound (1), ben-
zoxazinone compound, has been showed the lowest
potent compound. Likewise, arrangement of tested com-
pounds according to their activity toward Hela cell line
was the same to their arrangement for the previous cyto-
toxicity studies, HepG2 cell line. These results can be
summarized in Figure 9.
2.2.3 | Anticancer activity against
human amnion (WISH)
To further investigate their toxicity of these tested com-
pounds on normal human, the cytotoxicity effect of the
tested compounds was performed against human amnion
(WISH) cell line using tetrazolium (MTT) assay. This cell
line has been selected as normal human cell of human
epithelioid carcinoma (Hela) cell line. This selection is
aiming to study toxicity of tested compounds upon uterus
cells which represented by WISH cell line. The obtained
results were indicated in Table 5.
2.2.2 | Anticancer activity against
human epithelioid carcinoma (Hela)
Moreover, tested compounds were further tested against
human epithelioid carcinoma (Hela) cell line using tetra-
zolium (MTT) assay. The obtained results were indicated
in Table 4.
From the above data, compounds 5 (quinazolinone)
and 6 (quinazolinone fused with benzofuranone) were
less toxic toward WISH cell line. So, they are more safety

8
RADWAN ET AL.
TABLE 4 In vitro cytotoxic activity of tested compounds
against human epithelioid carcinoma (Hela) cell line using MTT
assay
Quinazolinone fused with Benzofuranone (6)> Quinazolinone (5) >
Benzoxazinone (1)> Benzoimidazole (2)> Acid> Quinazolinone
(hydrazide) (4) > Quinazolinone (ester) (3).
IC50 (μM)a
58.60 3.4
62.91 3.6
13.90 1.2
31.04 2.2
8.14 0.7
19.78 1.6
22.16 1.8
5.57 0.4
FIGURE 10 Illustrated the order of investigated compounds,
represented heterocyclic classes, according to their activity against
human amnion (WISH) cell line using MTT assay
Compound
Acidb
1
2
80
3
4
60
Hela
5
40
WISH
6
20
0
DOXc
aIC50 (μM): (mean SD), 1 to 10 (very strong), 11 to 20 (strong), 21 to
50 (moderate), 51 to 100 (weak), and above 100 (noncytotoxic).
bAcid: 2-(2-phenylacetamido)benzoic acid.
cDOX: doxorubicin.
FIGURE 11 Comparative toxicity of investigated compounds
against cancerogenic cell line (Hela) and healthy cell line (WISH)
Quinazolinone (hydrazide) > Benzoimidazole > Quinazolinone > Quinazolinone
fused with Benzofuranone> Quinazolinone (ester) > Acid > Benzoxazinone.
compounds, except compound 6 (quinazolinone fused
with benzofuranone) which is the lowest toxic com-
pound. But, compound 5 was more potent against can-
cerogenic cell line (Hela) than compound 6.
FIGURE 9 Illustrated the order of investigated compounds,
represented heterocyclic classes, according to their activity against
human epithelioid carcinoma (Hela) cell line using MTT assay
TABLE 5 In vitro toxicity activity of tested compounds against
human amnion (WISH) cell line using MTT assay
2.3 | Antioxidant activity screening
assay; ABTS method
Compound
IC50 (μM)a
24.90 1.9
30.26 2.3
29.15 2.3
19.17 1.7
22.48 1.9
48.35 3.2
51.26 3.5
Acidb
The antioxidant activities of tested compounds were eval-
uated. The obtained results were compared with ascorbic
acid as antioxidant compounds (Table 6).
1
2
3
4
5
6
The results showed clearly that, compound 4,
quinazolinone (hydrazide), had the best activity, while
compound 1, benzoxazinone compound, exhibited the
lowest activity. Introduction hydrazide moiety may be
enhancing the antioxidant property of compound 4. Like-
wise, arrangement of tested compounds according to
their antioxidant activities the same to their arrangement
for the previous cytotoxicity studies, human epithelioid
carcinoma (Hela) and human hepatocellular carcinoma
(HepG2) cell lines. These results can be summarized in
Figure 12.
aIC50 (μM): (mean SD), the highest value indicate that the lowest toxic
compounds because it expresses the cell viability of normal, healthy,
cell line.
bAcid: 2-(2-phenylacetamido)benzoic acid.
than other tested compounds. While, compound (3) was
more active against WISH cell line. These results can be
summarized in Figure 10.
