Induction of apoptosis, cytotoxicity and radiosensitization by novel 3,4-dihydroquinazolinone derivatives

Article abstract of DOI:10.1016/j.bmcl.2021.128308

Twenty new quinazolinone derivatives bearing a piperonyl moiety were designed and synthesized. The structures of the target compounds were in agreement with the microanalytical and spectral data. Compounds 4-10, 13, 14 and 17-27 were screened for their cytotoxic activity against HepG-2 and MCF-7 cancer cell lines. The target compounds showed IC₅₀ in the range of 2.46–36.85 μM and 3.87–88.93 μM for HepG-2 and MCF-7, respectively. The promising compounds 7, 19, 26 and 27 were selected to measure their EGFR inhibitory activity. The IC₅₀ values of the promising compounds were in the range of 146.9–1032.7 nM for EGFR in reference to Erlotinib (IC₅₀ = 96.6 nM). In further studies on compounds 7, 19, 26 and 27 using HepG-2 cell line, there was significant overexpression of p21 and downregulation of two members of IAPs protein family; Survivin and XIAP, relative to their controls. Annexin V-FITC and caspase-3 analyses have established a significant increase in early apoptosis. Moreover, the four selected compounds have impaired cell proliferation by cell cycle arrest at the G2/M phase compared to their respective control. Considering radiotherapy as the primary treatment for many types of solid tumors, the radiosensitizing abilities of compounds 7, 19, 26 and 27 were measured against HepG-2 and MCF-7 cell lines combined with a single dose of 8 Gy gamma radiation. Measurement of the IC₅₀ of the promising compounds after irradiation revealed their ability to sensitize the cells to the lethal effect of gamma irradiation (IC₅₀ = 1.56–4.32 μM and 3.06–5.93 μM for HepG-2 and MCF-7 cells, respectively). Molecular docking was performed to gain insights into the ligand-binding interactions of 7, 19, 26 and 27 inside the EGFR binding sites and revealed their essential interactions, explaining their good activity towards EGFR.

Full text of DOI:10.1016/j.bmcl.2021.128308

Bioorg. Med. Chem. Lett. 49 (2021) 128308
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Induction of apoptosis, cytotoxicity and radiosensitization by novel
3
,4-dihydroquinazolinone derivatives
a
b
c
a,*
Aiten M. Soliman , Amira Khalil , Eman Ramadan , Mostafa M. Ghorab
a
Department of Drug Radiation Research, National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), Cairo 11765,
Egypt
b
The Center for Drug Research and Development (CDRD), Pharmaceutical Chemistry Department, Faculty of Pharmacy, The British University in Egypt (BUE), El-
Sherouk City, Cairo 11837, Egypt
c
Pharmacology and Biochemistry Department, Faculty of Pharmacy, The British University in Egypt (BUE), El-Sherouk City, Cairo 11837, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Twenty new quinazolinone derivatives bearing a piperonyl moiety were designed and synthesized. The structures
of the target compounds were in agreement with the microanalytical and spectral data. Compounds 4-10, 13, 14
and 17-27 were screened for their cytotoxic activity against HepG-2 and MCF-7 cancer cell lines. The target
compounds showed IC50 in the range of 2.46–36.85 µM and 3.87–88.93 µM for HepG-2 and MCF-7, respectively.
The promising compounds 7, 19, 26 and 27 were selected to measure their EGFR inhibitory activity. The IC50
values of the promising compounds were in the range of 146.9–1032.7 nM for EGFR in reference to Erlotinib
3
,4-Dihydroquinazolinone
Sulfonamide
Apoptosis
EGFR
XIAP
Docking
(
IC50 = 96.6 nM). In further studies on compounds 7, 19, 26 and 27 using HepG-2 cell line, there was significant
overexpression of p21 and downregulation of two members of IAPs protein family; Survivin and XIAP, relative to
their controls. Annexin V-FITC and caspase-3 analyses have established a significant increase in early apoptosis.
Moreover, the four selected compounds have impaired cell proliferation by cell cycle arrest at the G2/M phase
compared to their respective control. Considering radiotherapy as the primary treatment for many types of solid
tumors, the radiosensitizing abilities of compounds 7, 19, 26 and 27 were measured against HepG-2 and MCF-7
cell lines combined with a single dose of 8 Gy gamma radiation. Measurement of the IC50 of the promising
compounds after irradiation revealed their ability to sensitize the cells to the lethal effect of gamma irradiation
(
IC50 = 1.56–4.32 µM and 3.06–5.93 µM for HepG-2 and MCF-7 cells, respectively). Molecular docking was
performed to gain insights into the ligand-binding interactions of 7, 19, 26 and 27 inside the EGFR binding sites
and revealed their essential interactions, explaining their good activity towards EGFR.
