Synthesis, in silico and in vitro assessment of new quinazolinones as anticancer agents via potential AKT inhibition

Article abstract of DOI:10.3390/molecules25204780

A series of novel quinazolinone derivatives (2–13) was synthesized and examined for their cytotoxicity to HepG2, MCF-7, and Caco-2 in an MTT assay. Among these derivatives, compounds 4 and 9 exhibited significant cytotoxic activity against Caco-2, HepG2, and MCF-7 cancer cells. Compound 4 had more significant inhibitory effects than compound 9 on Caco-2, HepG2, and MCF-7 cell lines, with IC50 values of 23.31 ± 0.09, 53.29 ± 0.25, and 72.22 ± 0.14μM, respectively. The AKT pathway is one of human cancer’s most often deregulated signals. AKT is also overexpressed in human cancers such as glioma, lung, breast, ovarian, gastric, and pancreas. A molecular docking study was performed to analyze the inhibitory action of newly synthetic quinazolinone derivatives against Homo sapiens AKT1 protein. Molecular docking simulations were found to be in accordance with in vitro studies, and hence supported the biological activity. The results suggested that compounds 4 and 9 could be used as drug candidates for cancer therapy via its potential inhibition of AKT1 as described by docking study.

Full text of DOI:10.3390/molecules25204780

Article
Synthesis, In Silico and In Vitro Assessment of New
Quinazolinones as Anticancer Agents via Potential
AKT Inhibition
1
2
3
4,
Ahmed A. Noser , Mohamed El-Naggar , Thoria Donia and Aboubakr H. Abdelmonsef *
1
Organic Chemistry, Chemistry Department, Faculty of Science, Tanta University, Tanta 31527, Egypt;
ahmed.nosir@science.tanta.edu.eg
Chemistry Department, Faculty of Sciences, University of Sharjah, Sharjah 27272, UAE;
2
melnagrr@sharjah.ac.ae
3
Biochemistry Division, Chemistry Department, Faculty of Science, Tanta University, Tanta 31527, Egypt;
thoria_donia@science.tanta.edu.eg
Chemistry Department, Faculty of Science, South Valley University, Qena 83523, Egypt
4
* Correspondence: aboubakr.ahmed@sci.svu.edu.eg
Received: 10 September 2020; Accepted: 17 October 2020; Published: 18 October 2020
Abstract: A series of novel quinazolinone derivatives (2–13) was synthesized and examined for their
cytotoxicity to HepG2, MCF-7, and Caco-2 in an MTT assay. Among these derivatives, compounds
4 and 9 exhibited significant cytotoxic activity against Caco-2, HepG2, and MCF-7 cancer cells.
Compound 4 had more significant inhibitory effects than compound 9 on Caco-2, HepG2, and MCF-
7 cell lines, with IC50 values of 23.31 ± 0.09, 53.29 ± 0.25, and 72.22 ± 0.14µM, respectively. The AKT
pathway is one of human cancer’s most often deregulated signals. AKT is also overexpressed in
human cancers such as glioma, lung, breast, ovarian, gastric, and pancreas. A molecular docking
study was performed to analyze the inhibitory action of newly synthetic quinazolinone derivatives
against Homo sapiens AKT1 protein. Molecular docking simulations were found to be in accordance
with in vitro studies, and hence supported the biological activity. The results suggested that
compounds 4 and 9 could be used as drug candidates for cancer therapy via its potential inhibition
of AKT1 as described by docking study.
Keywords: AKT1; cancer; quinazolinone; docking study; cytotoxic activity
1. Introduction
Cancer is one of the most widespread illnesses in the world [1]. In 2030, the number of new
cancer diagnoses is expected to be 21 million worldwide annually, with 17 million deaths because of
cancer every year and 75 million people living with cancer diagnoses [2]. Due to the resistance to
current cancer drugs and a lack of selectivity in tumor cells, the chemical design has become
increasingly sophisticated over the years [3–5]. Targeted cancer therapies are designed to improve
efficacy and selectivity by interfering with specific molecular targets and preventing the growth,
development, and spread of cancer [6]. The importance of PKB/AKT protein kinase for cellular
survival has been investigated in various cell types and animal systems and in response to several
stress factors. The serine-threonine kinase AKT, also known as PKB, is a proto-oncogenic key
player in cell proliferation, apoptosis, metabolism of glucose, and cell migration processes [7,8].
AKT activated by phosphorylation, which includes the binding of PI3K-phosphorylated
phosphoinositides (PI) called PIP3 AKT pleckstrin homology domain and subsequent
translocation to the plasma membrane and phosphorylation at two phosphorylation sites Thr308
and Ser473 by PDK1 and PDK2, respectively, resulting in its activation in tumor cells, so it was
Molecules 2020, 25, 4780; doi:10.3390/molecules25204780
www.mdpi.com/journal/molecules

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found that direct degradation of AKT deregulated AKT activity and promoted an apoptotic
process [9–11]. Three AKT isoforms have been identified in the human genome (AKT1, AKT2, and
AKT3) with a highly conserved pleckstrin homology domain. Under physiological conditions, AKT1
and AKT2 are ubiquitously expressed, whereas AKT3 expression is restricted, predominantly to
heart, kidney, brain, testis, lung, and skeletal muscle [12].
The inhibition of AKT has therefore been regarded as a promising therapeutic approach in
oncology for several years, and significant efforts have been made to find new effective and selective
anti-cancer drugs for AKT [6,13–16].
The quinazolinone moiety is founded in many natural products and pharmaceutical drugs. The
quinazolinone analogues have widespread applications ranging from biomedical science to material
science [17], such as antioxidant [18,19], antidiabetic [20], anti-inflammatory [21], antibacterial [22],
anticoagulant [23], antifibrotic [24,25], antiproliferative [26], anticonvulsant [27], and antituberculosis
[28]. Moreover, the quinazolinone moiety is considered to be a major class of promising structural
scaffold with PI3K inhibition [29]. Prompted by the aforementioned findings, and in attempt to
develop potent anticancer agents targeting AKT protein, a new series of novel quinazolinone
derivatives (2–13) was synthesized and investigated for their cytotoxic activity on cancer cell lines,
i.e., HepG2 (human liver cancer), MCF-7 (human breast cancer), and Caco-2 (human colon cancer).
As a part of our work, computer-based docking studies were performed, and absorption,
distribution, metabolism, excretion and toxicity (ADMET) properties, as well as the Structure Activity
Relationship (SAR) of the compounds, were determined to investigate their binding mode of
interactions with the active site of the target AKT1.
In this study, the synthesis and in silico and in vitro evaluation of a series of quinazoline
analogues as potential anticancer agents targeting AKT1 protein is described, as it is hyperactivated
in many cancers.
2. Results and Discussion
2.1. Chemistry of the Synthesized Compounds
The 2-phenyl-4H-benzo[d][1,3]oxazin-4-one (1) was synthesized and characterized as previously
described in the literature [30]. In the present study, 4-(4-oxo-2-phenyl quinazolin-3(4H)-yl)benzoic
acid (2) and 3-(4-oxo-2-phenylquinazolin-3(4H)-yl)benzoic acid (3) were obtained successfully by
refluxing compound 1 with p-amino benzoic acid and m-amino benzoic acid respectively in the
presence of pyridine, to give high yields of 88% and 87% respectively. Further chemical modification
of compounds 2 and 3 with hexadecanol in the presence of methanesulfonic acid and benzene as
solvent under reflux conditions using dean stark trap lead to formation of hexadecyl 4-(4-oxo-2-
phenylquinazolin-3(4H)-yl)benzoate (4) and hexadecyl 3-(4-oxo-2-phenyl quinazolin-3(4H)-
yl)benzoate (5), respectively, as shown in Scheme 1, with 90% and 88% yield. The structures of
compounds 2–5 were elucidated via NMR spectroscopy, FT-IR, and elemental analysis as shown in
Supplementary Materials.
The treatment of compound 1 with p-amino phenol leads to the formation of 3-(4-
hydroxyphenyl)-2-phenylquinazolin-4(3H)-one (6) with 91% yield. The chemical modification of
compound 6 with butyric acid and propionic acid in the presence of methanesulfonic acid and
benzene as solvent under reflux conditions using dean stark trap leads to the formation of 4-(4-oxo-
2-phenylquinazolin-3(4H)-yl)phenyl butyrate (7) and 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)phenyl
propionate (8) with 89% and 88% yield, respectively. The deprotonation of compound 7 using lithium
cyclohexylisopropylamine (LICHIPA) at −96 °C followed by alkylation using methyl iodide in the
presence of tetramethylethylenediamine (TMEDA) as catalyst lead to formation of (S)-4-(4-oxo-2-
phenylquinazolin-3(4H)-yl)phenyl 2-methylbutanoate (9) with 90% yield. While the alkylation of
compound 8 lead to the formation of (R)-4-(4-oxo-2-phenylquinazolin-3(4H)-yl)phenyl 2-
methylbutanoate (10) with 90% yield (the change of the configuration occur according to the principle
of order of addition, as represented in Scheme 2. The structures of compounds 6, 7, 8, 9 and 10 were

