Design, synthesis and assessment of new series of quinazolinone derivatives as EGFR inhibitors along with their cytotoxic evaluation against MCF7 and A549 cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2021.127987

New acetamide (IV a-e) and 1,3-thiazolidinone derivatives (VII a-e) were designed, synthesized and assessed for their cytotoxic activity against MCF-7 and A549 cell lines along with their lead compounds (erlotinib and gefitinib). The newly designed compou

Full text of DOI:10.1016/j.bmcl.2021.127987

Bioorg. Med. Chem. Lett. 41 (2021) 127987
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and assessment of new series of quinazolinone derivatives
as EGFR inhibitors along with their cytotoxic evaluation against MCF7 and
A549 cancer cell lines
,
,*
Marian W. Aziza, Aliaa M. Kamalb c, Khaled O. Mohamedb, Adel A. Elgendya
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr International University, Cairo, Egypt
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, October University for Modern Science and Arts (MSA), Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
New acetamide (IV a-e) and 1,3-thiazolidinone derivatives (VII a-e) were designed, synthesized and assessed for
their cytotoxic activity against MCF-7 and A549 cell lines along with their lead compounds (erlotinib and
gefitinib). The newly designed compounds were prepared according to the adopted procedures in schemes 1 and
2 from their quinazolinone parents. 3D QSAR pharmacophore and docking molecular modeling protocols were
conducted using Discovery Studio program, beside a full biological assay for these compounds. The cytotoxicity
evaluation demonstrated that compounds IVb, IVc, VIIa, VIIb, VIId exhibited potent cytotoxic activities against
both MCF-7 and A549 cell lines. Moreover, the molecular modeling studies corroborated to the affinity of the
compounds towards EGFR. Consequently, these five compounds were then screened for their EGFR inhibition
and evaluated as well for their toxicity to normal cells, which revealed that the acetamide derivative IVc and the
thiazolidinone derivative VIIa were the most potent and least toxic. DNA flow cytometry analysis was conducted
for compounds IVc and VIIa, which indicated that they both induced arrest at G2/M phase of the cell cycle.
Quinazolinone
Thiazolidinone
3DQSAR pharmacophore
Cytotoxic activity
EGFR
Cancer is considered as a general term that represents a wide range of
malignancies.1 Those malignancies are generally characterized by un-
controlled cellular proliferation and growth as well as affected cell
migration and spreading to other tissues and organs.2 Various factors
may lead to the change of a normal cell to tumor-affected one through
the alteration of wide range of regulatory, signal transduction and
apoptotic pathways. The characteristic feature of carcinogenic cells is,
mostly, the extensively uncontrolled cellular proliferation.3 Two of
highly spreading types of cancers are breast and lung cancers.4,5
Breast cancer is considered the most common malignant tumor and
the second known cause of death in women that results from numerous
genetic alterations that affect many genes,6,7 while lung cancer is
thought of as another leading death cause, especially non-small cell lung
cancer (NSCLC) that accounts for 80% of all lung cancer cases.8–10
The epidermal growth factor receptor (EGFR) physiological function
is the regulation of the development and homeostasis of epithelial tis-
sues.11 The widely expressed in lung and breast cancer EGFR is a driver
of tumorigenesis.12,13 Amplification and potent mutation of the genomic
locus usually result in inappropriate activation of the EGFR in cancer.
The EGFR is also highly recognized as a resistance biomarker in tumors
since its secondary mutation or amplification has been known to rise
under drug pressure.14,15
The EGFR pathway is inhibited by tyrosine kinase inhibitors (TKIs) as
erlotinib and gefitinib,16,17 they perform their action intracellularly as
they competitively bind to the EGFR adenosine triphosphate pocket,
which consequently inhibits EGFR auto-phosphorylation and down-
stream signal transduction.18,19
Quinazoline is a heterocyclic scaffold that exerts a wide range of
biological activities20–22 and is well known to exhibit anti-tumor activity
through inhibition of EGFR-TK and multiple other targets,23,24 as a
result lead EGFR inhibitors were early developed at Zeneca pharma-
ceuticals (now Astrazeneca) as a 4-(3-chloroanilino) quinazoline scaf-
fold was found to act as a potent enzyme inhibitor. Further investigation
of the structure and catalytic mechanism of the EGFR tyrosine kinase
wild type led to the identification of the new class of anilinoquinazoline
tyrosine kinase inhibitors. erlotinib and gefitinib were the first two
EGFR inhibitors followed by the development of lapatinib and afatinib
as other class members.25
* Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Misr International University, Cairo, Egypt.
E-mail address: adel.gendy@miuegypt.edu.eg (A.A. Elgendy).
https://doi.org/10.1016/j.bmcl.2021.127987
Received 22 December 2020; Received in revised form 6 March 2021; Accepted 10 March 2021
Available online 23 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
Our work was based on modification in the structure of erlotinib and
aldehydes yielding the final compounds VII a-e. The structures of com-
pounds VII a-e were also depicted by analytical and spectral data, IR
spectrum showed the presence of peaks of NH groups at 3468–3251
gefitinib as lead compounds, in which their aniline moiety in position 4
was changed to anilinoacetamide moiety (IV a-e derivatives), or ami-
nothiazolidinone moiety (VII a-e derivatives) in position 3 instead, as
shown in Fig. 1, the logic behind the introduction of those moieties was
based on their abilities to enhance the cytotoxic activities of various
reported anticancer agents.26–29
cmꢀ 1. In addition, the C O groups were found at 1735–1651 cmꢀ 1, Its
–
–
1H NMR revealed the presence of signals at 11.11–12.59 ppm, due to NH
moiety along with the disappearance of the CH2 moiety, plus the
confirmed appearance of extra aromatic protons at 7.02–8.74 ppm and
the development of the peak for the olefinic carbon at 134.92–158.86
ppm in the 13C NMR.30,31,43–59
A comparison between the effect of the suggested structural changes
in our derivatives on the cytotoxic activity against the reference (erlo-
tinib and gefitinib) was conducted through a detailed cytotoxic evalu-
ation in which the new developed molecules were found to have
superior activity compared to reference compounds.
The newly synthesized compounds IV a-e and VII a-e along with their
standards erlotinib and gefitinib were screened for their cytotoxicity in
vitro against breast cancer cell line (MCF7) as well as non-small cell lung
cancer cell line (A549). Promising compounds IVb, IVc, VIIa, VIIb, VIId,
concluded from the cytotoxicity screen as well as from molecular
modeling (3DQSAR pharmacophore and docking) protocols that were
conducted to configure the scientific potentials of these compounds,
were then selected to be subjected to EGFR-TK inhibitory assay (Wild-
type EGFR) in addition to being tested for their toxicity to normal cell
line. Compounds IVc and VIIa that conclusively showed superior EGFR
inhibitory activity as well as least toxicity to normal cell were then
evaluated through cell cycle analysis to elucidate their effect on the cell
cycle progression and the percent of apoptosis exerted by these
compounds.60
Furthermore, to comprehend the cytotoxic activity of the synthesized
compounds, 3DQSAR pharmacophore and docking molecular modeling
protocols were carried out, which supports the anticancer activity claim
based on results.