7
Cancer initiation and progression are due to several external envi-
cellular inflammation and death. During tumor progression, there is an
accumulation of various inhibitors of apoptosis proteins (IAPs) within
ronmental risk factors combined with internal genetic mutations.1
Despite the presence of various types of cancer treatments, limitations in
8
the cells. IAP family of proteins could inhibit the enzymatic activity of
2
current therapies exist as chemo-resistance, relapses and toxic effects.
caspases. Different IAPs, including XIAP, c-IAP1, c-IAP2 and Survivin,
were identified to inhibit initiator caspase-9 and effector caspases 3 and
This occurs due to poor selectivity resulting in off-target side effects that
3
9
seriously affect the patient’s quality of life. Targeted chemotherapies
7. Both XIAP and Survivin expression have been linked to both tumor
usually aim at the tumor microenvironment, regulate the tumor cell
cycle and induce apoptosis.4
recurrence and poor prognosis.
Moreover, apoptosis is triggered by selective crosslinking and inhi-
bition of receptor tyrosine kinases (RTKs).10 TKs are considered the most
Apoptosis is a genetically programmed mechanism critical for the
5
11
survival and normal functioning of the cells. Cancer is characterized by
prevalent therapeutic targets in anticancer drug development. The
suppression of the apoptotic mechanisms resulting in treatment resis-
epidermal growth factor receptor (EGFR) belongs to protein tyrosine
6
tance. Among the essential regulators of apoptosis is a family of en-
kinases and plays a critical role in cellular proliferation, differentiation
and survival of normal as well as cancer cells.1
2,13
Overexpression of
zymes known as caspases that play a significant role in balancing
*
Corresponding author.
E-mail address: mmsghorab@yahoo.com (M.M. Ghorab).
https://doi.org/10.1016/j.bmcl.2021.128308
Received 21 May 2021; Received in revised form 27 July 2021; Accepted 31 July 2021
Available online 4 August 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
inhibitors, mostly EGFR inhibitors as; Erlotinib, Afatinib, Lapatinib,
Vandetanib and Gefitinib.25
Similarly, Sulfonamide moiety represents the core of many clinically
used drugs comprising different pharmacological activities, including
anticancer activity. 26 They exert their anticancer effect through a va-
2
7,28
riety of mechanisms, mainly by carbonic anhydrase inhibition.
Furthermore, compounds having piperonyl ring exhibited a wide range
of biological activities. They act as a potential pharmacophore with
2
9
30
31
various activities as COX-2 inhibitors, antiviral, anticancer, and
apoptosis inducers.32
In this current work, we designed our library based on many repre-
sentative quinazolinones possessing interesting anticancer and
apoptotic inducer activity (Fig. 2) as follows; the 3-((5-chloro-2-
hydroxybenzylidene)amino)-2-(5-chloro-2-hydroxyphenyl)-2,3-dihy-
droquinazolin-4(1H)-one (A) reported by Zahedifard and co-workers
was found to cause significant inhibition on MCF-7 and MDA-MB-231
cell viability and to induce extrinsic and intrinsic apoptosis pathways
by cytochrome C activated caspase-9 expression.33 Wani et al. synthe-
sized the quinazolinone chalcone (B), that induced mitochondrial
dependent apoptosis, inhibited PI3K/Akt/mTOR signaling pathway in
human colon cancer cells and arrested the cell cycle at the S and G2/M
phases of the HCT-116 cells.34 Nowar and her colleagues reported the
quinazolinone-benzenesulfonamide derivative (C) to act as an anti-
proliferative and apoptosis inducer through the activation of caspases 3,
8
and 9 in HCT-116 cell line.35 Idelalisib (D), an FDA-approved selective
PI3Kδ inhibitor used for lymphoma treatment, can promote Bim-
dependent apoptosis in hepatocellular carcinoma.36 The natural 2-
chloro-6-phenyl-8H-quinazolino[4,3-b]quinazolin-8-one (E) isolated
from marine sponge, deemed to induce apoptosis in breast cancer cells
via ROS production.37 Moreover, the 2-(3-fluorophenyl)-6-morpholi-
noquinazolin-4(3H)-one (F) induced cell cycle arrest at the G2/M phase.