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characterized via NMR spectroscopy, FT-IR and elemental analysis as shown in Supplementary
Materials.
Formation of 3-hydroxy-4-(4-oxo-2-phenylquinazolin-3(4H)-yl)naphthalene-1-sulfonic acid (11)
with 91% yield was achieved by treatment of compound 1 with 4-amino-3-hydroxynaphthalene-1-
sulfonic acid.
The chemical modification of compound 11 with butyric acid and pentanoic acid in the presence
of methanesulfonic acid and benzene as solvent under reflux conditions using dean stark trap with
the same conditions mentioned above for the synthesis of compounds 7 and 8 afforded the formation
of 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)3-(butyryloxy)naphthalene-1-sulfonic acid (12) and 4-(4-
oxo-2-phenylquinazolin-3(4H)-yl)3-(pentanoxy)naphthalene-1-sulfonic acid (13) with 88% and 87%
yield, respectively, as shown in Scheme 3. The structures of compounds 11, 12, and 13 were
characterized using NMR spectroscopy, FT-IR and elemental analysis as represented in
Supplementary Materials.
O
O
N
Ph
p-aminobenzoic acid
(1)
m-aminobenzoic acid
pyridine
pyridine
COOH
O
N
O
N
N
COOH
Ph
(2)
N
Ph
(3)
hexadecanol, CH3SO3H
benzene
hexadecanol, CH3SO3H
benzene
O
(CH2)15
O
N
O
N
O
CH3
O
CH3
(CH2)15
N
O
Ph
(5)
N
Ph
(4)
Scheme 1. The synthesis pathway of compounds 2–5 from compound 1.

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OH
O
O
p-aminophenol
pyridine
O
N
N
Ph
N
Ph
(6)
(1)
butyric acid, CH3SO3H
benzene
propionic acid, CH3SO3H
benzene
O
O
O
O
O
O
N
N
N
Ph
(8)
N
Ph
(7)
LICHIPA,THF
LICHIPA,THF
HMPA
HMPA
EtI
MeI
Me
Et
O
Et
O
Me
O
N
O
N
O
O
N
N
Ph
Ph
(9)
(S form)
(10)
(R form)
Scheme 2. The synthesis pathway of compounds 6–10 from compound 1.

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SO3H
O
O
O
N
4-amino-3-hydroxy
naphthalene-1-
sulfonic acid
N
OH
Ph
N
Ph
pyridine
(1)
(11)
butyric acid, CH3SO3H
benzene
pentanoic acid, CH3SO3H
benzene
SO3H
O
SO3H
O
N
O
N
O
N
Ph
N
Ph
O
O
(12)
(13)
Scheme 3. The synthesis pathway of compounds 11–13 from compound 1.
2.2. In Silico Docking Study
AKT pathway plays an important role in multiple cell signaling mechanisms implicated in cell
metabolism, growth and division. Therefore, AKT1 has been selected as a promising target in cancer
treatment [13–16]. Compound 2–13 exhibited significant cytotoxic activities against Caco-2, HepG2,
and MCF-7 cancer cells, as represented in In Vitro Activity section. Based on these results, in silico
docking technique was executed to identify the potential inhibitors with high efficiency against
human AKT1 protein.
2.2.1. The Crystal Structure and Active Site of the Target
The crystallographic structure of molecular target AKT1 obtained from the PDB database (PDB
ID: 5WBL) was used for docking studies. Computational prediction tools declared that TYR46,
ARG59, PRO176, GLN178, TYR180, TRP191, PHE193, LYS196, and ASP204 are the binding pockets
of the target.
2.2.2. Molecular Docking Analysis
For in silico docking, the approach was performed using the PyRx virtual screening 3D tool. In
screening against AKT1, the synthesized compounds were docked to a three-dimensional model of
the target protein. Nine conformers are considered for each ligand–protein complex and the most
energetically favorable binding mode is chosen to identify the best-docked compound against human
AKT1 protein. The docking study exhibited nice fitting of the new synthetic compounds into the
active site of the target, as tabulated in Table 1. The quinazoline derivatives have significant
interaction poses with the modeled AKT1 through hydrogen bonds, π–π, π–cation, and π–σ

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interactions. Greater values of negative binding free energy ΔGb (reported in kcal/mol) indicate better
matching between the ligand molecule and target [31–34]. The reference compound doxorubicin had
the lowest binding free energy (ΔGb = −7.6 kcal/mol) and exhibited two π–π stacking with TRP191.
Compound 2 had a binding free energy ΔGb = −7.9 kcal/mol and exhibited π–π stacking with TRP191
at distances of 4.07 and 3.90 A°, respectively. Tryptophan (TRP) contains two phenyl and pyrrole
rings involved in forming two π–π interactions with phenyl ring of compound 2. Compound 3
interacts with protein at ARG59 and TYR46 through one hydrogen bond (O----H---O) plus two π–π
interactions with distances of 2.33, 4.04 and 5.10 A°, respectively. Tyrosine (TYR) contains a phenyl
ring involved in forming two π–π interactions with phenyl and pyrimidine rings of compound 3.
Compound 4, with the highest binding free energy (ΔGb = −10.2 kcal/mol), interacts with protein at
TYR180 forming one π–σ interaction at a distance of 2.84 A°. This is due to the interaction between
the pyrimidine ring of the compound and the aromatic side chain of Tryptophan. Compound 5
possesses π–π and π–cation interactions with the target protein through TRP191 and LYS196. This is
due to lysine (LYS) containing a positively charged ε-amino group that is involved in forming π–
cation interaction with phenyl moiety in the compound. In addition, compound 6 forms two π–π
interactions with the target through TRP191 at the distances of 4.41 and 3.94 A°, respectively.
Compound 9 (S-isomer), with a binding free energy ΔGb = –9.8 kcal/mol, showed π–π interaction with
the target protein through PHE193. The results showed that phenylalanine (PHE) contains an
aromatic ring that is involved in forming π–π interactions with the phenyl moiety of the compound.
Meanwhile, compound 10 (R-isomer) with a dock score of -7.7 kcal/mol exhibited two π–σ
interactions through TRP191 and TYR180. In addition, compound 11 showed three hydrogen bonds
with GLN178 and PRO176 at distances of 2.10, 2.09 and 2.07 A°, respectively. Compound 12 showed
rich network interactions, such as hydrogen bonds, π–π and π–cation interactions, with the target
through TRP191, ASP204 and LYS196. Finally, compound 13 showed π–π interaction with TRP191
and PHE193. Figure 1 represents 2D (Left side) and 3D (Right side) docking interactions between
molecules 2–13 and the active site of AKT1 protein.
Table 1. The binding free energy ΔGb (kcal/mol) of the newly synthetic compounds with AKT after
molecular docking.
Binding
Energy
(kcal/mol)
Docked Complex (Amino Acid-
Ligand) Interactions
Distance
(A°)
Structure
π–π interactions
compound 2—TRP191
COOH
O
4.07
3.90
2
3
−7.9
−9.3
N
compound 2---TRP191
N
O
Ph
H-bonds
compound 3---ARG59:O
π–π interactions
compound 3---TYR46
compound 3--- TYR46
π–σ interactions
2.33
N
COOH
(CH2)15
4.04
5.10
N
Ph
O
O
O
CH3
4
5
−10.2
−8.5
compound 4---TYR180:HE1
2.84
N
N
N
O
Ph
π–π interactions
compound 5---TRP191
compound 5---TRP191
compound 5---TRP191
compound 5---TRP191
π–cation interactions
3.92
3.91
3.99
4.51
O
CH3
(CH2)15
O
N
Ph