Schemes 1 and 2 discussed the synthetic pathways applied in pre-
paring the targeted compounds. Scheme 1 utilized the synthesis of
benzoxazinone derivative (I) and its amino derivative (II) by the reac-
tion with hydrazine hydrate, followed by nucleophilic substitution with
chloroacetyl chloride, and finally by synthesis of aminoacetamide de-
rivatives (IV a-e). The structures of compounds IV a-e were confirmed by
analytical and spectral data, IR spectrum showed the presence of peaks
of NH groups at 3325–3197 cmꢀ 1, while the CH2 moiety appeared at
All targeted compounds IV a-e and VII a-e were inspected in vitro
against two EGFR-over expressing cell lines, which are the non-small cell
lung carcinoma cell line (A549) (well known to exert wild-type EGFR
expression) and the breast cancer cell line (MCF-7) that also shows
numerous growth factor receptors expression including wild-type EGFR,
in order to evaluate the new compounds cellular efficiency. Throughout
this in-vitro testing, erlotinib and gefitinib were used as reference. The
data for the in-vitro cytotoxicity of the tested compounds is shown in
Table 1. The results revealed that most of the tested compounds showed
2962–2904 cmꢀ 1. Furthermore, C O groups were found at 1701–1543
–
–
cmꢀ 1. Its 1H NMR revealed the presence of signals at 3.83–4.27 ppm,
due to NH moiety and significant increase in aromatic protons at range
5.83–8.21 ppm and the 13C NMR confirmed as well the synthesized
compounds. Scheme 2 showed the reaction of compound II with
ammonium thiocyanate forming thiourea derivative (V), subsequently
cyclized to thiazolidinone (VI) by the reaction with chloroacetyl chlo-
ride, which finally went through condensation with different aromatic
R1
O
H
N
N
N
H
O
N
ph
IV a-e
O
N
H
N
N
N
O
S
ph
R2
VII a-e
Fig. 1. Structure of proposed EGFR inhibitors.
2

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
Scheme 1. Synthesis of intermediates
(I-III, V and VI) and targeted compounds
(IV a-e) and (VII a-e). Reagents and
conditions: Benzoyl chloride, pyridine.
(ii) Hydrazine hydrate (80%), ethanol,
8–10 h; (iii) Chloroacetyl chloride, room
temperature, 2–3 h; (iv) Aromatic ani-
O
N
COOH
NH2
O
i
lines, ethanol, reflux, 6–8 h; (v) Ammo-
ph
nium thiocyanate, ethanol, reflux, 4–8 h;
I
(vi) Chloroacetyl chloride, DMF, rt,
10–12 h, then anhydrous Na acetate,
ethanol, reflux, 5 h; (vii) Aromatic al-
dehydes, glacial acetic acid, anhydrous
Na acetate, reflux, 8–10 h.
ii
O
N
O
H
N
NH2
N
Cl
N
iii
O
ph
N
ph
III
II
iv
R1
O
H
N
N
N
H
O
N
ph
IV a-e
cytotoxic activity against both A549 (0.72–187 µM) and MCF-7
(1.003–15.25 µM) cell lines, respectively. Five compounds IVb, IVc,
VIIa, VIIb, VIId displayed superior cytotoxic activity against both cell
lines compared to the reference compounds, However, the thiazolidi-
none derivative (VIIa) was the most active in (A549) cell line, while the
thiazolidinone derivative (VIIe) was the one for (MCF-7) cell line (IC50
= 0.72 µM, 1.003 µM, respectively).40,61
established by studying their cytotoxic potential against normal cells, as
manifested in Table 3.32 The results demonstrated that these compounds
exhibited high selective cytotoxicity against malignant cells, especially
compound IVc (51.23 µM) and VIIa (44.34 µM), which correlates with
the EGFR activity profile.
The two compounds, IVc and VIIa, that showed superior cytotoxic
activity against both MCF-7 and A549 cell lines as well as EGFR
enhanced inhibition and also exhibited good cytotoxicity profile over
normal cell relative to erlotinib and gefitinib, were subjected to cell
cycle analysis and apoptotic assay in order to figure out their effect on
progression of cell cycle and the percent of apoptosis they induced. For
results of the cell cycle analysis for compounds IVc and VIIa, cf. Table 4
and Fig. 2.40,63
After being evaluated for their cytotoxic activity, five compounds
IVb, IVc, VIIa, VIIb, VIId of the ten synthesized compounds that elicited
superior cytotoxic activity against both MCF-7 and A549 cell lines were
then investigated for their EGFR inhibitory activity, using erlotinib and
gefitinib as references, as illustrated in Table 2. All the tested com-
pounds were found to exert EGFR inhibitory activity, with IC50 values
ranging from 65.63 to 178.1 nM. Two compounds IVc and VIIa exhibited
relatively good activity at sub-molecular level, with IC50 = 81.07 nM
and 65.63 nM, respectively, compared to erlotinib (54.34 nM) and
gefitinib (166 nM).40,62
The results obtained clearly revealed that both compounds IVc and
VIIa led to Pre G1 apoptosis with cell cycle arrest at G2/M phase, which
was confirmed by an increase in the percentage of DNA content
(36.21%) after addition of compound IVc and (29.17%) after addition of
compound VIIa, compared to the control cell (10.49%). This
Compounds IVb, IVc, VIIa, VIIb, VIId selectivity profiles were then
3

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
O
N
H
N
NH2
N
v
II
S
ph
V
vi
O
N
H
N
N
N
O
S
ph
VI
vii
O
H
N
N
N
O
S
N
ph
R2
VII a-e
Scheme 2. Synthesis of intermediates (I-III, V and VI) and targeted compounds (IV a-e) and (VII a-e). Reagents and conditions: Benzoyl chloride, pyridine. (ii)
Hydrazine hydrate (80%), ethanol, 8–10 h; (iii) Chloroacetyl chloride, room temperature, 2–3 h; (iv) Aromatic anilines, ethanol, reflux, 6–8 h; (v) Ammonium
thiocyanate, ethanol, reflux, 4–8 h; (vi) Chloroacetyl chloride, DMF, rt, 10–12 h, then anhydrous Na acetate, ethanol, reflux, 5 h; (vii) Aromatic aldehydes, glacial
acetic acid, anhydrous Na acetate, reflux, 8–10 h.
demonstrates the role of these compounds in EGFR inhibition, as EGFR
activation is required for progression from G2 to M phase in the second
mitotic wave cells, in part through activation of the phosphatase cdc25/
string.43
increased (11.03%) after treatment with compound IVc and (9.27%)
after treatment with compound VIIa, compared to the control cells
(1.19%), which represents a prominent marker of apoptosis.