It can be combined with 5-fluorouracil (5-FU) to cause a synergistic toxic
effect on oral squamous cell carcinoma (OSCC).38 Also, the (E)-2-(2,4-
dichlorostyryl)-3-(3-(dimethylamino)propyl) quinazolin-4(3H)-one (G)
synthesized by Zhang et al.39 exerted its anticancer effect by arresting
the mutant p53 function to trigger the deregulation of Cdk2 caused Bim-
mediated apoptosis.
Fig. 1. Chemical structures of FDA-approved TK inhibitors showing quinazo-
line scaffold.
In this article, the target compounds were designed based on an
interesting hybrid drug approach to have a quinazoline ring as the main
building block. We also wanted to explore the effect of incorporating
piperonyl group and sulfonamide moiety in the designed compounds.
We will explore the chemical synthesis of the novel 3, 4 dihy-
droquinazoline piperonyl derivatives and their cytotoxic effect against
breast cancer (MCF-7) as well as hepatic cancer (HepG-2) cell lines and
their enzymatic inhibitory activity against the EGFR. Also, to study the
potential effects of the promising compounds on cell cycle, caspases,
XIAP and their use as promising apoptosis inducers. Furthermore, mo-
lecular docking was carried out inside the active site of EGFR to deter-
mine the possible binding interactions between the promising
compounds and the receptor.
Schemes 1-3 show our synthetic strategy for preparing the desired
quinazoline derivatives 4-10, 13, 14 and 17-27. The methyl 2-amino-
benzoate
1 was reacted with thiophosgene to yield the iso-
Fig. 2. The design strategy of our target compounds.
4
0
thiocyanatobenzoate derivative 2, which was further reacted with
piperonylamine 3 in dimethylformamide (DMF) and few drops of trie-
thylamine (TEA) to yield our starting material the 3-(benzo[d][1,3]
dioxol-5-ylmethyl)-2-mercaptoquinazolin-4(3H)-one 4. The synthesis of
a key hydrazide compound 6 was illustrated in Scheme 1 and proceeded
by the reflux of compound 4 with ethyl bromoacetate in acetone con-
EGFR is evident in different types of tumors such as breast, ovarian and
squamous cell cancers, 1 leading to irregular apoptosis and uncon-
trolled cellular proliferation.15 Seeking EGFR inhibition using small
molecules novel agents has been an interesting and promising area for
selective cancer treatment.
4
2 3
taining K CO forming the acetate derivative 5. A solution of compound
Quinazoline is an attractive chemical scaffold displaying a wide
5
was refluxed in ethanol containing hydrazine hydrate to produce our
spectrum of biological activities. 1
6–20
In our previous work, we have
paid great interest in investigating quinazolines as anticancer
1
target hydrazide compound 6 in a decent yield. H NMR of 6 confirmed
the disappearance of the ethyl protons of compound 5 and the appear-
2
1–23
agents.
Quinazoline as a chemical scaffold is a common pharma-
2
ance of NH protons of 6 (see experimental section).
2
4
cophore among many reported EGFR inhibitors. Fig. 1 shows exam-
ples of FDA-approved quinazolines that acts as multi-target TK
The hydrazide compound 6 was used for the synthesis of compounds
–10, 13, 14, 17 and 18 according to Schemes 1 & 2. Nucleophilic
7
2

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Scheme 1. Synthesis of compounds 4-10.
reaction between compound 6 and different sulfonyl chloride de-
rivatives in the presence of pyridine afforded 7–10 in good yield in the
under reflux. The resulted candidates 13 and 14 were produced in good
yield, Scheme 2. Structure elucidation for 13 and 14 was confirmed
1
1
range of 72–80 %. The H NMR spectrum of compound 7 showed
mainly through H -NMR. The proton NMR spectrum for 13 revealed the
′
characteristic triplet and quartet at 1.21 and 4.13 ppm due to the ethyl
obvious ethyl protons, whereas compound 14 s spectrum showed a
1
3
group, which corresponds to 14.61 and 61.57 ppm in C NMR. A
3
singlet peak for COCH protons at 2.05 ppm. The hydrazinecarbo-
representative singlet appeared in the 1H NMR of 9 at 2.26 ppm
thioamide derivatives 17 and 18 were produced by the reaction of the
hydrazide 6 and the corresponding isothiocyanate benzenesulfonamide
derivatives 15 and 16 in pyridine under reflux conditions as illustrated
in Scheme 2. The open-chain derivatives were believed to be formed
through nucleophilic substitution.