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compound 5---LYS196:NZ
π–π interactions
compound 6---TRP191
6.19
OH
O
N
4.41
3.94
6
−8.0
−9.8
N
compound 6---TRP191
Ph
π–π interactions
Et
O
O
Me
Et
O
9
O
compound 9---PHE193
4.00
N
N
O
Ph
π–σ interactions
compound 10---TRP191
Me
O
3.65
2.72
10
11
−7.7
−8.9
N
compound 10---TYR180:HE1
N
Ph
N
H-bonds
compound 11---GLN178:HE22
compound 11---GLN178:HE22
2.10
2.09
SO3H
O
N
compound 11----PRO176:O
2.07
OH
Ph
H-bonds
compound 12---TRP191:HE1
compound 12---ASP204:OD1
π–π interactions
2.48
2.24
SO3H
O
N
compound 12---TRP191
compound 12--- TRP191
compound 12--- TRP191
compound 12--- TRP191
π–cation interactions
compound 12---LYS196:NZ
compound 12---LYS196:NZ
π–π interactions
3.90
3.99
3.87
5.10
12
−9.2
N
O
Ph
O
4.67
5.24
compound 13--- TRP191
compound 13--- TRP191
compound 13--- PHE193
4.01
4.41
5.34
SO3H
O
13
−9.1
N
O
compound 13--- PHE193
4.80
N
Ph
O
All synthesized molecules with the best binding energy are represented with docking interactions in
the table showing H-bonding, π–π, π -cation, and π–σ interactions.

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Compound 2
Compound 3
Compound 4
Compound 5

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Compound 6
Compound 9
Compound 10
Compound 11

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Compound 12
Compound 13
Figure 1. Binding modes/interactions of the synthetic compounds with active sites of AKT1 protein.
(Left side) 2D representations demonstrating the molecular interactions between compounds and the
active site region of AKT1 protein. The amino acid residues are shown in three-letter code, H-bonds
are in pink doted lines, and π-interactions are in yellow lines. (Right side) 3D representations
demonstrating the molecular interactions between compounds and the active site region of AKT1
protein. The compounds are represented by blue stick models, while the active site regions are shown
by green stick models. H-bond contacts are shown in pink dotted lines, while ꢀ-stacking is shown in
yellow lines.
2.2.3. Docking Simulation
In 2D docking simulations, the amino acid residues are shown in three-letter code, H-bonds are
in pink doted lines, and π-interactions are in yellow lines. Meanwhile, in 3D docking simulations, the
binding residues of AKT1 protein are shown in green colored stick models and the ligands in blue
one. The hydrogen bonds are represented by pink dotted lines, and π-interactions are shown by
yellow lines. Hetero moieties like pyrimidine are observed to be a common pharmacophore group
that interacts with the functional residues of the cancer target protein AKT1 through various
interactions like hydrogen bonds and π-stacking, as shown in Table 2.
2.2.4. ADMET Property Evaluation
As a part of our study, the in silico absorption, distribution, metabolic, excretion (ADME) and
toxicity (T) of the newly synthesized quinazoline compounds were identified using the admetSAR
+
tool, as shown in Table 2. Interestingly, all the newly synthesized compounds had good BBB values,

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which describe the ability of the compounds to cross the blood–brain barrier; these values were in
the acceptable range. Additionally, the values show that the compounds can be absorbed by the
human intestines and are non-carcinogenic. The results show that these compounds show better
inhibition properties against AKT1 protein. Drug-likeness parameters of compounds were calculated
using Mol inspiration software, as summarized in Table 3. The results show interesting values for the
compounds, which obey Lipinski’s rule, whereas all the compounds have topological surface areas
in the acceptable range. Furthermore, the numbers of H-bond acceptors and donors in the tested
compounds are in an acceptable range. Finally, the compounds possess high numbers of rotatable
bonds. The bioavailability radar gives an overview of the drug-likeness of molecule 9 as an example
(see Supplementary Materials). The region in pink color indicates the range for each property. The
boiled-egg plot between WLOGP and TPSA is used to predict gastrointestinal and brain penetration
of the selected compound 9, as shown in Supplementary Materials. The plot shows that the
probability of a good BBB crossing is high. From all these results, we can conclude that all molecules
exhibit good absorption and distribution within the body. These molecules can be considered potent
antagonists against human AKT1 protein and can be used as anti-cancer agents.
Table 2. List of ADMET properties of the newly synthesized molecules.
Molecular Weight Blood–brain Human Intestinal
Caco-2
AMES Toxicity Carcinogenicity
(g/mol)
342.35
342.35
566.79
566.79
314.34
398.46
398.46
444.47
514.56
528.59
Barrier (BBB+) Absorption (HIA+) Permeability (Caco-2)
2
3
4
5
6
0.958
0.958
0.979
0.979
0.975
0.977
0.977
0.615
0.670
0.692
0.969
0.969
0.995
0.995
0.997
0.997
0.997
0.859
0.840
0.856
0.619
0.619
0.531
0.531
0.548
0.600
0.600
0.586
0.601
0.596
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Non carcinogenic
Non carcinogenic
Non carcinogenic
Non carcinogenic
Non carcinogenic
Non carcinogenic
Non carcinogenic
Non carcinogenic
Non carcinogenic
Non carcinogenic
9
10
11
12
13
The pharmacokinetic properties of the synthesized molecules are evaluated by admetSAR.
Table 3. Physicochemical properties of the synthesized compounds.
2
3
TPSA (A ) HBA HBD N rotatable Volume (A )
2
3
4
5
6
72.20
72.20
61.20
61.20
55.12
61.20
61.20
109.50
115.57
115.57
5
5
5
5
4
5
5
7
8
8
1
1
0
0
1
0
0
2
1
1
3
3
19
19
2
6
6
3
7
298.05
298.05
567.60
567.60
279.06
365.77
365.77
362.51
432.62
449.42
9
10
11
12
13
8
TPSA, topological polar surface area; HBA, number of hydrogen bond acceptors; HBD, number of
hydrogen bond donors; N rotatable, number of rotatable bonds.
2.3. In Vitro Activity
It was found, as shown in Figure 2, that the 50% ABTS scavenging activities of compounds 4 and
9 are 62.3 ± 0.09 and 18 ± 1.2, respectively.

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Figure 2. ABTS radical scavenging antioxidant activity of compounds 4 and 9. The IC50 values of each
drug are expressed as mean ± SE of three independent experiments performed in triplets calculated
and plotted using Graphpad Prism software 6 (San Diego, CA).
In the research for new anticancer agents, the most common screening methods are screening
tests against a panel of different cancer cell lines. In this study, (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was carried out to determine the cytotoxic effects of the
compounds on HepG2, MCF-7, and Caco-2 cancer cell lines (Figure 3 and Table 4). Compounds 4 and
9 exhibited significant cytotoxic activity against Caco-2, HepG2, and MCF-7 cancer cells. Compound
4 had more significant inhibitory effects than compound 9 on Caco-2, HepG2, and MCF-7 cell lines,
with IC50 values of 23.31 ± 0.09, 53.29 ± 0.25, and 72.22 ± 0.14 µM, respectively. This compound was
also as effective as doxorubicin (IC50 = 49.38 ± 0.15) on HepG2 cells. The IC50 values of compound 9
against HepG2, MCF-7, and Caco-2 cell lines were 171.4 ± 0.12, 96.58 ± 0.17 and 73.87 ± 0.13,
respectively, which is less than doxorubicin (49.38 ± 0.15, 58.1 ± 0.07 and 5.7 ± 0.12, respectively).
Introducing the substituents on phenyl ring affected the activity of the compounds, in addition, the
position of the substituents on the phenyl ring affected the biological activity of the compound
[35,36]. As can be seen in compound 4, the substituent at the para position increased anticancer
activity against the selected cancer cells than meta-position in compound 5; additionally, the
configuration of the compound affected its biological activity, as it was found that the S configuration
in compound 9 was better than the R configuration in compound 10.
Table 4. IC50 of compound 4 and compound 9 as well as doxorubicin (as a reference drug) on different
cancer cell lines.
IC50
Compound
HepG2
MCF-7
Caco-2
4
53.29 ± 0.25 72.22 ± 0.14 23.31 ± 0.09
9
171.4 ± 0.12 96.58 ± 0.17 73.87 ± 0.13
Doxorubicin 49.38 ± 0.15 58.1 ± 0.07
5.7 ±1.2
Data are expressed as mean ± SE for three independent experiments using Graphpad Prism software
6 (San Diego, CA, USA).