Ethyl 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acetate (3),
2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide
(4),
Figure 11 shows the effect of the tested compounds
on the cancerogenic cell, human epithelioid carcinoma
(Hela), and compared them to their effect on the healthy
cell line, human amnion (WISH).
From the competitive studies, compound 5,
quinazolinone derivative, was found the best bioactive
compound among tested compounds because it was less
toxic upon healthy cell line (WISH) than other tested
3-amino-2-benzylquinazolin-4(3H)-one (5) represented
quinazolinone class and 2-benzyl-3-((3-oxoisobenzofuran-1
(3H)-ylidene)amino) quinazolin-4(3H)-one (6) are deriva-
tives for quinazolinone (all contain, quinazolinone moiety).
Compound (4) has three hydrogen atoms available for for-
mation of a hydrogen bond with nitrogenous bases of DNA,
while compound (5) has only two hydrogen atoms. While,
compounds (3) and (6) have no hydrogen atoms available

RADWAN ET AL.
9
TABLE 6 Antioxidant activity assay (ABTS) of tested
compounds
develop an appropriate efficient strategic treatment for
patients having hepatic and/or uterus cancers. In the
future, we will focus upon compounds belong to the
quinazolinone class according to their promising activi-
ties against different cell lines.
Compound
Absorbance of samples
% Inhibition
Control of ABTSa
0.518
0.058
0.421
0.442
0.337
0.392
0.254
0.358
0.368
0
Ascorbic-acid
Acidb
88.8
18.7
14.7
34.9
24.3
51.0
30.9
28.9
1
2
3
4
5
6
4 | EXPERIMENTAL
4.1 | Chemistry
4.1.1 | General
Melting points were determined on an electric melting
point apparatus and are uncorrected. FTIR spectra (KBr
disk) were performed in the microanalytical center, Cairo
University, Egypt. The infrared spectra were recorded with
FTIR-4100 spectrophotometer product of Jasco, Japan.
The spectra were recorded from 4000 cm−1 to 450 cm−1.
13C NMR spectra were determined on JNM-ECA II
aABTS: 2,2'-azino-bis (3-ethyl benzothiazoline-6-sulfonic acid).
bAcid: 2-(2-phenylacetamido)benzoic acid.
Quinazolinone (hydrazide) (4) > Benzoimidazole (2) > Quinazolinone
(5) > Quinazolinone fused with Benzofuranone (6)> Quinazolinone
(ester) (3) > Acid > Benzoxazinone (1).
1
500 MHz at NMR unit, Mansoura University. H NMR
FIGURE 12 Illustrated the order of investigated compounds,
represented heterocyclic classes, according to their antioxidant
activities
spectra were determined on Bruker Avance (III) 400 MHz
at Center for Drug Discovery research and development,
1
Ain Shams University, while H NMR spectrum of com-
for hydrogen bond formation. Compound (6) might be react
with nitrogen bases of DNA via acylation more than com-
pound (3), thus the order of reactivity 4 > 5 > 6 > 3. This
agreed well with the results obtained in Tables 2–4, and 6.
Reactivity of compound (2) might be due to the presence of
benzimidazole moiety and compound (1) could be due to
oxazinone moiety.
pound (1) was determined on JNM-ECA II 500 MHz at
NMR unit, Mansoura University. The chemical shifts are
in ppm. The solvent was DMSO. We used dimethyl sulfox-
ide (DMSO) as solvent for NMR analysis because we used
it for anticancer assays. The splitting patterns (multiplici-
ties) in 1H NMR were designated as singlet (s), doublet (d),
triplet (t), quarter (q), and multiplet (m). Mass spectra
were recorded using GC MS—Qp-2010 Plus Shimadzu at
microanalytical center, Cairo University, Egypt. Mass spec-
tra were recorded at 70 ev.
3 | CONCLUSIONS
In conclusion, in vitro, 2-(2-benzyl-4-oxoquinazolin-3
(4H)-yl)acetohydrazide (4) is the most potent antioxidant
and anticancer agents against human epithelioid carci-
noma (Hela). Moreover, it showed anticancer activity
against human HepG2 in which it was proved using
DNA binding affinity. Although, 3-amino-2-ben-
zylquinazolin-4(3H)-one (5) is less potent anticancer
agent against (Hela) than compound (4) but it is more
safe against normal cell, human amnion (WISH). In spite
of compound 2-benzyl-3-((3-oxoisobenzofuran-1(3H)-
ylidene) amino)quinazolin-4(3H)-one (6) is the most safe,
but compound (5) was more potent against cancerogenic
cell lines, Hela and HepG2. So, compound (5),
quinazolinone derivative, was the best compound among
other tested compound.