3
attributed to the CH C
hydrogens and corresponds to 21.44 pm at 13
NMR. Also, the acetamide group of 10 was peculiar in the NMR spectra.
Furthermore, compound 6 was allowed to react with acetophenone
derivatives 11 and 12 in the presence of ethanol and glacial acetic acid
3

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Scheme 2. Synthesis of compounds 13, 14, 17 and 18.
Scheme 3. Synthesis of compounds 19–27.
4

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Table 1
by spectroscopic data. IR spectra of 19–27 showed NH groups and 2CO
bands at their identified region. 1H NMR spectra revealed two singlets
The IC50 values ( M) of compounds 4-10, 13, 14, 17-27 against HepG-2 and
μ
attributed to the S-CH
2
and NH protons. 13C NMR spectra exhibited an
MCF-7 cell lines using MTT assay.
Compound no.
HepG-2 IC50 (µM)*
MCF-7 IC50 (µM)*
up-field signal for the S-CH
carbons.
2
and two downfield signals for the 2CO
4
5
6
7
8
9
27.26 ± 0.31
–
26.64 ± 0.42
–
The potential cytotoxic effect of the synthesized quinazolinone 4-10,
13, 14 and 17-27 on the viability of HepG-2 and MCF-7 were detected
by MTT assay. The cell viability is expressed as percent of viable cells
8.15 ± 0.17
8.76 ± 0.20
22.20 ± 0.31
28.16 ± 0.20
33.94 ± 0.28
9.08 ± 0.13
4.21 ± 0.17
36.85 ± 0.25
9.34 ± 0.34
2.46 ± 0.27
8.51 ± 0.33
7.18 ± 0.21
17.87 ± 0.10
12.09 ± 0.26
–
20.69 ± 0.35
15.32 ± 0.33
88.93 ± 4.01
49.77 ± 0.14
11.41 ± 0.32
13.92 ± 0.25
15.10 ± 0.13
3.87 ± 0.10
46.03 ± 0.08
5.86 ± 0.184
7.60 ± 0.31
14.54 ± 0.33
10.33 ± 0.49
26.12 ± 0.56
–
(
mean ± SD) compared to untreated cells (taken as 100 % viable) for 24
hr by serial dilution. The estimated IC50 value (concentration that in-
hibits growth by 50%) of the target compounds against HepG-2 and
MCF-7 cell lines are listed in Table 1. The initial cytotoxicity screening
showed that compounds 4, 6-10, 13, 14, 17-23 and 25-27 were effective
against both HepG-2 cells and MCF-7 cells except for compounds 5 and
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
3
4
7
8
9
0
1
2
3
4
5
6
7
2
4 due to irregular concentration–response relationship. Table 1 shows
that compounds 4, 6-10, 13, 14, 17-23 and 25-27 have significant
cytotoxic effects on HepG-2 and MCF-7 with IC50 values in the range of
2
.46–36.85 µM and 3.87–88.93 µM, respectively. Doxorubicin and
11.13 ± 0.15
4.01 ± 0.14
13.17 ± 0.36
8.90 ± 0.39
10.17 ± 0.32
20.36 ± 0.33
14.93 ± 0.23
6.84 ± 0.15
9.34 ± 0.24
12.40 ± 0.37
Doxorubicin
Erlotinib
*The values represent the mean ± SD of three independent experiments.
Table 2
EGFR inhibitory activities of compounds 7, 19, 26 and 27
compared to Erlotinib (IC50, nM).
a
Compound no.
IC50 (nM)
7
146.9 ± 3.6
207.1 ± 4.9
1032.7 ± 31.8
754.3 ± 22.0
96.6 ± 2.0
1
2
2
9
6
7
Erlotinib
a
The values represent the mean of three independent
experiments ± SD.
The synthesis of compounds 19–27 was performed by the reaction of
compound 4 41 with the corresponding series of N-chloro substituted
2 3
acetamides in acetone containing K CO to produce a series of haloge-
nated quinazolinone derivatives 19–27 in excellent yield. These hybrid
molecules were synthesized by introducing halogenated phenyl groups
on the acetamide tail to provide a set of compounds having variable
lipophilic and electronic nature to study the structure–activity rela-
tionship (SAR). The structures of the target compounds were confirmed
Fig. 4. Effect of compounds 7, 19, 26 & 27 on the secretion of caspase-3 in
HepG-2 cells. The level of caspase-3 in cultural medium was assessed by ELISA
showing significant increase compared to untreated control. Values are pre-
sented as means ± SD, ****, <0.0001, Vs respective control (n = 3).