In the current study, we synthesized number of acetamide and 1,3-
thiazolidinone derivatives for quinazolinone compounds as novel
EGFR inhibitors via consecutive protocols of molecular modeling. First,
we studied the docking of erlotinib in the biologically active binding site
of EGFR, then we generated predictive pharmacophore model through
3D quantitative relationship between structure and activity, using
To investigate IVc and VIIa apoptotic induction effect and quantifi-
cation of the percentage of apoptosis. Annexin V-FITC/Propidium iodide
dual staining assay was carried out according to the mechanism re-
ported. The effect of compounds IVc and VIIa on the apoptotic induction
is shown in Table 5.41,64
The results revealed that the total apoptotic cells percentage
3DQSAR pharmacophore generation protocol from a set of 30
4

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
Table 1
structure obtained in complex with erlotinib as an inhibitor (PDB code:
1M17), to predict their binding mode and binding interactions.33,34 The
compatibility between the pharmacophore model fit values and the
docking affinities and interactions offered a relevant overview of the
developed new EGFR inhibitors promising activity.65,66
Cytotoxicity of the tested compounds, erlotinib and gefitinib against A549 and
MCF-7 cell lines.
Compounds
IC50
µM
MCF-7
A549
The molecular docking of erlotinib was based on the crystal structure
of EGFR (PDB code: 1M17), which was the crystal structure of EGFR
complexed with erlotinib as a ligand.34 Prepared protein parameters
were used to prepare the protein that would eliminate common prob-
lems in the provided protein structure undergoing preparation for more
processing. Ligand preparation tool was used for the preparation of
erlotinib (AQ4999) and Docking was carried out using the CDocker
protocol. The CDocker energy of erlotinib was ꢀ 19.0663, its CDocker
interaction energy was ꢀ 42.5609, it formed 4 hydrogen bonds with
amino acids (MET769, ASP831, GLY772, GLU738), the planar regions of
quinazoline moiety formed hydrophobic interaction with amino acids
(ALA719, CYS751, LEU820, LEU694, VAL702) of the hydrophobic
pocket. These interactions resembled those reported for erlotinib (PDB
code: 1M17) with additional hydrogen and hydrophobic interaction as
the reported results emphasized the importance of the MET769 and
CYS751 interactions only (Fig. 3).34
IVa
15.25 ± 0.33
2.42 ± 0.05
1.74 ± 0.03
4.41 ± 0.09
15.12 ± 0.33
4.05 ± 0.09
3.76 ± 0.08
2.30 ± 0.05
7.49 ± 0.16
1.003 0.02
11.18 ± 0.24
3.57 ± 0.09
13.3 ± 0.5
2.1 ± 0.1
IVb
IVc
7.29 ± 0.3
187 ± 7.5
25 ± 1.1
IVd
IVe
VIIa
VIIb
VIIc
0.72 0.03
5.31 ± 0.2
21.3 ± 0.9
3.22 ± 0.1
44.7 ± 1.8
10.0 ± 0.4
3.53 ± 0.1
VIId
VIIe
erlotinib
gefitinib
Table 2
EGFR inhibitory activity of the target compounds,
compared to erlotinib and gefitinib as reference
standards.
In order to carry out a 3D QSAR pharmacophore generation, a set of
30 EGFR inhibitors with known activity (IC50) ranging from 0.36 to 204
nM was collected from literature for the generation of EGFR inhibitors
library (Fig. 4).18,35 The ligand library was prepared using Accelrys
Discovery Studio 4.0 software.
Compounds
EGFR
IC50
nM
VIId
178.1 ± 9.1
81.07 4.1
136 ± 6.9
IVc
The collected ligand library was divided into training set of 24 Li-
gands and test set of 6 ligands. For developing the protocol, hydrogen
bond acceptors (HBAs), hydrogen bond donor (HBDs), hydrophobic
moieties (HYPs), positive ionizable groups (PIs) and aromatic rings
(ARs) were selected as the chemical features based on the result of a
conducted feature mapping protocol for the training set ligands. The
uncertainty was set to 1.5 and IC50 was selected as the activity measure.
The generated pharmacophore models (10 hypothesis) were then vali-
dated based on their statistical significance and their ability to predict
the biological activity of unknown ligands. Based on cost difference and
accuracy of estimated activity and the reference mapping results, the
validity of the model was proven.42
VIIb
VIIa
65.63 3.3
143.4 ± 7.3
54.34 ± 1.9
166 ± 8.3
IVb
erlotinib
gefitinib
Table 3
In-vitro cytotoxicity screening against normal cell
line.
Compounds
Cytotoxicity
PCS-800-011
IC50
µM
The analysis of the 10 hypothesis revealed that the best was the first
model, it showed the highest cost difference of 134.01, which indicated
statistical significance of the model with high predictive power > 90%,
its RMS value was 2.14 and correlation coefficient of 0.857, revealing
the suitability of the model to predict novel compounds activity, as
shown in Table 6. Besides the cost analysis, the predicted activity of each
compound by the model was compared to their experimental reported
IC50 values, as shown in Table 7. The differences between both values
were quite small, which confirmed the validity of the model. The valid
model showed 1 hydrogen bond acceptor and 2 ring aromatic features
(Fig. 5a).
VIIa
44.34 ± 2.79
17.28 ± 1.09
36.85 ± 2.32
31.35 ± 1.97
51.23 ± 3.22
32.24 ± 2.03
40.78 ± 1.28
VIId
IVb
VIIb
IVc
erlotinib
gefitinib
compounds having reported activities over our target EGFR. The
generated pharmacophores were then validated in order to select the
best model to map our proposed compounds to evaluate their fitting and
estimated activity, then the most promising proposed compounds
derived from both the Pharmacophore modeling and the biological
evaluation were then docked into the binding site of the EGFR crystal
Mapping of erlotinib into the valid model was carried out, using
ligand-pharmacophore mapping protocol of Accelrys Discovery Studio
4.0 software, which revealed fit value of 6.46472 and estimated IC50 of
Table 4
Cell cycle analysis result of compounds IVc and VIIa on MCF-7 cell line, after 24 h.
Compounds
Code
Results
DNA content
MW
Conc.
%G0-G1
%S
%G2/M
%Pre-G1
Comment
µM
IVc/MCF7
VIIa/MCF7
Cont.MCF7
404.85
424.478
—
1.74
4.05
—
34.32
42.95
52.94
29.47
27.88
36.57
36.21
29.17
10.49
37.24
26.38
1.63
cell growth arrest@G2/M
cell growth arrest@G2/M
—
5

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
Fig. 2. Effect of compounds IVc and VIIa on the cell cycle of MCF-7 cells after 24 hr.
Furthermore, the promising proposed analogues (IVc and VIIa)
were docked into the crystal structure of EGFR found in complex
with the inhibitor erlotinib (PDB code: 1M17).34
Table 5
Effect of compounds IVc and VIIa on apoptotic induction compared to the
control cells after 24 h.
Docking studies of the designed proposed compound IVc. The
docking score of compound IVc was of ꢀ 20.6858, it showed a CDock-
er_Interaction_Energy of ꢀ 39.4273, and it formed two hydrogen bonds
with amino acid MET769 and THR830. Moreover, the planar aromatic
rings of the compound formed Van der Waals interaction with amino
acids LEU694, VAL702, LEU820, LYS721 (Fig. 6).