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Figure 3. Compounds 4 and 9 and doxorubicin inhibit proliferation in different cancer cell lines. Cells
were treated with various concentrations of each compound for 48 h and cell viability was plotted
against drug concentration to calculate IC50. The IC50 values of each drug are expressed as mean ± SE
of three independent experiments performed in triplets calculated and plotted using Graphpad Prism
software 6 (San Diego, CA).
2.4. Structure Activity Relationship (SAR)
In connection with the activity values found with the structural moieties of the newly synthetic
compounds, it was declared that compounds 4 and 9 possessed the greatest activity. The cytotoxic
activity of both compounds 4 and 9 against Caco-2, HepG2, and MCF-7 cancer cells was further
investigated by the in silico molecular docking technique. Compound 4, with the highest binding free
energy (ΔGb = −10.2 kcal/mol), interacted with the target due to the interaction between the
pyrimidine ring of the compound and the aromatic side chain of Tryptophan. Moreover, compound
9 (S-isomer), with a binding free energy ΔGb = –9.8 kcal/mol, showed π–π interaction with the target
protein through PHE193. Compound 4 had more significant inhibitory effects than compound 9 on
Caco-2, HepG2, and MCF-7 cell lines, with IC50 values of 23.31 ± 0.09, 53.29 ± 0.25, and 72.22 ± 0.14µM,
respectively. This compound was also as effective as doxorubicin (IC50 = 49.38 ± 0.15) on HepG2 cells.
The IC50 values of compound 9 against HepG2, MCF-7, and Caco-2 cell lines were 171.4 ± 0.12, 96.58
± 0.17 and 73.87 ± 0.13, respectively, which is less than doxorubicin (49.38 ± 0.15, 58.1 ± 0.07 and 5.7 ±
0.12, respectively). Introducing substituents on the phenyl ring affected the activity of the
compounds; additionally, the position of the substituents on the phenyl ring affected the biological
activity of the compound. As represented in compound 4, the substituent at the para position
increased anticancer activity against the selected cancer cells than meta-position in compound 5;
additionally, the configuration of the compound affected its biological activity, as it as found that the
S configuration in compound 9 was better than the R configuration in compound 10.

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3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
Anthranilic acid, benzoyl chloride, p-aminobenzoic acid, m-aminobenzoic acid, p-aminophenol,
cyclohexylisopropylamine (CHIPA), N,N,N’,N’-tetramethylethylenediamine (TMEDA), n-
butyllithium solution 2.5 M in hexane (n-BuLi), hexadecanol, methyliodide (MeI), ethyliodide (EtI),
propionic acid, butyric acid, pentanoic acid, tetrahydrofuran (THF), methanesulfonic acid
(CH3SO3H), pyridine and benzene were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo,
USA).
Reactions were monitored by TLC performed on precoated plates Merck Kieselgel 60 F254 (EMD
Millipore corporation, Billerica, MA, USA). Infrared spectra were recorded at Tanta University by
central laboratory using a Perkin Elmer 1420 spectrophotometer (Waltham, MA, USA), and the
spectra were carried out by using KBr disc technique, the samples were dried in oven then mounted
on a sample holder with a large cavity. Melting points were determined in degrees centigrade by the
open capillary method using Gallenkamp melting point and were reported uncorrected.
The elemental analyses of compounds were performed at the micro analytical center, Cairo
1
13
University using Perkin-Elmer 240 CHN Elemental analyzer, H NMR and C NMR spectra were
collected at resonance frequencies of 400 MHz at Kafr El-sheikh university. NMR spectra were
performed on a Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA)
operating at 400 MHz using dimethyl sulfoxide (DMSO) as a solvent and tetramethylsilane as an
1
internal standard. The chemical shifts for H NMR are reported in ppm from tetramethylsilane (0
13
ppm) or referenced to the solvent (DMSO-d6, δ2,50). Chemical shifts (δ) for C NMR spectra refer to
the signals for residual deuterated solvents (DMSO-d6, 37.5). Multiplicities are reported by the
following abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet).
3.1.2. Synthesis of 2-phenyl-4H-benzo[d][1,3]oxazin-4-one (1)
Compound 1 was prepared as described by Tiwary [30] with 86% Yield.
3.1.3. Synthesis of 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)benzoic acid (2)
In a 50 mL two-neck round-bottom flask a mixture of compound 1 (2.23 g, 10 mmol) and para
amino benzoic acid (1.64 g, 12 mmol) in pyridine (30 mL) was refluxed for 6 h (TLC control) then
poured into ice/water. The resulting solid was filtered, washed with water, and dried. Yield: 88%;
1
13
mp: 270 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.21–8.11 (m, 13H, Ar-H), 11 (s, 1H, COOH); C
NMR (400 MHz, DMSO-d6) δ (ppm): 169.3, 126, 131, 139, 122, 165, 161, 121, 129, 127.5, 177.7, 122.6,
151.7, 126.4; IR (KBr) ν: 3330 (OH), 3063 (Ar-H), 1715 (CO); Anal. Calcd for C21H14N2O3 (342.35): C,
73.68%; H, 4.12%; O, 14.02%; N, 8.18%. Found: C, 73.36%; H, 4.04%; N, 8.09%.
3.1.4. Synthesis of 3-(4-oxo-2-phenylquinazolin-3(4H)-yl)benzoic acid (3)
In a 50 mL two-neck round-bottom flask a mixture of compound 1 (2.23 g, 10 mmol) and meta
amino benzoic acid (1.64 g, 12 mmol) in pyridine (30 mL) was refluxed for 6 h (TLC control) then
poured into ice/water. The resulting solid was filtered, washed with water, and dried. Yield: 87%;
1
13
mp: 256 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.21–8.51 (m, 13H, Ar-H), 11 (s, 1H, COOH); C
NMR (400 MHz, DMSO-d6) δ (ppm): 169.3, 130.7, 120, 132.9, 127, 128.7, 128.8, 128.9, 129, 126, 126.1,
164, 161, 121, 127.7, 133.5, 122.5, 151.5, 126.3, 130.2.
3.1.5. Synthesis of hexadecyl 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)benzoate (4)
In a 50 mL two-neck round-bottom flask a mixture of compound 2 (3.42 g, 10 mmol),
hexadecanol (2.42 g, 10 mmol) and 1 mL of methanesulphonic acid in benzene (50 mL) was refluxed
for 8 h under N2-gas using dean stark trap (TLC control) then the product was cooled and neutralized