Fig. 3. Effect of compounds 7, 19, 26 & 27 on transcriptional level of Survivin (A), XIAP (B) and p21 (C). The relative mRNA expression of each gene was measured
using real-time RT-PCR on HepG-2 cells treated with compounds 7, 19, 26 & 27 after normalizing the cycle thresholds (Ct) of each triplicate against their corre-
sponding GAPDH. Data are expressed as mean ± S.E. ***, <0.0005, ****, <0.0001, Vs respective control (n = 3).
5

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Fig. 5. Effect of compounds 7, 19, 26 & 27 on cell cycle distribution. Treatment of HepG-2 cells by compounds 7, 19, 26 & 27 have significantly increased the
proportion of G2/M phase while significant decrease of G1-G0 and S phases compared to control. A) DMSO treated HepG-2 cells used as control. B) Treated HepG-2
with 7. C) Treated HepG-2 with 19. D) Treated HepG-2 with 26. E) Treated HepG-2 with 27. E) Percentage of cell populations. Values are presented as means ± SD,
**, p < 0.0022 Vs respective control (n = 3).
6

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Fig. 6. Annexin V-FITC/PI double staining analysis of apoptosis in compounds 7, 19, 26 & 27 treated HepG-2 cells (A) HepG-2 control cells (B) HepG-2 cells treated
with 7C) HepG-2 cells with 19 (D) HepG-2 cells with 26. (E) HepG-2 cells with 27. Top right quadrant, dead cells in a late stage of apoptosis (Q2); bottom right
quadrant (Q3), cells are undergoing apoptosis; bottom left quadrant, viable cells (Q1). (E): Chart comparison of cell distribution after treatment with compounds 7,
1
9, 26 & 27. Values are presented as means ± SD, with significant, **** p < 0.000 Vs. respective control (n = 3).
Erlotinib were used as reference drugs, with IC50 8.90, 10.17 µM for
HepG-2 cells and 9.34, 12.40 µM for MCF-7 cells, respectively. The 2-((3-
range of 2.46–17.87 µM and 5.86–26.12 µM for MCF-7 cells except for
compound 24. Based on our results, we choose to conduct further
investigation on the quinazolinone sulfonamide derivative 7 and the
quinazolinone derivatives 19, 26 & 27 displaying promising cytotox-
icity on both cell lines. The HepG-2 cells were chosen to conduct the
apoptosis and cell cycle analysis tests as the compounds showed sig-
nificant cytotoxicity compared to the MCF-7 cells. IC50 values of the
(
benzo[d][1,3]dioxol-5-ylmethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)
thio)-N-(2-fluorophenyl)acetamide 19 has shown a potent cytotoxic ef-
fect on both HepG-2 and MCF-7 with IC50 values of 2.46 and 5.86 µM,
respectively. It was also observed that the acetamide derivatives bearing
halogens 19–27 showed very promising activity on HepG-2 cells in the
7

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Table 3
inhibition values of these compounds against EGFR. The IC50 values
were in the range of 146.9–1032.7 nM versus 96.6 nM for Erlotinib.
Regarding the N’-(2-((3-(benzo[d][1,3]dioxol-5-ylmethyl)-4-oxo-3,4-
The cytotoxicity of the promising compounds 7, 19, 26 and 27 after irradiation.
b
b
Compound no.
HepG-2 C50 (µM)
MCF-7 IC50 (µM)
dihydroquinazolin-2-yl)thio)acetyl)ethanesulfonohydrazide
7
was
7
2.25 ± 0.04
1.56 ± 0.05
1.76 ± 0.07
4.32 ± 0.23
5.93 ± 0.16
3.06 ± 0.02
4.91 ± 0.21
4.69 ± 0.32
found to be the most potent in this study. The 2-((3-(benzo[d][1,3]
dioxol-5-ylmethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(2-fluo-
rophenyl)acetamide 19 displaying the most potent cytotoxic activity
towards the HepG-2 cells comes in the second place in EGFR inhibitory
activity (IC50 = 207.1 nM). The promising candidates were further
subjected to cell cycle analysis and apoptotic assay.
1
2
2
9
6
7
b
Represents the mean of three independent experiments ± SD.
most potent compounds are in the range of 2.46–13.17 µM for HepG-2
cell line.