Compound
Apoptosis
Necrosis
Total
Early
Late
IVc/MCF7
VIIa/MCF7
Cont.MCF7
37.24
26.38
1.63
2.56
4.95
0.32
23.65
12.16
0.12
11.03
9.27
1.19
Docking studies of the designed proposed compound VIIa. The
docking score of compound VIIa was of ꢀ 21.8412, it showed a CDock-
er_Interaction_Energy of ꢀ 40.5207, and it formed two hydrogen bonds;
–
one between the C O of the thiazolidinone moiety and amino acid
–
MET769 and one between the N of the quinazolidinone moiety and
amino acid LYS721. Moreover, the planar aromatic rings of the com-
pound formed Van der Waals interaction with amino acids LEU694,
VAL702, PHE699, LEU820, LYS721 (Fig. 6).
The docking study showed that the binding mode of the designed
compounds inside the 1M17 resembled that reported of erlotinib along
with additional Van der Waals or hydrophobic interactions in the pro-
posed molecules, due to the variation in substituents.
In conclusion, ten compounds IV a-e, VII a-e were synthesized and
evaluated for their cytotoxic activity. Four acetamide derivatives (IVa,
IVb, IVc, IVe) and four 1,3-thiazolidinone derivatives (VIIa, VIIb, VIIc,
VIId) exhibited significant cytotoxic activity against both MCF-7 and
A549 cell lines, while two compounds IVd and VIIe showed significant
cytotoxic activity against MCF-7, only compared to erlotinib (11.18, 10
µM, respectively) and gefitinib (3.57, 3.53 µM, respectively). Five
compounds, IVb, IVc, VIIa, VIIb, VIId, that elicited superior cytotoxicity
against both MCF-7 and A549 cell lines, were subjected to EGFR
inhibitory assay as well as to evaluation for their toxicity against normal
cells. Two compounds IVc, VIIa showed promising inhibitory activity
against EGFR (81.07, 65.63 nM, respectively) as well as good normal cell
cytotoxicity profile (51.23, 44.34 µM, respectively), compared to erlo-
tinib and gefitinib. The two compounds IVc, VIIa were subjected to cell
cycle analysis and apoptotic assay that revealed significant inhibition at
G2/M phase, with good necrosis activity. The results of molecular
docking and pharmacophore studies confirmed that the fitting and the
binding mode were consistent with the EGFR inhibitory activity of the
tested compounds. According to the observed results in both cytotoxicity
assay against MCF-7 and A549 cell lines and the EGFR inhibition assay,
Fig. 3. Docking of erlotinib inside the binding site of EGFR (PDB code: 1M17)
in 3D style.
1.42653 nM (Fig. 5b).
Pharmacophore mapping of the proposed compounds was carried
out using same ligand-pharmacophore mapping protocol used for the
reference erlotinib to determine their fit values and estimated IC50
(Table 8), which showed their compatibility with the pharmacophore
model, as displayed by their fit values. Furthermore, most of the pro-
posed analogues were observed to have promising estimated IC50
values.
6

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
N
N
N
N
HN
N
Cl
HN
Br
Cl
HN
N
O
N
H
O
H
6b=81
5c=18
Cl
N
N
N
N
HN
N
Cl
HN
Cl
HN
Cl
N
O
N
N
O
N
O
O
8b=204
7c=47
O
O
N
S
O
NH
N
N
H
O
HN
N
N
16j=0.36
57b=14
Br
O
N
O
O
N
N
H
HN
N
HN
Cl
NH
O
O
Cl
O
O
OH
4m=33.1
12b=0.39
Fig. 4. Some representative compounds with their EGFR inhibitory activity in nM for the library of reported active ligand used for 3D QSAR pharmacophore
model generation.
7

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
Table 6
The ten generated hypotheses with their maximum fit, total cost, cost difference, RMS and correlation coefficient.
Hypothesis
Maximum fit
Total cost
Cost difference
RMS
Correlation coefficient
1
7.71
7.86
6.39
5.98
6.18
6.03
6.3
131.09
148.83
151.24
156.05
162.64
168.13
168.54
170.63
171.24
175.68
134.01
116.26
113.85
109.05
102.45
96.959
96.548
94.464
93.852
89.413
2.14
2.49
2.53
2.61
2.71
2.79
2.8
0.857
0.8
2
3
0.79
0.77
0.75
0.74
0.739
0.732
0.731
0.71
4
5
6
7
8
8.08
6.7
2.83
2.839
2.9
9
10
5.28
Table 7
The training set along with its fit value, experimental IC50 and estimated IC50
.
Training set
Fit value
Experimental IC50
Estimated IC50
12b
7.02084
6.91646
6.50226
6.46472
6.06563
6.04728
5.85794
5.80685
5.50433
5.48903
5.4625
0.39
0.36
4.4
2
0.396428
0.504123
1.3084
16j
9a
erlotinib
1.42653
3.57575
3.73008
5.7684
21b
0.38
2
52b
58b
5
gefitinib
37
6.4886
dacomitinib
6
13.0218
13.4887
14.3382
17.8045
30.4846
31.6792
32.6788
33.15
afatinib
10
osimertinib
12.92
14
57b
8b
5.36847
5.13492
5.11823
5.10473
5.09852
5.09632
5.09151
5.08823
5.08561
5.08484
5.08415
5.06265
5.06056
204
22
5b
6b
81
4m
13j
13e
4d
33.1
50
33.3177
33.6892
33.9447
34.1498
34.2103
34.2652
36.0043
36.1772
14
52.6
20.6
35.5
66.2
14.5
30.7
4n
4u
4e
4v
4h
Table 8
Mapping results of the proposed EGFR inhibitors to pharmacophore hypothesis 1.
Compounds
Fit value
Estimated activity (nM)
IVd
VIIe
IVa
7.65749
7.63027
7.63012
7.62963
7.62878
7.49281
7.49275
7.49161
7.48818
7.48354
0.0915196
0.0974389
0.097471
0.0975823
0.0977731
0.133719
0.133735
0.134089
0.135152
0.136602
IVe
IVb
VIIc
VIId
IVc
VIIa
VIIb
the para-monosubstituted acetamide derivatives with electron with-
drawing groups (e.g. Cl) as in compound IVc showed higher anti-cancer
activity of IC50 = 1.74 µM on MCF-7 cell line and IC50 = 7.29 µM on
A549 cell line and more EGFR inhibition at 81.07 nM, while in the
thiazolidinone derivatives (VII a-e), due to their bulky structure, it was
found that the unsubstituted compound VIIa exhibited higher cytotoxic
activity against A549 cell line, with IC50 = 0.72 µM, and more EGFR
inhibition at 65.63 nM, while in MCF-7, the 3,4-dimethoxy substituted
derivative (VIIe) was found to exert higher cytotoxicity, due to the
presence of the electron donating groups that enhanced the activity.
Declaration of Competing Interest
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.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127987.
8

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
Fig. 5. (a) The best generated pharmacophore model with the features, considering constraint distances and angles between features, considering hydrogen bond
donor (HBA) colored in green and ring aromatic (RA) colored in orange. (b) Erlotinib viewed as fit reference compound with fit value of 6.46472 and estimated IC50
of 1.42653 nM.