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by 5% sodium carbonate. The resulting solid was filtered, washed with water, and dried. Yield: 90%;
1
H NMR (400 MHz, DMSO-d6) δ (ppm): 7.08–8.21 (m, 13H, Ar-H), 0.96 (t, 3H, CH3), 1.23–1.75 (m, 28H,
13
CH2), 4.11 (t, 2H, CH2); C NMR (400 MHz, DMSO-d6) δ (ppm): 166, 125.9, 130.2, 121.7, 137.3, 164.2,
161, 121, 128.9, 127.6, 133.7, 122.6, 151.5, 128.9, 126.3, 129, 130.4, 65, 29.2, 26, 29.6, 29.9, 32, 22.9, 14.3;
IR (KBr) ν: 1675 (CO), 3063 (Ar-H), 2994 (aliph-H); Anal. Calcd for C37H46N2O3 (566.77): C, 78.41%; H,
8.18%; O, 8.47%; N, 4.94%. Found: C, 78.21%; H, 8.11%; N, 4.72%.
3.1.6. Synthesis of hexadecyl 3-(4-oxo-2-phenyl quinazolin-3(4H)-yl)benzoate (5)
In a 50 mL two-neck round-bottom flask a mixture of compound 3 (3.42 g, 10 mmol),
hexadecanol (2.42 g, 10 mmol) and 1 mL of methanesulphonic acid in benzene (50 mL) was refluxed
for 8 h under N2-gas using dean stark trap (TLC control) then the product was cooled and neutralized
by 5% sodium carbonate. The resulting solid was filtered, washed with water, and dried. Yield: 88%;
1
H NMR (400 MHz, DMSO-d6) δ (ppm): 7.08–8.52 (m, 13H, Ar-H), 0.96 (t, 3H, CH3), 1.23–1.75 (m, 28H,
13
CH2), 4.11 (t, 2H, CH2); C NMR (400 MHz, DMSO-d6) δ (ppm): 166, 130.2, 119.8, 132.9, 126, 129, 125.7,
164.2, 161, 121, 128.9, 127.6, 133.7, 122.6, 151.5, 128.9, 126.3, 130.4, 126.5, 65, 29.1, 26, 29.6, 29.9, 32, 22.9,
14.3.
3.1.7. Synthesis of 3-(4-hydroxyphenyl)-2-phenylquinazolin-4(3H)-one (6)
In a 50 mL two-neck round-bottom flask a mixture of compound 1 (2.23 g, 10 mmol) and para
amino phenol (1.30 g, 12 mmol) in pyridine (30 mL) was refluxed for 6 h (TLC control) then poured
into ice/water. The resulting solid was filtered, washed with water, and dried. Yield: 91% ; mp: 220
1
13
°C; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.21–7.91 (m, 13H, Ar-H), 5 (s, 1H, OH); C NMR (400
MHz, DMSO-d6) δ (ppm): 164, 161, 121, 128.8, 127.6, 133.7, 122.6, 151.6, 128.9, 126.3, 129, 130.3, 125.6,
123, 116.3, 154.3; IR (KBr) ν: 3320 (OH), 3063 (Ar-H), 1735 (CO); Anal. Calcd for C20H14N2O2 (314.34):
C, 76.42%; H, 4.49%; O, 10.18%; N, 8.91%. Found: C, 76.22%; H, 4.32%; N, 8.66%.
3.1.8. Synthesis of 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)phenyl butyrate (7)
In a 50 mL two-neck round-bottom flask a mixture of compound 6 (3.14 g, 10 mmol), butyric
acid (0.91 g, 10 mmol) and 1 mL of methanesulphonic acid in benzene (50 mL) was refluxed for 8 h
under N2-gas using dean stark trap (TLC control) then the product was cooled and neutralized by 5%
sodium carbonate. The resulting solid was filtered, washed with water, and dried. Yield: 89%; mp:
1
230 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.08–8.21 (m, 13H, Ar-H), 0.96 (t, 3H, CH3), 1.66 (m,
13
2H, CH2), 2.44 (t, 2H, CH2); C NMR (400 MHz, DMSO-d6) δ (ppm): 127.6, 133.7, 122.6, 151.5, 121,
129, 164, 161, 129.8, 121.8, 147, 122, 172.5, 35.9, 18.5, 13.7, 128.9, 126.3, 129,130.2; IR (KBr) ν: 1675 (CO),
3063 (Ar-H), 2994 (aliph-H); Anal. Calcd for C24H20N2O3 (384.43): C, 74.98%; H, 5.24%; O, 12.49%; N,
7.29%. Found: C, 74.68%; H, 5.14%; N, 7.19%.
3.1.9. Synthesis of 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)phenyl propionate (8)
In a 50 mL two-neck round-bottom flask a mixture of compound 6 (3.14 g, 10 mmol), propionic
acid (1.09 mL, 10 mmol) and 1 mL of methanesulphonic acid in benzene (50 mL) was refluxed for 8 h
under N2-gas using dean stark trap (TLC control) then the product was cooled and neutralized by 5%
sodium carbonate. The resulting solid was filtered, washed with water, and dried. Yield: 88%; mp:
1
260 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.08–8.21 (m, 13H, Ar-H), 1.11 (t, 3H, CH3), 2.14 (m,
13
2H, CH2); C NMR (400 MHz, DMSO-d6) δ (ppm): 172.3, 27.4, 9.5, 147, 121.8, 122, 129,8, 121.8, 164,
161, 121, 129, 127.6, 133.7, 122.6, 151.5, 128.9, 126.3, 130.5; IR (KBr) ν: 1675 (CO), 3063 (Ar-H), 2994
(aliph-H); Anal. Calcd for C23H18N2O3 (370.40): C, 74.58%; H, 4.90%; O, 12.96%; N, 7.56%. Found: C,
74.38%; H, 4.64%; N, 7.29%.

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3.1.10. Synthesis of (S)-4-(4-oxo-2-phenylquinazolin-3(4H)-yl)phenyl 2-methylbutanoate (9)
In a 100 mL two-neck round-bottom flask(3.84 g, 10 mmol) of compound 7 was swelled in 20 mL
THF overnight, then (3.32 mL, 20 mmol) of CHIPA was added at -96 °C (using methanol and liquid
nitrogen) as cooling bath, then (3 mL, 15 mmol) of n-BuLi was added drop wise then the reaction
mixture was stirred for 1h under N2-gas, then 1 mL of TMEDA was added as catalyst and finally (1.24
mL, 20 mmol) of methyl iodide was added drop wise at -40°C, then the reaction mixture was stirred
1
overnight. The resulting solid was filtered, washed with water, and dried [37,38]. Yield: 90%; H NMR
(400 MHz, DMSO-d6) δ (ppm): 7.05–8.22 (m, 13H, Ar-H), 0.98 (t, 3H, CH3), 1.20 (d, 3H, CH3), 1.60 (m,
13
2H, CH2), 2.55 (m, 1H, CH); C NMR (400 MHz, DMSO-d6) δ (ppm): 127.6, 133.5, 122.5, 151.5, 121,
128.8, 164, 161, 128.7, 129.8, 122, 121.9, 147, 175.5, 41, 16.5, 126, 129, 130, 26.5, 12; IR (KBr) ν: 1725 (CO),
3069 (Ar-H), 2989 (aliph-H); Anal. Calcd for C25H22N2O3 (398.45): C, 75.36%; H, 5.57%; O, 12.05%; N,
7.03%. Found: C, 75.13%; H, 5.37%; N, 6.92%.
3.1.11. Synthesis of (R)-4-(4-oxo-2-phenylquinazolin-3(4H)-yl)phenyl 2-methylbutanoate (10)
In a 100 mL two-neck round-bottom flask(3.70 g, 10 mmol) of compound 8 was swelled in 20 mL
THF overnight, then (3.32 mL, 20 mmol) of CHIPA was added at -96°C (using methanol and liquid
nitrogen) as cooling bath, then (3 mL, 15 mmol) of n-BuLi was added drop wise then the reaction
mixture was stirred for 1h under N2-gas, then 1 mL of TMEDA was added as catalyst and finally (1.60
mL, 20 mmol) of ethyl iodide was added drop wise at -40 °C; then, the reaction mixture was stirred
overnight. The resulting solid was filtered, washed with water, and dried. Yield: 90%.
3.1.12. Synthesis of 3-hydroxy-4-(4-oxo-2-phenylquinazolin-3(4H)-yl)naphthalene-1-sulfonic acid
(11)
In a 50 mL two-neck round-bottom flask a mixture of compound 1 (2.23 g, 10 mmol) and 4-
amino-3-hydroxynaphthalene-1-sulfonic acid (2.87 g, 12 mmol) in pyridine (30 mL) was refluxed for
6 h (TLC control) then poured into ice/water. The resulting solid was filtered, washed with water,
1
and dried. Yield: 91%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.21–7.95 (m, 14H, Ar-H), 2 (s, 1H, S-
13
OH), 5 (s, 1H, C-OH); C NMR (400 MHz, DMSO-d6) δ (ppm): 133.4, 117.5, 135.3, 126.7, 126.4, 120,
125.8, 124.5, 127, 125.4, 164, 161, 121, 129, 127.5, 133.5, 122.5, 151.5, 128.7, 126.3, 129.2, 130.5; IR (KBr)
ν: 3320 (OH), 3065 (Ar-H), 1735 (CO); Anal. Calcd for C24H16N2O5S (444.46): C, 64.86%; H, 3.63%; O,
18.00%; S, 7.21%. Found: C, 64.56%; H, 3.34%; S, 7.11%.
3.1.13. Synthesis of 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)3-(butyryloxy)naphthalene-1-sulfonic acid
(12)
In a 50 mL two-neck round-bottom flask a mixture of compound 11 (4.44 g, 10 mmol), butyric
acid (0.91 mL, 10 mmol) and 1 mL of methanesulphonic acid in benzene (50 mL) was refluxed for 8 h
under N2-gas using dean stark trap (TLC control) then the product was cooled and neutralized by 5%
1
sodium carbonate. The resulting solid was filtered, washed with water, and dried. Yield: 88%; H
NMR (400 MHz, DMSO-d6) δ (ppm): 7.08–7.91 (m, 14H, Ar-H), 2 (s, 1H, S-OH), 0.96 (t, 3H, CH3), 1.55
13
(m, 2H, CH2), 2.36 (t, 2H, CH2); C NMR (400 MHz, DMSO-d6) δ (ppm): 132.9, 121, 130.5, 135.6, 125.5,
121.2, 125.9, 126.5, 127, 127.7, 164, 161, 121, 130, 127.5, 133.5, 122.5, 151.5, 128.7, 126.3, 130.5, 129, 172.5,
35.9, 18.5, 13.5.; IR (KBr) ν: 3345 (OH), 1675 (CO), 3063 (Ar-H), 2994 (aliph-H); Anal. Calcd for
C28H22N2O6S (514.55): C, 65.36%; H, 4.31%; O, 18.66%; N, 5.44%; S, 6.23. Found: C, 65.16%; H, 4.21%;
N, 4.14; S, 6.11%.
3.1.14. Synthesis of 4-(4-oxo-2-phenylquinazolin-3(4H)-yl)3-(pentanoxy)naphthalene-1-sulfonic acid
(13)
In a 50 mL two-neck round-bottom flask a mixture of compound 11 (4.44 g, 10 mmol), pentanoic
acid (1.09 mL, 10 mmol) and 1 mL of methanesulphonic acid in benzene (50 mL) was refluxed for 8 h
under N2-gas using dean stark trap (TLC control) then the product was cooled and neutralized by 5%
1
sodium carbonate. The resulting solid was filtered, washed with water, and dried. Yield: 87%; H