Inhibitors of apoptosis proteins (IAP) are a family of proteins that
4
2
inhibit apoptosis induced by several pro-apoptotic stimuli. The IAP
family includes XIAP and Survivin; their expression has been linked to
poor prognosis, increased tumor recurrence and radioresistance in many
The promising compounds 7, 19, 26 and 27 were subjected to EGFR-
TK inhibitory assay compared to Erlotinib. Table 2 demonstrated the
Fig. 7. A: 2D docking pose of compound 7, B: 3D interactions of 7 (brown), superimposed on the native ligand erlotinib (magenta).
8

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Fig. 8. A: 2D interaction map of compound 19, B: 3D interaction pose of 19 (green), superimposed on the native ligand erlotinib (magenta).
cancer types.43 Accordingly, blocking of Survivin and XIAP expression is
becoming a promising strategy in cancer therapy.44 HepG-2 cells were
treated with compounds 7, 19, 26 & 27 to measure relative mRNA
expression of Survivin, XIAP and p21 mRNA using real-time RT-PCR
after normalizing the cycle thresholds (Ct) of each triplicate against their
corresponding control GAPDH. In this study, treatment of HepG-2 cells
with the selected compounds has decreased the relative mRNA expres-
sion of XIAP and Survivin by almost 50% compared to control (Fig. 3A
and 3B).
suggesting that these novel quinazolinones could be a potent radio-
sensitizing agent for hepatocellular carcinoma.
P21 has a well-described molecular role in carcinogenesis of tumors,
not only in proliferation arrest but also during apoptosis.4 Compounds
7, 19, 26 & 27 treatment of HepG-2 have upregulated the cyclin-
dependent kinase inhibitor p21 mRNA expression (~500%, 400%,
400% and 200%, respectively) in comparison to control (Fig. 3C). P21 is
a key cell cycle regulator that plays an important role in growth inhi-
5
4
6
1
bition and overexpression leads to G , G2, or S-phase arrest. Our cell
Moreover, many studies have proven that increased expression of
IAP proteins, especially XIAP and Survivin are associated with radio-
resistance.43 Our data show significant downregulation of XIAP and
Survivin expression in HepG-2 treated cells relative to untreated control,
cycle analysis data have shown significant G2/M arrest which may be
related to p21 overexpression (Fig. 3C, Fig. 5). Taken together, these
results suggest that 7, 19, 26 & 27 treatment is responsible for over-
coming apoptosis resistance and arresting the proliferation of HepG-2 by
9

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Fig. 9. A: 2D docking pose of compound 26, B: 3D interactions of 26 (blue), superimposed on the native ligand erlotinib (magenta).
suppressing Survivin and XIAP and overexpressing p21 (Fig. 3).
Active caspase-3 is a critical executioner of apoptosis. It is an inactive
proenzyme activated by extrinsic (death ligand) and intrinsic (mito-
chondrial) pathways leading to cell death.47 In many types of cancer, the
over-expressed IAPs interact with caspase-9 preventing the activation of
caspase-3.48 So IAPs employ their antiapoptotic activity through direct
comparison to DMSO treated HepG-2 cells used as control (Fig. 4). Our
data suggests that the decreased level of caspase-3 is due to decreased
expression of XIAP and Survivin upon treatment of HepG-2 by the
selected compounds.
Progression of eukaryotic cells through the cell cycle is controlled by
successive stimulation and inhibition by cyclin-dependent kinases
(CDKs) and associated cyclin subunits at specific checkpoints to ensure
correct cell division.50 The checkpoints G1/S and G2/M of the cell cycle
are responsible for preventing inappropriate cell cycle progression due
to DNA damage or inadequate DNA replication.51 Cell cycle parameters
of HepG-2 cells treated with compounds 7, 19, 26 & 27 were compared
with DMSO treated HepG-2 used as control. As shown in Fig. 5,
following treatment with 7, 19, 26 & 27 there was a significant decrease
4
9
inhibition of active caspases. HepG-2 cells were treated with com-
pounds 7, 19, 26 & 27 and caspase-3 level was evaluated using ELISA
Kit. The compounds have decreased the relative mRNA expression of
XIAP and Survivin by almost 50% compared to control (Fig. 3A and 3B).