Fig. 6. 3D image of the proposed compounds IVc and VIIa inside 1M17, using Accelrys Discovery Studio.
13 Cairns RA, et al. Cancer cell metabolism. Cold Spring Harb Symp Quant Biol. 2011;76:
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quinazolinone), 13.49 (C-,6 benzene), 17.4 (C-6 quinazolinone), 18.51 (C-4 aniline),
18.78 (C-3,5 aniline), 19.43 (C-1 benzene), 13.15 (C-5 quinazolinone), 13.63 (C-3,5
benzene), 133.34 (C-4 benzene), 134.87 (C-7 quinazolinone), 139.87 (C-1 aniline),
146.71 (C-8a quinazolinone), 164.9 (C=O quinazolinone), 168.5 (C- quinazolinone),
170.51 (C=O acetamide). Anal. Calcd for C4HN4O (398.46): C, 7.7; H, 5,5; N, 14.05;
Found: C, 71.98; H, 5,68; N, 14.31 %.
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50 2-(4-chlorophenylamino)-N-(4-oxo–phenylquinazolin-3(4H)-yl) acetamide (IVc).
yield 80%, m.p. 134-136 ◦C; IR (KBr), cm-1: 330,336 (-NH), 306 (arom.CH), 981
(aliph.CH), 1697-1566 (C=O); 1 H NMR: δ 3.77 (q,H, CHCO), 11.06 (s,1H, NHCO,
DO exchange), 4.16 (s, 1H, NH, DO exchange), 6.37 (d,H, arom.H), 7.015 (d,H,
arom.H), 7.45 (s,H, arom.H), 7.49 (d,3H, arom.H), 7.59 (s,1H, arom.H), 7.65 (d,H,
arom.H), 8.1 (s,1H, arom.H), 13C-NMR (100MHz, DMSO-d6) δ (ppm): 45.91 (CH
acetamido), 114.09 (C-,6 aniline), 10.35 (C-4a quinazolinone), 11. (C-8
quinazolinone), 17.0 (C-4 aniline), 17.93 (C-,6 benzene), 18.16 (C-6 quinazolinone),
18.37 (C-1 benzene), 18.88 (C-5 quinazolinone), 18.91 (C-3,5 benzene), 130.56 (C-
3,5 aniline), 133.86 (C-4 benzene), 135.73 (C-7 quinazolinone), 147.06 (C-1 aniline),
147.48 (C-8a quinazolinone) , 156.73 (C=O quinazolinone), 159.74 (C-
quinazolinone), 170.45 (C=O acetamido). Anal. Calcd for CH17ClN4O (404.85): C,
65.; H, 4.19; N, 13.83; Found: C, 65.04; H, 4.31; N, 14.06 %.
29 Dawood KM, et al. Synthesis of some new pyrazole-based 1,3-thiazoles and 1,3,4-
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51 2-(2,4-dichlorophenylamino)-N-(4-oxo–phenylquinazolin-3(4H)-yl) acetamide (IVd).
yield 56%, m.p. 7-74 ◦C; IR (KBr), cm-1: 348,305 (-NH), 3066 (arom.CH), 935 (aliph.
CH), 1678-1543 (C=O); 1H NMR: δ 4.87 (s,H, CHCO), 11.85 (s,1H, NHCO, DO
exchange), 11.65 (s, 1H, NH, DO exchange), 7.1 (t,1H, arom.H), 7.3 (t,1H, arom.H),
7.55 (d,1H, arom.H), 7.57 (s,3H, arom.H), 7.53 (d,H, arom.H), 5.59 (s,1H, arom.H),
7.6 (d,H, arom.H), 7.98 (d,1H, arom.H), 13C-NMR (100MHz, DMSO-d6) δ (ppm):
65.5 (CH acetamido), 114.09 (C-6 aniline), 10.66 (C-4a quinazolinone), 13.63 (C-8
quinazolinone), 14.55 (C-1 aniline), 16.9 (C-4 aniline), 17.71 (C-,6 benzene), 18.66
(C-6 quinazolinone), 19.66 (C-5 aniline), 130.36 (C-1 benzene), 13.06 (C-5
quinazolinone), 13.59 (C-4 benzene), 134.5 (C-7 quinazolinone), 137.95 (C-1
aniline), 148.71 (C-8a quinazolinone) , 161.65 (C=O quinazolinone), 164.99 (C-
quinazolinone), 170.64 (C=O acetamido). Anal. Calcd for CH16ClN4O (439.9): C,
60.09; H, 3.64; N, 1.74; Found: C, 60.3; H, 3.87; N, 13.01 %.
35 Yu-Jing YJ, Zhang CM, Liu ZP. Recent developments of small molecule EGFR
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52 2-(4-bromophenylamino)-N-(4-oxo–phenylquinazolin-3(4H)-yl) acetamide (IVe).
yield 85%, m.p. -4 ◦C; IR (KBr), cm-1: 335,3197 (-NH), 306 (arom.CH), 935 (aliph.
CH), 1678-1543 (C=O); 1H NMR: δ 3.84 (s,H, CHCO), 10.75 (s,1H, NHCO, DO
exchange), 4.87 (s, 1H, NH, DO exchange), 6.6 (d,H, arom.H), 7.4 (d,H, arom.H), 7.6
(d,3H, arom.H), 7.53 (d,3H, arom.H), 7.58 (q,1H, arom.H), 5.66 (d,H, arom.H), 7.93
(d,1H, arom.H), 13C-NMR (100MHz, DMSO-d6) δ (ppm): 65.5 (CH acetamido),
107.55 (C-4 aniline), 114.91 (C-,6 aniline), 117.16 (C-4a quinazolinone), 119.0 (C-8
quinazolinone), 10.66 (C-,6 benzene), 13.63 (C-6 quinazolinone), 17.71 (C-1
benzene), 18.66 (C-5 quinazolinone), 19.44 (C-3,5 benzene), 131.71 (C-4 benzene),
13.59 (C-3,5 aniline), 133.38 (C-7 quinazolinone), 148.10 (C-1 aniline), 148.71 (C-
8a quinazolinone) , 161.65 (C=O quinazolinone), 164.99 (C- quinazolinone), 170.01
(C=O acetamido). Anal. Calcd for CH17BrN4O (449.31): C, 58.76; H, 3.78; N, 1.46;
Found: C, 58.9; H, 3.85; N, 1.63 %.
41 Allam HA, et al. Design and Synthesis of some new 2,4,6-trisubstituted quinazoline
EGFR inhibitors as targeted anticancer agents. Bioorg Chem. 2020;98, 103726.
42 Gupta AK, et al. Pharmacophore modelling, molecular docking and virtual screening
for EGFR (HER 1) tyrosine kinase inhibitors. SAR QSAR Environ Res. 2011;22:
239–263.
43 Baker NE, et al. The EGF receptor defines domains of cell cycle progression and
survival to regulate cell number in the developing Drosophila eye. Cell. 2001;104:
699–708.