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NMR (400 MHz, DMSO-d6) δ (ppm): 7.08–7.91 (m, 14H, Ar-H), 2 (s, 1H, OH), 0.96 (t, 3H, CH3), 1.33–
13
1.55 (m, 4H, 2CH2), 2.36 (t, 2H, CH2); C NMR (400 MHz, DMSO-d6) δ (ppm): 133, 120.7, 130.5, 135.8,
125, 121, 125.5, 126.3, 127, 128, 164, 161, 121.2, 128.8, 127.5, 133.5, 122.5, 151.5, 128.9, 126, 129, 130.3,
172.5, 33.5, 27.5, 22.5, 14; IR (KBr) ν: 3375 (OH), 1695 (CO), 3083 (Ar-H), 2974 (aliph-H); Anal. Calcd
for C29H24N2O6S (528.58): C, 65.90%; H, 4.58%; O, 18.16%; N, 5.30%; S, 6.07. Found: C, 65.56%; H,
4.41%; N, 5.14; S, 5.91%.
3.2. In Silico Study
A three-dimensional structure of human AKT1 protein was downloaded from the RSCB protein
Data Bank [39]. An in-house database of ten quinazolinone compounds was created in SDF (Standard
file format). All the compounds were energy minimized and used in the virtual screening study [40–
42]. The binding pockets of the target were identified using 3DLigandSite and MetaPocket2.0 tools
[43,44]. A grid is created around the binding pockets of AKT1 to perform screening. The docking was
carried out by PyRx screening tool [45]. Finally, in silico pharmacokinetic and molecular properties
of the synthesized quinazoline derivatives are predicted using various software’s such as admetSAR
[46] http://lmmd.ecust.edu.cn/admetsar1/, Mol inspiration https://www.molin spiration.com/and
SwissADME http://swissadme.ch/ web-based tools to select the compounds having optimum drug-
likeness.
3.3. In Vitro ABTS Radical Scavenging Antioxidant and Anticancer Activities of Predicted Compounds
2,2 azino-bis3-ethylbenthiazoline-6-sulfonic acid (ABTS), ascorbic acid and DMSO were
purchased from Sigma-Aldrich Chemical Co. USA. MTT solution was obtained from BIO BASIC
CANADA INC.
3.3.1. Evaluation of ABTS Radical Scavenging Activity
Total antioxidant activity was estimated according to the method described by Re et al. [47].
Briefly, 0.1 mL of different concentration of compound 4 (10–100 µg/mL) and 2.5–25 µg/mL for
compound 9 was mixed with ABTS (pregenerated by adding 5ml of 4.9 mM potassium persulphate
solution to 5 mL of a 14 mM ABTS solution and incubate 16h in dark). The mixture was shaken
vigorously and allowed to stand in the dark at room temperature for 6 min. Absorbance of the
resulting solution was monitored at 734 nm spectrophotometrically within 6 min. of reaction. The
percentage of scavenged ABTS radical was calculated from the following equation:
ABTS scavenging % = (Ao – As/Ao) × 100
(1)
where Ao is the absorbance of the blank. As is the absorbance of sample and standard at 734 nm.
IC50, which denotes the amount (µg) of a sample in 1 mL solution required to reduce the initial
concentration of ABTS radicals by 50%, was calculated using Graphpad Prism 5.
3.3.2. Cell Culture
Three cancer cell lines namely HepG2 (human liver cancer), MCF-7 (human breast cancer) and
Caco-2 (human colon cancer), were included in the study. Cell lines were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and maintained in a
95% humidified incubator at 37 °C supplied with 5% CO2. All the reagents were purchased Gibco-
BRL, USA.
3.3.3. Determination of Compounds Cytotoxicity on Cells (MTT Protocol)
Cell proliferation (viability) was evaluated by MTT assay to establish IC50 (concentration to
inhibit 50% of cells) according to the method of Denizot and Lang [48]. In brief, cells were seeded in
5
4
96-well plates to final count 1× 10 cells/mL (3 × 10 /well) and incubated at 37 °C in 5% CO2 humidified
incubator then left overnight for adhesion. The cells were treated by replacing the old media with
another medium containing different concentration of investigated compounds (6.25–200 µM) and