4.1.2 | Synthesis tested compound 1 to 6
Synthesis of 2-(2-phenylacetamido)benzoic acid
To a solution of anthranilic acid (36.46 mmol, 5 g) in pyr-
idine (10 mL), phenylacetyl chloride (54.67 mmol,
7.23 mL) was added gradually with continuous stirring.
After the addition complete, the reaction mixture was
stirred in an ice bath for two hours and then poured upon
hydrochloric acid containing crushed ice. The solid prod-
uct that deposited was collected by filtration and washed
with water, dried and recrystallized from ethanol to give
2-phenylacetylamino benzoic acid as beige crystals, m.
p. 166 to 168ꢀC, yield 91.43%.
A further in-depth study to elucidate the underline
mechanism (s) involved in such promising activities
against tested cell lines will be performed in order to
Synthesis of 2-benzyl-3,1-benzoxazin-4-one (1)
A
solution of 2-phenylacetylamino benzoic acid
(7.83 mmol, 2 g) in acetic anhydride (1.5 mL) was heated

10
RADWAN ET AL.
under reflux in a water bath for one hour. The reaction
mixture was left until solidify to give benzoxazinone (1).
Benzoxazinone compound (1) has been prepared and
divided it into different lots for physical analysis (IR,
mass, 1H NMR and 13C NMR) and biological assays.
These lots were stored under control condition (desicca-
tor) until use.
Yield 95.76%, yellow crystals, m.p. 152ꢀC, IR (KBr),
υ/cm−1: 1760 (C=O) of δ-lactone, and lacked any band
for υNH. 1H NMR (500 MHz): δ = 4 (s, 2H, CH2Ph), 7.3 to
8.5 (m, 9H, ArH) and devoid any band for a hydroxyl
group. 13C NMR (500 MHz): δ = 169.536(2C), 161.085,
159.149, 145.890, 136.839, 134.702 (2C), 131.049, 129.523
(2C), 128.607 (2C), 127.892 (2C), 126.976 (2C), 119.765,
116.503, 44.698, 40.425.
Synthesis of 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)
acetohydrazide (4)
A mixture of ester (3) (6.21 mmol, 2 g) and hydrazine
hydrate (63.91 mmol, 3.1 mL) in ethanol (30 mL) was
heated under reflux for eight hours, left to cool. The solid
product that deposited was collected by filtration, dried and
recrystallized from ethanol to give the pure product (4).
White crystals, m.p. 260 to 262ꢀC, IR (KBr), υ/cm−1:
1
1669 (C=O), 3170 (NH), 3323 (NH). H NMR (400 MHz):
δ = 3.4 (s, 2H, CH2Ph), 5 (s, 2H, CH2), 7.2 to 8.8 (m, 9H,
ArH), 9.3 (s, 1H, NH). 13C NMR (500 MHz): δ = 180.934,
166.131, 161.409, 155.992, 146.901, 135.541, 134.654,
128.779 (2C), 127.005, 126.833 (2C), 119.870, 44.403, 41.017.
Synthesis of 3-amino-2-benzylquinazolin-4(3H)-one (5)
A mixture of benzoxazinone (1) (8.47 mmol, 2 g) and
hydrazine hydrate (12.99 mmol, 0.63 mL) in ethanol
(30 mL) was heated under reflux for three hours, left to
cool. The solid product that deposited was collected by
filtration, dried and recrystallized from ethanol to give
the pure product (5).
Synthesis of N-(2-(1H-benzo[d]imidazol-2-yl)phenyl)-
2-phenyl acetamide (2)
O-Phenylenediamine (12.65 mmol, 1.3674 g) was added
to a solution of benzoxazinone (1) (8.43 mmol, 2 g) in
ethanol (30 mL). The reaction mixture was heated under
reflux for three hours, left to cool. The solid product that
deposited was collected by filtration, dried and rec-
rystallized from benzene to give pure product (2).
Yield 48.80%, grey crystals, m.p. 158 to 160ꢀC, IR
(KBr), υ/cm−1: 1674 (C=O), 3283 (nonbonded NH), 3208
(bonded NH). 1H NMR (400 MHz): δ = 3.4 (s, 2H,
CH2Ph), 7.1 to 8.5 (m, 9H, ArH), 11.1 (s, 1H, NH). 13C
NMR (500 MHz): δ = 169.612 (2C), 140.797, 134.855
(2C), 131.096, 129.551, 128.626, 127.033, 122.760, 119.860,
44.698. HRMS (ESI): m/z calcd for C21H17N3O: 327.38
[M + H]+; found: 327.00.