53 3-(4-oxo-4,5-dihydrothiazol-2-ylamino)-2-phenylquinazolin-4(H)-one (VI). yield
65%, m.p.120 ◦C in acetic acid; IR (KBr), cm-1: 441 (-NH), 059,028 (arom.CH), 299
(aliph.CH), 175-1712 (C=O), 1647-1546 (C=C); 1H NMR: δ 11.14 (s, 1H, NH, D2O
exchange), 2.50 (s,2H, aliphatic H). 7.6 (d,2H, arom.H), 7.75 (t,2H, arom.H), 7.77
(d,1H, arom.H), 7.90 (d,1H, arom.H), 7.95 (d,2H, arom.H), 8.05 (t,1H, arom.H),
8.21 (d,1H, arom.H), 1C-NMR (100MHz, DMSO-d6) δ (ppm): 20.72 (C-5
thiazolidinone), 121.0 (C-4a quinazolinone), 127.88 (C-8 quinazolinone), 128.86 (C-
2,6 benzene), 128.97 (C-6 quinazolinone), 129.72 (C-1 benzene), 10.57 (C-5
quinazolinone), 1.17 (C-,5 benzene), 1.99 (C-4 benzene), 15.67 (C-7 benzene),
147.08 (C-8a quinazolinone) , 156.72 (C-2 thiazolidinone), 159.87 (C-4
quinazolinone), 167.89 (C-2 quinazolinone), 169.12 (C-4 thiazolidinone). Anal.
Calcd for C17H12N4O2S (6.7): C, 60.64; H, .56; N, 16.64; Found: C, 58.17; H, .75; N,
16.46 %.
44 Compounds I, II, III, V were prepared according to reported methods.36–39
45 For the preparation of compound (VI), a solution of the thiourea derivative V (1g, 6
mmol: 1 equiv.) in DMF was stirred with chloroacetyl chloride (6 mmol: 1 equiv.) at
room temperature for 10-12 h. The resulting mixture was poured over ice cold water
then filtered and was subsequently refluxed in ethanol in the presence of sodium
acetate for 5 h. The resulting mixture was poured over water then filtered and the
filtered solid was then washed twice with water then crystallized from ethanol to
yield derivatives (VI).
46 For the preparation of compounds (IV a-e), a solution of the acetamide derivative III
(1g, 6mmol: 1 equiv.) in absolute ethanol was stirred in the presence of sodium
acetate with the appropriate substituted amine (6mmol: 1 equiv.) and refluxed for 12
h. The resulting mixtures were filtered and the filtered solids were then washed with
water then crystallized from ethanol to yield derivatives (IVa-e).
54 3-(5-benzylidene-4-oxo-4,5-dihydrothiazol-2-ylamino)-2-phenylquinazolin-4(H)-one
(VIIa). Yield 87%, m.p. 194-196 ◦C; IR (KBr), cm-1: 441 (-NH), 059,028 (arom.CH),
299 (aliph.CH), 175-1712 (C=O), 1647-1546 (C=C); 1H NMR: δ 12.59 (s, 1H, NH,
D2O exchange), 7.02 (d,1H, arom.H). 7.14 (d,1H, arom.H), 7.24 (d,2H, arom.H),
7.29 (d,H, arom.H), 7.4 (d,2H, arom.H), 7.4 (d,2H, arom.H), 5.56 (d,1H, arom.H),
7.74 (d,2H, arom.H), 8.15 (d,1H, arom.H), 1C-NMR (100MHz, DMSO-d6) δ (ppm):
117.11 (C-4a quinazolinone), 120.6 (C-8a quinazolinone), 12.44 (C-2,6 benzelidine),
125.21 (C-2,6 benzelidine), 127.49 (C-6 quinazolinone), 128.84 (C-1 benzene),
128.89 (C-,5 benzelidine), 129.48 (C-,5 benzene), 129.48 (C-5 quinazolinone), 11.78
(C-4 benzene), 11.78 (C-5 thiazolidinone), 12.67 (C-7 quinazolinone), 14.78 (C-1
benzelidine), 15.02 (olefinic carbon), 15.02 (C-8a quinazolinone) , 141.58 (C-4
quinazolinone), 165.18 (C-2 thiazolidinone), 165.18 (C-2 quinazolinone), 170.48 (C-
4 thiazolidinone). Anal. Calcd for C24H16N4O2S (424.47): C, 67.84; H, .76; N, 1.19;
Found: C, 68.12; H, .79; N, 1.27 %.
47 For the preparation of compounds (VIIa-e), a solution of the thiazolidinone
derivative VI (1g, 6mmol: 1 equiv.) in glacial acetic acid was stirred with the
appropriate substituted aromatic aldehyde (6mmol: 1 equiv.) and refluxed for 48 h.
The resulting mixtures were filtered and washed twice with water then crystallized
from ethanol to yield derivatives (VIIa-e).
48 N-(4-oxo-2-phenylquinazolin-3(4H)-yl)-2- (phenyl amino) acetamide (IVa). yield
72%, m.p. 197-200 ◦C; IR (KBr), cm-1: 3298,3236 (-NH), 3051 (arom.CH), 2904
(aliph.CH), 1701,1604 (C=O); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.76 (s,2H,
CHCO), 11.17 (s,1H, NHCO, D2O exchange), 10.91 (s, 1H, NH, D2O exchange), 6.32
(d,2H, arom.H), 7.02 (d,2H, arom.H), 6.59 (s,1H, arom.H), 7.45 (d,3H, arom.H),
7.49 (s,1H, arom.H), 7.58 (s,1H, arom.H), 7.66 (d,2H, arom.H), 8.1 (s,1H, arom.H).
13C-NMR (100MHz, DMSO-d6) δ (ppm): 46.19 (CH2 acetamido), 112.80 (C-2,6
aniline), 117.05 (C-4 aniline), 121.24 (C-4a quinazolidinone), 127.02 (C-8
quinazolidinone), 127.91 (C-2,6 benzene), 128.15 (C-6 quinazolidinone), 128.37 (C-
1 benzene), 128.92 (C-5 quinazolidinone), 129.22 (C-3,5 benzene), 130.57 (C-3,5
aniline), 133.89 (C-4 benzene), 135.70 (C-7 quinazolidinone), 147.07 (C-1 aniline),
148.61 (C-8a quinazolinone), 156.81 (C=O quinazolidinone), 159.73 (C-2
quinazolinone), 170.83 (C=O acetamide). Anal. Calcd for C22H18N4O2 (370.41): C,
71.27; H, 4,86; N, 15.12; Found: C, 71.03; H, 4,97; N, 15.38 %.