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doxorubicin as reference drug, ranging from (6.25–200 µM) in triplicate for each concentration. The
microplate was incubated in CO2 incubator for 48 h. At the end of treatment, the supernatant from
each well was discarded and 20 µL of MTT (5 mg/mL) were added into each well for additional 4 h
incubation at 37 °C. After that, the supernatant from each well was removed and the formazan
crystals formed by viable cells were dissolved with DMSO (200 µL/well) with shaking (at highest
speed) for 15 min at room temperature. The absorbance was read using Bio-RAD micro plate reader
(Japan) at 570 nm. The OD measurements for control wells were considered to correspond 100%
growth, their relative OD then calculated the percentage growth in other wells.
The percentage of viability was calculated as follow:
%Viability= Sample absorbance/Control absorbance × 100
(2)
The potent compounds in docking studies were selected to study ABTS antioxidant activity as
well as anticancer effect using MTT assay on three cancer cell lines.
4. Conclusions
In the present work, the synthesis and in silico and in vitro evaluation of a series of quinazoline
analogues were described as potential anticancer agents targeting AKT1 protein. Among them,
compounds 4 and 9 were the most effective anticancer agents on the tested cancer cell lines and the
docking results were fundamentally in agreement with the biological data. According to the in vitro
and in silico studies, compound 4 stands out as a promising orally bioavailable anticancer drug
candidate for further in vivo study. The need for evaluation of their effect on AKT isoforms and
downstream substrates is important to elucidate the molecular mechanisms. Furthermore, in vivo
studies in cancer models are important for confirming its efficacy and additional mechanisms as
anticancer agents. Additionally, toxicity to host cells is an important characteristic to assess the safety
of drug candidates early in the drug discovery process. In summary, the results indicated that the
quinazolinone derivatives could be used as potential inhibitors for cancer treatment via AKT1
inhibition.
1
Supplementary Materials: The following are available online. H and 13C NMR spectra of the synthesized
compound. The oral bioavailability radar of the molecule 9 and the Boiled-egg plot of the selected compound 9.
Author Contributions: All the authors designed the research. A.A.N. and M.E.-N. performed the synthesis of
the compounds, contributed to the characterization of compounds. T.D. suggest the kinase for docking studies,
performed the ABTS antioxidant experiment and anticancer activity of the most effective compounds as
predicted by docking study. A.H.A. contributed to docking studies and their interpretation and shared in
writing the paper. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Nepali, K.; Sharma, S.; Sharma, M.; Bedi, P.M.S.; Dhar, K.L. Rational approaches, design strategies,
structure activity relationship and mechanistic insights for anticancer hybrids. Eur. J. Med. Chem. 2014, 77,
422–487, doi:10.1016/j.ejmech.2014.03.018.
2.
3.
4.
5.
Popat, K.; McQueen, K.; Feeley, T.W. The global burden of cancer. Best Pract. Res. Clin. Anaesthesiol. 2013,
27, 399–408, doi:10.1016/j.bpa.2013.10.010.
Nussbaumer, S.; Bonnabry, P.; Veuthey, J.-L.; Fleury-Souverain, S. Analysis of anticancer drugs: A review.
Talanta 2011, 85, 2265–2289, doi:10.1016/j.talanta.2011.08.034.
Holohan, C.; Van Schaeybroeck, S.; Longley, D.B.; Johnston, P.G. Cancer drug resistance: An evolving
paradigm. Nat. Rev. Cancer 2013, 13, 714–726, doi:10.1038/nrc3599.
Foo, J.; Michor, F. Evolution of acquired resistance to anti-cancer therapy. J. Theor. Biol. 2014, 355, 10–20,
doi:10.1016/j.jtbi.2014.02.025.

Molecules 2020, 25, 4780
19 of 21
6.
Nitulescu, G.M.; Margina, D.; Juzenas, P.; Peng, Q.; Olaru, O.T.; Saloustros, E.; Fenga, C.; Spandidos, D.A.;
Libra, M.; Tsatsakis, A.M. Akt inhibitors in cancer treatment: The long journey from drug discovery to
clinical use. Int. J. Oncol. 2015, 48, 869–885, doi:10.3892/ijo.2015.3306.
7.
8.
9.
Brazil, D.P.; Hemmings, B.A. Ten years of protein kinase B signalling: A hard Akt to follow. Trends Biochem.
Sci. 2001, 26, 657–664, doi:10.1016/s0968-0004(01)01958-2.
Brazil, D.P.; Park, J.; Hemmings, B.A. PKB Binding Proteins: Getting in on the Akt. Cell 2002, 111, 293–303,
doi:10.1016/s0092-8674(02)01083-8.
Bachelder, R.E.; Ribick, M.J.; Marchetti, A.; Falcioni, R.; Soddu, S.; Davis, K.R.; Mercurio, A.M. P53 Inhibits
α6β4 Integrin Survival Signaling by Promoting the Caspase 3–Dependent Cleavage of Akt/PKB. J. Cell Biol.
1999, 147, 1063–1072, doi:10.1083/jcb.147.5.1063.
10. Basso, A.D.; Solit, D.B.; Chiosis, G.; Giri, B.; Tsichlis, P.; Rosen, N. Akt Forms an Intracellular Complex with
Heat Shock Protein 90 (Hsp90) and Cdc37 and Is Destabilized by Inhibitors of Hsp90 Function. J. Biol. Chem.
2002, 277, 39858–39866, doi:10.1074/jbc.m206322200.
11. Xu, J.; Liu, D.; Songyang, Z. The Role of Asp-462 in Regulating Akt Activity. J. Biol. Chem. 2002, 277, 35561–
35566, doi:10.1074/jbc.m203805200.
12. Manning, B.D.; Cantley, L.C. AKT/PKB Signaling: Navigating Downstream. Cell 2007, 129, 1261–1274,
doi:10.1016/j.cell.2007.06.009.
13. Morrow, J.K.; Du-Cuny, L.; Chen, L.; Meuillet, E.J.; Mash, E.A.; Powis, G.; Zhang, S. Recent development
of anticancer therapeutics targeting Akt. Recent Pat. Anti Cancer Drug Discov. 2011, 6, 146–159,
doi:10.2174/157489211793980079.
14. McDowell, K.A.; Riggins, G.J.; Gallia, G.L. Targeting the AKT pathway in glioblastoma. Curr. Pharm. Des.
2011, 17, 2411–2420, doi:10.2174/138161211797249224.
15. Cassinelli, G.; Zuco, V.; Gatti, L.; Lanzi, C.; Zaffaroni, N.; Colombo, D.; Perego, P. Targeting the Akt Kinase
to Modulate Survival, Invasiveness and Drug Resistance of Cancer Cells. Curr. Med. Chem. 2013, 20, 1923–
1945, doi:10.2174/09298673113209990106.
16. Brown, J.S.; Banerji, U. Maximising the potential of AKT inhibitors as anti-cancer treatments. Pharmacol.
Ther. 2017, 172, 101–115, doi:10.1016/j.pharmthera.2016.12.001.
17. Pannipara, M.; Al-Sehemi, A.G.; Kalam, A.; Musthafa, T.N.M. Photophysics of Dihydroquinazolinone
Derivatives: Experimental and Theoretical Studies. J. Fluoresc. 2017, 27, 1160–1170, doi:10.1007/s10895-017-
2051-0.
18. Zaghaghi, Z.; Mirjalilib, B.B.; Monfared, A. Synthesis of 2,3-Dihydroquinazolin-4(1H)-ones Promoted by
Polystyrene Sulfonic Acid. Org. Chem. Res. 2019, 5, 80–86.
19. Wang, H.-X.; Liu, H.-Y.; Li, W.; Zhang, S.; Wu, Z.; Li, X.; Li, C.-W.; Liu, Y.-M.; Chen, B.Q. Design, synthesis,
antiproliferative and antibacterial evaluation of quinazolinone derivatives. Med. Chem. Res. 2019, 28, 203–
214, doi:10.1007/s00044-018-2276-8.
20. Ghaleno, M.R.; Ghaffari-Moghaddam, M.; Khajeh, M.; Oveisi, A.R.; Bohlooli, M. Iron species supported on
a mesoporous zirconium metal-organic framework for visible light driven synthesis of quinazolin-4(3H)-
ones through one-pot three-step tandem reaction. J. Colloid Interface Sci. 2019, 535, 214–226,
doi:10.1016/j.jcis.2018.09.099.
21. Sakr, A.; Kothayer, H.; Ibrahim, S.M.; Baraka, M.M.; Rezq, S. 1,4-Dihydroquinazolin-3(2H)-yl benzamide
derivatives as anti-inflammatory and analgesic agents with an improved gastric profile: Design, synthesis,
COX-1/2 inhibitory activity and molecular docking study. Bioorg. Chem. 2019, 84, 76–86,
doi:10.1016/j.bioorg.2018.11.030.
22. Gatadi, S.; Gour, J.; Shukla, M.; Kaul, G.; Das, S.; Dasgupta, A.; Madhavi, Y.V.; Chopra, S.; Nanduri, S.
Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives as
potent antibacterial agents effective against multidrug resistant Staphylococcus aureus. Bioorg. Chem. 2019,
83, 569–579, doi:10.1016/j.bioorg.2018.11.007.
23. Sajjadifar, S.; Hamidi, H.; Pal, K. Revisiting of Boron Sulfonic Acid Applications in Organic Synthesis: Mini-
Review. J. Chem. Rev. 2019, 1, 35–46, doi:10.33945/sami/jcr.2019.1.3546.
24. Piotrowska, D.G.; Andrei, G.; Schols, D.; Snoeck, R.; Grabkowska-Drużyc, M. New Isoxazolidine-
Conjugates of Quinazolinones—Synthesis, Antiviral and Cytostatic Activity. Molecules 2016, 21, 959,
doi:10.3390/molecules21070959.