Yield 50.52%, white crystals, m.p. 160 to 162ꢀC, IR
(KBr), υ/cm−1: 1672 (C=O), 3198 (NH), 3315 (NH). H
1
NMR (400 MHz): δ = 3.7 (s, 2H, CH2Ph), 7.1 to 8.5 (m,
9H, ArH), 11.2 (t, 2H, NH2). 13C NMR (500 MHz):
δ = 169.603 (2C), 140.797, 134.845 (2C), 131.087, 129.551,
128.617 (2C), 127.024, 122.760, 119.851, 116.407, 44.689.
HRMS (ESI): calcd for C15H13N3O: 251.28 [M + H]+;
found: 251.10.
Synthesis of 2-benzyl-3-((3-oxoisobenzofuran-1(3H)-
ylidene) amino)quinazolin-4(3H)-one (6)
A mixture of quinazolinone derivative (5) (4.78 mmol,
1.2 g) and phthalic anhydride (7.16 mmol, 1.06 g) in etha-
nol (30 mL) was heated under reflux for three hours, left
to cool. The solid product that deposited was collected by
filtration, dried and recrystallized from ethanol to give
the pure compound (6).
Yield 39.09%, white crystals, m.p. 162 to 164ꢀC, IR
(KBr), υ/cm−1: 1660 (C=O), 1747 (C=O). 1H NMR
(400 MHz): δ = 3.7(s, 2H, benzylic proton), 7.1 to 8.5 (m,
13H, ArH). HRMS (ESI): calcd for C23H15N3O3: 381.38
[M + H]+; found: 381.35.
Synthesis of ethyl 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)
acetate (3)
A mixture of benzoxazinone (1) (8.43 mmol, 2 g) and gly-
cine ethyl ester hydrochloride (16.94 mmol, 2.3558 g) in
ethanol/anhydrous sodium acetate mixture (30 mL/0.5 g)
was refluxed for three hours and then left to cool. The
solid product that deposited was collected by filtration,
dried and recrystallized from ethanol to give the pure
product (3).
Yield 65.28%, yellow crystals, m.p. 94 to 96ꢀC, IR
(KBr), υ/cm−1: 1672 (C=O), 1752 (C=O (ester)) and
devoid any band for υNH. 1H NMR (400MHz): δ = 3.4 (s,
2H, CH2Ph), 3.9 (t, 3H, CH3 (ethyl)), 4.1 (q, 2H, CH2
(ethyl)), 4.8 (s, 2H, CH2), 7.1 to 8.5 (m, 9H, ArH). 13C
NMR (500 MHz): δ = 169.593 (2C), 167.685 (2C),
161.343, 155.524, 140.787, 134.902 (3C), 131.087, 129.551,
129.551, 128.769 (3C), 127.1 (2C), 126.213, 122.741,
119.832 (2C), 61.600 (2C), 45.519, 44.708, 40.988,
14.014 (2C).
4.2 | Anticancer activity
4.2.1 | For SRB assay
Cell line
Hepatocellular carcinoma (HepG2) cell lines were
obtained from Nawah Scientific Inc. (Mokatam, Cairo,

RADWAN ET AL.
11
Egypt). Cells were maintained in DMEM (Dulbecco's
Modified Eagle Medium) media supplemented with
100 mg/mL of streptomycin, 100 units/mL of penicillin
and 10% of heat-inactivated fetal bovine serum in humid-
ified, 5% (vol/vol) CO2 atmosphere at 37ꢀC.
to test the inhibitory effects of compounds upon cell
growth using the MTT test, cell lines mentioned above
were used. The conversion of the yellow tetrazolium bro-
mide (MTT) to a purple formazan derivative by mito-
chondrial succinate dehydrogenase in viable cells is the
basis of the colorimetric assay. The cells were cultured in
RPMI-1640 medium with 10% fetal bovine serum. Antibi-
otics were added 100 units/mL penicillin and 100 μg/mL
streptomycin at 37ꢀC in a 5% CO2 incubator. The cells
Cytotoxicity assay
Cytotoxicity assays were performed at Nawah Scientific
Inc. (Mokatam, Cairo, Egypt). Cell viability was assessed
by SRB assay. Aliquots of 100 μL cell suspension
(5 × 103 cells) were in 96-well plates and incubated in
complete media for 24 hours. Lung cancer cells were
treated with another aliquot of 100 μL media containing
six compounds at two concentrations 10 μm and 100 μm
for quick screening while tested compound (1) at various
concentrations ranging from 0.01, 0.1, 1, 10, and 100 μg/
mL for routine analysis. Tested compounds were
completely soluble in DMSO for anticancer assay. After
72 hours of drug exposure, cells were fixed by replacing
media with 150 μL of 10% trichloroacetic acid (TCA) (act
as acidic medium) and incubated at 4ꢀC for one hour.