55 3-(5-(4-methoxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)-2-
phenylquinazolin-4(H)-one (VIIb). yield 65%, m.p. 2-4 ◦C; IR (KBr), cm-1: 468 (-NH),
167 (arom.CH), 2904,2819 (aliph.CH), 1724-1685 (C=O), 164-159 (C=C); 1H NMR:
δ 11.11 (s, 1H, NH, D2O exchange), .5 (s,H, OCH), 7.21 (d,2H, arom.H). 7.24 (d,1H,
arom.H), 7.6 (d,2H, arom.H), 7.76 (d,H, arom.H), 7.91 (d,2H, arom.H), 7.98 (d,1H,
arom.H), 8.09 (d,2H, arom.H), 8.74 (d,1H, arom.H), 1C-NMR (100MHz, DMSO-d6) δ
(ppm): 20.72 (OCH), 117.0 (C-,5 benzelidine), 120.7 (C-4a quinazolinone), 121.29
(C-8 quinazolinone), 12.45 (C-2,6 benzene), 127.01 (C-2,6 benzelidine), 127.49 (C-6
quinazolinone), 127.90 (C-1 benzelidine), 128.15 (C-1 benzene), 128. (C-5
quinazolinone), 129.48 (C-5 thiazolidinone), 10.59 (C-4 benzene), 1.98 (C-7
quinazolinone), 141.59 (olefinic carbon), 147.08 (C-8a quinazolinone) , 156.71 (C-4
benzelidine), 159.87 (C-4 quinazolinone), 165.19 (C-2 thiazolidinone), 169.11 (C-2
quinazolinone), 170.49 (C-4 thiazolidinone). Anal. Calcd for C25H18N4OS (454.5): C,
66; H, .96; N, 12.2; Found: C, 65.89; H, 4.12; N, 12.45 %.
49 2-(4-ethylphenylamino)-N-(4-oxo–phenylquinazolin-3(4H)-yl) acetamide (IVb).
yield 66%, m.p. 199-01 ◦C; IR (KBr), cm-1: 331,390 (-NH), 304 (arom.CH), 96 (aliph.
CH), 1697-1589 (C=O); 1H NMR: δ 3.8 (s,H, CHCO), 10.74 (s,1H, NHCO, DO
exchange), 10.15 (s, 1H, NH, DO exchange), 5.83 (d,H, arom.H), 6.96 (d,H, arom.H),
7.56 (s,1H, arom.H), 7.6 (d,3H, arom.H), 7.61 (d,H, arom.H), 7.94 (s,1H, arom.H),
13C-NMR (100MHz, DMSO-d6) δ (ppm): 16.65 (C- ethyl), 7.8 (C-1 ethyl), 46.15 (CH
acetamido), 113.08 (C-,6 aniline), 119.07 (C-4a quinazolinone), 10.96 (C-8
10

M.W. Aziz et al.
Bioorganic & Medicinal Chemistry Letters 41 (2021) 127987
56 3-(5-(4-bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)-2-
then observed and the formazan resulting crystals were then dissolved in certain
amount of MTT Solubilization solution [M-8910] equivalent to the original volume
of the culture medium. The absorbance was then spectrophotometrically measured at
a wavelength 570 nm.
phenylquinazolin-4(H)-one (VIIc). yield 78%, m.p. 175-177 ◦C; IR (KBr), cm-1: 02-
255 (-NH), 194-02 (arom.CH), 2927-2781 (aliph.CH), 1697,1681 (C=O), 1589,159
(C=C); 1H NMR: δ 12.14 (s, 1H, NH, D2O exchange), 7.18 (t,1H, arom.H). 7.21 (t,2H,
arom.H), 7.55 (q,H, arom.H), 7.64 (t,2H, arom.H), 7.94 (d,2H, arom.H), 7.99 (d,1H,
arom.H), 8.07 (d,2H, arom.H), 8.7 (d,1H, arom.H), 1C-NMR (100MHz, DMSO-d6) δ
(ppm): 120.19 (C-4a quinazolinone), 122.7 (C-4 benzelidine), 12.28 (C-8
quinazolinone), 125.19 (C-2,6 benzene), 127.51 (C-6 quinazolinone), 127.84 (C-2,6
benzelidine), 129.0 (C-1 benzene), 129.4 (C-5 quinazolinone), 10.49 (C-,5 benzene),
11.50 (C-5 thiazolidinone), 11.76 (C-4 benzene), 12.58 (C-,5 benzelidine), 12.62 (C-
7 quinazolinone), 14.5 (C-1 benzelidine), 141.57 (olefinic carbon) , 158.85 (C-8a
quinazolinone), 162.81 (C-4 quinazolinone), 165.11 (C-2 thiazolidinone), 165.81 (C-
2 quinazolinone), 170.45 (C-4 thiazolidinone). Anal. Calcd for C24H15BrN4O2S
(50.7): C, 57.21; H, 2.97; N, 11,12; Found: C, 57.8; H, .2; N, 11.5 %.
62 Cells were grown in a 96 well plate and incubated at 37◦C and under 5% CO2
overnight. The test compounds were dissolved into cell culture medium free of
serum, then treated with 4 tested compounds concentrations (2, 10, 50, 250 nM).
Each well was washed by 200 µl of the prepared 1× wash buffer A. the plate was
washed 4 times with the buffer then was tapped upside down in order to remove the
wash buffer. Prepared 1× blocking buffer (200 µl) was then added and incubated at
37 ◦C for 1h to remove any excess wash buffer. 1× activated Anti-Phospho-EGFR (50
µl) were added to the wells and incubated at room temperature with shaking for 2h.
The plate was washed with 200 µl wash buffer B, 4 times. 50 µl of 1× Anti-Mouse IgG
(HRP-conjugated secondary antibody) were then added to the wells followed by
incubation while shaking at room temperature for 30 min in dark room. Addition of
50 µl of stop solution was done and the results was read t 450 nm and OD was
measured immediately.
57 3-(5-(2,4-dihydroxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)-2-
phenylquinazolin-4(H)-one (VIId). yield 92%, m.p. 221-22 ◦C; IR (KBr), cm-1: 02-251
(-NH), 190-02 (arom.CH), 2924-2785 (aliph.CH), 1701-1651 (C=O), 1589,159
(C=C); 1H NMR: δ 12.14 (s, 1H, NH, D2O exchange), 8.55,8.7 (d,2H, OH), 7.2 (s,1H,
arom.H). 7.24 (t,1H, arom.H), 7.55 (t,1H, arom.H), 7.6 (q,H, arom.H), 7.64 (t,1H,
arom.H), 7.66 (d,2H, arom.H), 7.95 (d,2H, arom.H), 7.98 (d,1H, arom.H), 8.06
(d,1H, arom.H), 1C-NMR (100MHz, DMSO-d6) δ (ppm): 120.06 (C-benzelidine),
120.5 (C-5 benzelidine), 122.74 (C-1 benzelidine), 12.4 (C-4a quinazolinone),
125.20 (C-8 quinazolinone), 127.49 (C-2,6 benzene), 127.84 (C-6 quinazolinone),
129.1 (C-1 benzene), 129.47 (C-5 quinazolinone), 10.49 (C-,5 benzene), 11.51 (C-6
benzelidine), 11.76 (C-4 benzene), 12.67 (C-5 thiazolidinone), 14.77 (C-7
quinazolinone), 14.92 (olefinic carbon) , 17.15 (C-8a quinazolinone), 141.58 (C-4
benzelidine), 158.86 (C-2 benzelidine), 162.80 (C-4 quinazolinone), 165.17 (C-2
thiazolidinone), 165.82 (C-2 quinazolinone), 169.4 (C-4 thiazolidinone). Anal. Calcd
for C24H16N4O4S (456.48): C, 6.6; H, .5; N, 12.26; Found: C, 62.87; H, .78; N, 12.4 %.