Molecules 2020, 25, 4780
20 of 21
25. Marzaro, G.; Castagliuolo, I.; Schirato, G.; Palu’, G.; Via, M.D.; Chilin, A.; Brun, P. Substituted
quinazolinones as kinase inhibitors endowed with anti-fibrotic properties. Eur. J. Med. Chem. 2016, 115, 416–
425, doi:10.1016/j.ejmech.2016.03.053.
26. Dohle, W.; Prota, A.E.; Menchon, G.; Hamel, E.; Steinmetz, M.O.; Potter, B.V.L. Tetrahydroisoquinoline
Sulfamates as Potent Microtubule Disruptors: Synthesis, Antiproliferative and Antitubulin Activity of
Dichlorobenzyl-Based Derivatives, and a Tubulin Cocrystal Structure. ACS Omega 2019, 4, 755–764,
doi:10.1021/acsomega.8b02879.
27. Saeedi, M.; Mohammadi-Khanaposhtani, M.; Pourrabia, P.; Razzaghi, N.; Ghadimi, R.; Imanparast, S.;
Faramarzi, M.A.; Bandarian, F.; Esfahani, E.N.; Safavi, M.; et al. Design and synthesis of novel
quinazolinone-1,2,3-triazole hybrids as new anti-diabetic agents: In vitro α-glucosidase inhibition, kinetic,
and docking study. Bioorg. Chem. 2019, 83, 161–169, doi:10.1016/j.bioorg.2018.10.023.
28. Das, S.; Bhattacharjee, J.; Panda, T.K. Guanylation/cyclisation of amino acid esters using an imidazolin-2-
iminato titanium initiator. Dalton Trans. 2019, 48, 7227–7235, doi:10.1039/c8dt04630a.
29. Shagufta, S.; Ahmad, I. An insight into the therapeutic potential of quinazoline derivatives as anticancer
agents. MedChemComm 2017, 8, 871–885, doi:10.1039/c7md00097a.
30. Tiwary, B.K.; Zirmire, R.K.; Pradhan, K.; Nanda, A.K. Innovare Academic Sciences Preparation and
Spectroscopic Characterization of Inclusion Complex of 2-Phenyl-4h-Benzo[D][1,3]Oxazin-4-One and Β -
Cyclodextrin. Int. J. Pharm. Pharm. Sci. 2014, 6, 7–10.
31. Henrich, S.; Feierberg, I.; Wang, T.; Blomberg, N.; Wade, R.C. Comparative binding energy analysis for
binding affinity and target selectivity prediction. Proteins Struct. Funct. Bioinform. 2010, 78, 135–153,
doi:10.1002/prot.22579.
32. El-Naggar, M.; Mohamed, M.E.; Mosallam, A.M.; Salem, W.; Rashdan, H.R.; Abdelmonsef, A.H. Synthesis,
Characterization, Antibacterial Activity, and Computer-Aided Design of Novel Quinazolin-2,4-dione
Derivatives as Potential Inhibitors Against Vibrio cholerae. Evol. Bioinform. 2020, 16,
doi:10.1177/1176934319897596.
33. Abdelmonsef, A.H.; Mosallam, A.M. Synthesis, in vitro biological evaluation and in silico docking studies
of new quinazolin-2,4-dione analogues as possible anticarcinoma agents. J. Heterocycl. Chem. 2020, 1–8,
doi:10.1002/jhet.3889.
34. Abdelmonsef, A.H. Computer-aided identification of lung cancer inhibitors through homology modeling
and virtual screening. Egypt. J. Med. Hum. Genet. 2019, 20, doi:10.1186/s43042-019-0008-3.
35. Metwally, N.H.; Abdelrazek, F.M.; Eldaly, S.M. Synthesis, Molecular Docking, and Biological Evaluation
of Some Novel Bis-heterocyclic Compounds Based N,N′-([1,1′-biphenyl]-4,4′-diyl)bis(2-cyanoacetamide) as
Potential Anticancer Agents. J. Heterocycl. Chem. 2018, 55, 2668–2682, doi:10.1002/jhet.3290.
36. Haredi Abdelmonsef, A.; Eldeeb Mohamed, M.; El-Naggar, M.; Temairk, H.; Mohamed Mosallam, A.
Novel Quinazolin-2,4-Dione Hybrid Molecules as Possible Inhibitors Against Malaria: Synthesis and in
silico Molecular Docking Studies. Front. Mol. Biosci. 2020, 7, 105.
37. Noser, A.; Selim, A.; Badawy, M. Asymmetric synthesis of α-alkylated acid in high enantiomeric purity
using poly (Methylmethacrylate) resins. Int. J. Chem. Appl. Biol. Sci. 2014, 1, 135, doi:10.4103/2348-
0734.146958.
38. Selim, A.; Baren, M.; Noser, A. Enantioselective Synthesis of (S) 3-Methyl-4-Octanol as Insect Pheromone
Using Polymeric Asymmetric Reagent. J. Appl. Chem. 2017, 6, 41–49.
39. Berman, H.M.; Battistuz, T.; Bhat, T.N.; Bluhm, W.F.; Bourne, P.E.; Burkhardt, K.; Feng, Z.; Gilliland, G.L.;
Iype, L.; Jain, S.; et al. The Protein Data Bank. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002, 58, 899–907,
doi:10.1107/s0907444902003451.
40. Abdelmonsef, A.H.; Dulapalli, R.; Dasari, T.; Padmarao, L.S.; Mukkera, T.; Vuruputuri, U. Identification of
Novel Antagonists for Rab38 Protein by Homology Modeling and Virtual Screening. Comb. Chem. High
Throughput Screen. 2016, 19, 875–892, doi:10.2174/1386207319666161026153237.
41. Dasari, T.; Kondagari, B.; Dulapalli, R.; Abdelmonsef, A.H.; Mukkera, T.; Padmarao, L.S.; Malkhed, V.;
Vuruputuri, U. Design of novel lead molecules against RhoG protein as cancer target—A computational
study. J. Biomol. Struct. Dyn. 2017, 35, 3119–3139, doi:10.1080/07391102.2016.1244492.
42. Rondla, R.; Padmarao, L.S.; Ramatenki, V.; Haredi-Abdel-Monsef, A.; Potlapally, S.R.; Vuruputuri, U.
Selective ATP competitive leads of CDK4: Discovery by 3D-QSAR pharmacophore mapping and molecular
docking approach. Comput. Biol. Chem. 2017, 71, 224–229, doi:10.1016/j.compbiolchem.2017.11.005.

Molecules 2020, 25, 4780
21 of 21
43. Wass, M.N.; Kelley, L.A.; Sternberg, M.J.E. 3DLigandSite: Predicting ligand-binding sites using similar
structures. Nucleic Acids Res. 2010, 38, 469–473, doi:10.1093/nar/gkq406.
44. Huang, B. MetaPocket: A Meta Approach to Improve Protein Ligand Binding Site Prediction. OMICS A J.
Integr. Biol. 2009, 13, 325–330, doi:10.1089/omi.2009.0045.
45. Dallakyan, S.; Olson, A.J. Small-Molecule Library Screening by Docking with PyRx. In Chemical Biology;
Springer: Berlin/Heidelberg, Germany, 2015; Volume 1263, pp. 243–250, ISBN 9780123944474.
46. Cheng, F.; Li, W.; Zhou, Y.; Shen, J.; Wu, Z.; Liu, G.; Lee, P.W.; Tang, Y. admetSAR: A Comprehensive
Source and Free Tool for Assessment of Chemical ADMET Properties. J. Chem. Inf. Model. 2012, 52, 3099–
3105, doi:10.1021/ci300367a.
47. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying
an improved ABTS radical cation decolorization assay. Free. Radic. Biol. Med. 1999, 26, 1231–1237,
doi:10.1016/s0891-5849(98)00315-3.
48. Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium
dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 1986, 89, 271–277,
doi:10.1016/0022-1759(86)90368-6.
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