As expected, on evaluation of caspase-3 level of treated HepG-2 with the
promising compounds, caspase-3 level has increased approximately
5
0% (for compounds 7, 26 and 27) and 100% (for compound 19) in
1
0

A.M. Soliman et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
Fig 10. A: 2D interaction pose of compound 27. B: 3 D interaction map of 27 (red), superimposed on the native ligand erlotinib (magenta).
in the fraction of G0-G1 phase (0.039%, 0%, 0.06 % and 0.042%
respectively compared to 71.6 % of control) and marked increase in the
proportion of cells in G2-M phase (94.2%, 92.9%, 85.8% and 87.8%,
respectively compared to 21.7% of control). Also, compounds 7, 19, 26
and 27 have arrested the S phase cells (2.45%, 0.071%, 2.32% and
p21 gene is a key regulator of the cell cycle that binds to cyclin/CDK
complexes and inhibits kinase activation, subsequently blocking cell
cycle progression.50 The observed G2/M arrest followed by induction of
apoptosis in treated HepG-2 cells can be explained by up-regulation of
p21 expression (Fig. 3C). Moreover, cell cycle phases reflect different
degrees of radiosensitivity of cells to radiation. The most sensitive
phases to radiation are G2 and M phases in contrast to the most radio-
resistant S phase point.51 An interesting finding in our work, G2/M
2
.73%, respectively) compared to control with 6.08%. We believe that
1
9 is superior relative to 7, 26 and 27 as there is almost no S phase arrest
with 0.071%, compared to 2.45%, 2.32% and 2.73%, respectively. The
1
1

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Bioorganic & Medicinal Chemistry Letters 49 (2021) 128308
phase arrest was significantly increased by using flow cytometry upon
treatment of HepG-2 cells with 7, 19, 26 & 27 treatments (Fig. 5).
Annexin V assay has revealed that compounds 7, 19, 26 & 27
treatment induced early stage of apoptosis in HepG-2 cells. The Annexin
V-FITC plots in Fig. 6 show HepG-2 cell distribution within four different
quadrants (Q1, Q2, Q3 and Q4). The data shows a decrease in the per-
centage of viable cells with a significant increase in the percentage of
cells undergoing early apoptosis. A reduction of viable cells was seen in
all treated cells in comparison to the untreated cells. The percentage of
viable cells has decreased from 94.4 % for untreated cells to 86.1%, 84.5
quinazolinone-sulfonamide derivatives 7–10, 13, 14, 17 and 18 were
synthesized based upon hybridization strategy between the quinazoline
ring that constitutes the core of many tyrosine kinase inhibitors and
sulfonamide moiety possessing potent anticancer properties. The cyto-
toxic activities of the newly synthesized compounds were evaluated
against HepG-2 and MCF-7 cell lines. The target compounds showed IC50
ranging from 2.46 to 36.85 µM for HepG-2 and 3.87 to 88.93 µM for
MCF-7 cell lines. The promising derivatives 7, 19, 26 and 27 were
selected to be screened as EGFR inhibitors and displayed IC50 ranging
from 146.9 to 1032.7 nM compared to Erlotinib (IC50= 96.6 nM).
Compounds 7, 19, 26 and 27 have decreased the expression level of
XIAP and Survivin compared to control, which in turn have increased
caspase-mediated apoptosis in HepG-2 cells through significant caspase-
3 activation. The increased level of caspase-3 suggests that the tested
quinazolinones have targeted XIAP to activate caspases, which in turn
have induced apoptosis in HepG-2 cells. Most probably, these four
compounds have increased and sensitized HepG-2 to apoptosis through
p21 overexpression. Taken together, these results suggest that com-
pounds 7, 19, 26 and 27 treatments have overcome apoptosis resistance
by suppressing Survivin, XIAP and overexpressing caspase-3 and p21.
The observed G2/M arrest followed by induction of apoptosis in treated
HepG-2 cells might be explained by up-regulation of p21 expression.
Moreover, flow cytometry analysis of Annexin-V/PI double stain has
confirmed compounds 7, 19, 26 and 27 effects on apoptosis through a
decrease in the percentage of viable cells with a significant concomitant
increase in the percentage of cells undergoing early apoptosis. The
radiosensitizing activities of 7, 19, 26 and 27 were studied on HepG-2
and MCF-7 cancer cell lines after being irradiated by a single dose of
8 Gy gamma radiation. The IC50 of the promising compounds after
irradiation ranges from 1.56 to 4.32 µM for HepG-2 cells and 3.06–5.93
µM for MCF-7 cell lines compared to 2.46–13.17 µM for HepG-2 and
5.86–15.32 µM for MCF-7 before irradiation. The radiosensitizing mo-
lecular mechanism of 7, 19, 26 and 27 might explain G2/M phase arrest,
p21 overexpression, downregulation of XIAP and Survivin expression in
HepG-2 treated cells. The compounds showed an increase in their
cytotoxic effect when combined with radiation, confirming their po-
tential radiosensitizing activity. Molecular docking of the promising
compounds in the binding site of EGFR showed their ability to reproduce
the fundamental interactions explaining the good activity on EGFR. The
cytotoxicity, radiosensitization and pro-apoptotic properties of the
promising compounds suggest that they could be considered as anti-
cancer and radiosensitizing agents.