58 3-(5-(,4-dimethoxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino)-2-
phenylquinazolin-4(H)-one (VIIe). Yield 75%, m.p. 219-221 ◦C; IR (KBr), cm-1: 02-
255 (-NH), 194-028 (arom.CH), 2954-2927 (aliph.CH), 1681 (C=O), 159,159 (C=C);
1H NMR: δ 12.1 (s, 1H, NH, D2O exchange), (s,6H, OCH), 7.2 (s,1H, arom.H). 7.24
(t,1H, arom.H), 7.5 (t,1H, arom.H), 7.58 (t,1H, arom.H), 7.65 (q,H, arom.H), 7.7
(t,2H, arom.H), 7.97 (t,1H, arom.H), 8.04 (d,2H, arom.H), 8.54 (d,1H, arom.H), 1C-
NMR (100MHz, DMSO-d6) δ (ppm): 22.90 (2 OCH), 117.05 (C-2 benzelidine),
120.09 (C-5 benzelidine), 120.7 (C-6 benzelidine), 122.76 (C-4a quinazolinone),
12.45 (C-8 quinazolinone), 125.21 (C-2,6 benzene), 127.49 (C-6 quinazolinone),
127.84 (C-1 benzelidine), 129.1 (C-1 benzene), 129.48 (C-5 quinazolinone), 10.49
(C-,5 benzene), 11.51 (C-5 thiazolidinone), 11.76 (C-4 benzene), 12.6 (C-7
quinazolinone) , 17.15 (C-4 benzelidine), 141.48 (C- benzelidine), 158.86 (olefinic
carbon), 162.80 (C-8a quinazolinone), 165.18 (C-4 quinazolinone) , 165.82 (C-2
thiazolidinone), 169.4 (C-2 quinazolinone) , 170.48 (C-4 thiazolidinone). Anal. Calcd
for C26H20N4O4S (484.5): C, 64.9; H, 4.12; N, 11.55; Found: C, 64.52; H, 4.; N, 11.74
%.
63 Breast MCF-7 cells collected from American type culture were seeded at a density of
1 × 106 cells/well in a RPMI 1640 with 10% fetal bovine serum for 24 h at 37◦C. cells
were then treated with the tested compounds (IVc, VIIa) respectively per well in a 6
well plate before enzyme assay for 24 h. 70% ethanol were used to fix the cells at 4◦C
for 30 min. cells were then centrifuged for 5 min at 1200 rpm, the supernatant was
discarded and the pellet was washed using PBS and cells was stained with propidium
iodide staining buffer (PI 200 mg). PBS with 0.1% (v/v) triton X-100, 2 mg DNase-
free RNAase A (Sigma) (10 ml) at ambient temperature for 15 min in absence of
light. Samples were then lately analyzed using BD-FACS caliber flow cytometry
reader for propidium iodide-DNA fluorescence from 10,000 events flow cytometry.
64 Incubation of cells was carried and treated in order to induce apoptosis as in cell
cycle analysis. Trypsinisation was used to collect the cells and the cells were washed
twice with ice cold PBS, followed by re-suspension in 500 ul of 1× binding buffer.
Annexin V-FITC (5 µl) and propidium iodide (5 µl) were added then cells were
incubated for 15 min at room temperature in absence of light. Cells were quantified
after incubation by analyzing Annexin V-FITC binding by flow cytometry
(wavelength 488 nm) using BD-FACS caliber flow cytometry reader.
65 All the molecular modeling studies were accomplished using Discovery studio 4.0
software. The X-ray crystallographic structure (PDB ID: 1M17) of EGFR enzyme co-
crystallized with Erlotinib as inhibitor was downloaded from protein data bank.33 All
ligands and water molecules that are not involved in binding were removed. The
protein structure was prepared for docking study using automatic prepare protein
protocol in Discovery Studio with default options. The co-crystallized ligand
(Erlotinib) was used to define the active site for the docking algorithm. C-Docker
protocol was used to perform the docking where the -C-Docker energy was used for
initial docking pose and refined by -C-Docker interaction energy. Docking protocol
was first validated by self-docking of the co-crystallized ligand (Erlotinib). The
validated docking protocol was then used to study the ligand-target interactions in
the active site for the newly synthesized compounds IV a-e and VII a-e to predict their
binding mode to rationalize their promising activity.
59 Melting points were determined on open glass capillaries using BUCHI B-540
apparatus, faculty of pharmacy, Misr International University and were uncorrected.
Infra-red spectra were measured on a Shimadzu FT-IR 8400s Infra-red
66 All compounds were build using Accelry’s Discovery Studio 4.0 window and
optimized using CHARMm force field. The compounds were divided randomly into
training and test set were the training set constitutes 80% of the total compounds
number. The training set molecules were submitted to the HypoGen algorithm of DS
4.0. Among the 11 features available for selection, the features H-bond acceptor, H-
bond donor, ring aromatic, positive ionizable and hydrophobic features present in
the training set were selected for pharmacophore generation. 3D QSAR generation
protocol was applied in Discovery Studio software. The outcome of HypoGen results
in a large number of hypotheses, the cost analysis was applied to evaluate the
hypotheses. Evaluation of the hypothesis was performed on the basis of three cost
values, null, fixed and total costs. The fixed cost represents the simplest model that
fits all data perfectly while the null cost represents a model with no features, whereas
the total cost is the summation of the weight, error and configuration costs. RMS
(root mean square difference) between the estimated and predicted activity of the
training set increases the error cost, the feature weights in the model as it deviates
from the ideal value of 2.0 increase the weight cost while the configuration cost
depends on the hypothetical space complexity to be analyzed. The hypothesis
statistical significance increase as the total cost approach the fixed cost value and the
cost difference between null and fixed cost is high.
spectrophotometer (cm-1) using KBr disks, faculty of pharmacy, Cairo University. 1
and 13CNMR spectra were determined in DMSO-d6 with Bruker Avance-400
spectrophotometer operating at 400MHz and 100 MHz respectively at Micro-
H
analytical unit, Faculty of pharmacy, Cairo University. Elemental analysis (C, H and
N) were performed using the ElMentar, Vario EL Germany Instrument at the Regional
Center for Mycology and Biotechnology, Faculty of Science, AL-Azhar University.
TLC for reaction monitoring was performed on 0.255 mm aluminum plates coated
with silica gel 60F-254 (Merck) in an appropriate solvent and visualized under U.V.
light (254 nm).
60 Biological evaluation was done in the Egyptian company for the production of
vaccines, sera and drugs laboratory (VACSERA, GIZA, Egypt).
61 Experiments were performed on two human tumor cell lines, MCF-7 (breast
carcinoma cell line) and A549 (small cell lung carcinoma cell lines). The cell lines
were collected from the American type. For ultimate results, cells in the log phase of
growth were used and each test involves a blank containing complete medium
lacking cells. Reconstituted MTT were added in equivalent amount to 10% of the
volume of the culture medium. Cultures were then incubated for 2-4 h depending of
the type and maximum density of the cell. After the incubation period, cultures were
11