TCA solution was removed, and the cells were washed
five times with distilled water. Aliquots of 70 μL SRB
solution (0.4% wt/vol) were added and incubated in a
dark place at room temperature for 10 minutes. Plates
were washed three times with 1% acetic acid and allowed
to air-dry overnight. Then, 150 μL of TRIS (10 mM) was
added to dissolve protein-bound SRB stain; the absor-
bance was measured at 540 nm using a BMG LABTECH
FLUOstar Omega microplate reader (Ortenberg, Ger-
many). The same assay was performed for compound (1)
toward HepG2 at different concentrations ranging from
0.01, 0.1, 1, 10, and 100 μg/mL for routine analysis.
were seeded in
a 96-well plate at a density of
1.0 × 104 cells/well at 37ꢀC for 48 hours under 5% CO2.
After incubation, the cells were treated with different
concentrations of compounds and incubated for 24 hours.
After 24 hours, of drug treatment, 20 μL of MTT solution
at 5 mg/mL was added and incubated for 4 hours. Tested
compounds were completely soluble in DMSO for anti-
cancer assay. DMSO in volume of 100 μL is added into
each well to dissolve the purple formazan formed. The
colorimetric assay is measured and recorded at absor-
bance of 570 nm using a plate reader (EXL 800). The rela-
tive cell viability in percentage was calculated as (A570 of
treated samples/A570 of untreated sample) × 100.[31,32]
4.3 | Colorimetric assay for compounds
that bind DNA
One hundred mL of 0.05 M Tris-HCl buffer (pH 7.5)
mixing with 7.5 mM MgSO4 was for suspending DNA
methyl green (20 mg); using a magnetic stirrer, the mix-
ture was stirred at 37ꢀC for 24 hours. 10, 100, 1000 mg of
samples have been dissolved in the ethanol, under vac-
uum solvent was removed, and 200 μL of the
DNA/methyl green solution were added to each tube.
Samples in the dark were incubated at ambient tempera-
ture. The samples were finally absorbed at 642 to 645 nm
after 24 hours. Readings for original absorption have
been fixed and normalized in the untreated standard.[33]
4.2.2 | For MTT assay
Cell line
Hepatocellular carcinoma (HePG-2), Epithelioid carci-
noma (Hela), and Human amnion (WISH). The cell lines
were obtained from ATCC via Holding company for bio-
logical products and vaccines (VACSERA), Cairo, Egypt.
4.4 | Antioxidant activity screening
assay using ABTS method
Chemical reagents
Antioxidant activity determinations were evaluated from
the bleaching of 2,20-azino-bis(3-ethylbenzothiazoline-
6-sulphonic acid (ABTS) derived radical cations. The
reaction of ABTS (60 mL) with MnO2 (3 mL, 25 mg/mL)
in phosphate buffer solution (10 mM, pH 7, 5 mL) was
performed for synthesize ABTS [2,20-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid)] for obtaining radical
cation. Centrifugation and filtration of the solution did
after shaking it for a few minutes. The resulting green-
blue solution (ABTS radical solution) of was absorbed
The reagents were Roswell Park Memorial Institute
(RPMI-1640) medium, MTT and DMSO (Sigma co.,
St. Louis, Missouri) and Fetal Bovine serum (GIBCO,
UK). Doxorubicin was used as a standard anticancer drug
for comparison.
Cytotoxicity assay
Cytotoxicity assays were performed at Faculty of Phar-
macy, Mansoura University, Mansoura, Egypt. In order

12
RADWAN ET AL.
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and recorded at λmax 734 nm to obtain A control. The
addition of (20 mL of 1 mg/mL) solution of the tested
sample in spectroscopic grade MeOH/buffer (1:1, vol/vol)
to the ABTS solution was for recording absorbance
(A test).[34] The inhibition has been determined
according to the following:
AðcontrolÞ−AðtestÞ
%Inhibition =
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AðcontrolÞ
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A standard antioxidant, ascorbic acid (20 mL, 2 mM)
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