%
, 86.6 %, 92.5 % for 7, 19, 26 & 27, respectively.
Radiotherapy is a curative treatment for cancer patients mainly
through damaging DNA by ionizing radiation.52,53 Exposing cells to
ionizing radiation results in the formation of free radicals by aqueous
radiolysis.5
4,55
On the other hand, radioresistance of cancer cells is the
primary cause of poor prognosis in cancer patients. So, effective radio-
sensitizers having low toxicity and high selectivity are a must to improve
the efficacy of radiotherapy. The quinazoline-based EGFR inhibitor,
Gefitinib was identified as an effective radiosensitizer with high selec-
5
6,57
tivity.
Similarly, in this study we aimed to develop a radiosensitizer
with high selectivity by maintaining the EGFR autophosphorylation
inhibitory activity. The cytotoxic activities of the most potent com-
pounds 7, 19, 26 and 27 on HepG-2 and MCF-7 cell lines were evaluated
after being exposed to a single dose of 8 Gy gamma irradiation. The IC50
of the compounds are reported in Table 3; the cytotoxic activities of the
compounds have increased after the cells containing the compounds
been subjected to irradiation and became in the range of 1.56–4.32 µM
for HepG-2 cells and 3.06–5.93 µM for MCF-7 cells. Also, significant
downregulation of XIAP and Survivin expression in HepG-2 treated cells
relative to untreated control was observed by these compounds as
mentioned above thus suggesting their radiosensitizing ability.
To study the binding features of the promising compounds, molec-
ular docking simulations were carried out. Compounds 7, 19, 26 and 27
were selected for evaluating their EGFR inhibitory activity and were
docked into the crystal structure of the target kinase in complex with
Erlotinib (PDB code: 1M17).58 Validation of the docking protocol was
initiated by self-docking the native ligand in the binding pocket of EGFR.
Self-docking of the native ligand reproduced the key interactions and the
binding patterns reported earlier,59 with an energy score of ꢀ 7.20 kcal/
mol and RMSD = 1.0352 Å. Energy score and RMSD values are within
acceptable limits.60 The active residues of 1M17 consist mainly of these
key amino acids; Met 769, Thr 766, Leu 694, Ala 719, Gln 767, Leu 764,
Leu 768, Phe 771, Pro 770, Thr 830, Gly 772, Leu 820 and Asp 831. The
2
D and 3D forms of interactions of compounds 7, 19, 26 and 27 were
Declaration of Competing Interest
given in Figs.7-10. The quinazolinone scaffold of 7, 19, 26 and 27 is
located in an area defined by Thr 766, Met 742, Val 702, Ala 719, Leu
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
7
64, Leu 768 and Lys 721 residues. The substituted phenyl group in the
acetamide moiety of 19, 26 and 27 were stabilized by the formation of
hydrophobic contacts with Leu 694, Leu 820, Pro 770, Gly 772. Com-
pound 7 showed the highest EGFR inhibitory potential with IC50= 207.1
nM, displaying the best binding affinity with the lowest energy score
recorded as ꢀ 7.45 kcal/mol, and RMSD = 1.1945 Å. Molecular docking
interactions of 7 were represented as three hydrogen bonding, two of
them are between CO and NH of hydrazide with Met 769 and the third
bond between CO of quinazolinone and Thr 830 residue of 2.57, 2.89
and 2.34 Å bond distance, respectively (Fig. 7A & B). In the second place
came compound 19, the binding score was computed as ꢀ 7.24 kcal/mol
with RMSD = 1.3921 Å. It showed two hydrogen bonds between CO of
acetamide with Met 769 and CO of quinazolinone with Thr 830 residue
with bond distances of 3.02 and 2.93 Å (Fig. 8A & B). Compounds 26
and 27 showed the same interactions exerted by 19. Compound 26
displayed a binding score of ꢀ 6.58 kcal/mol and RMSD = 1.5461 Å
Acknowledgment
The authors appreciate the staff members of gamma irradiation unit
at the National Center for Radiation Research and Technology (NCRRT)
for carrying out the irradiation process.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128308.
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