Discovery of new quinazolin-4(3H)-ones as VEGFR-2 inhibitors: Design, synthesis, and anti-proliferative evaluation

Article abstract of DOI:10.1016/j.bioorg.2020.104380

Sixteen novel quinazoline-based derivatives were designed and synthesized via modification of the VEGFR-2 reported inhibitor 7 in order to increase the binding affinity of the designed compounds to the receptor active site. The designed compounds were evaluated for their VEGFR-2 inhibitory effects. Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer. The bioactivity of the new compounds was performed against HepG-2, MCF-7 and HCT-116 cell lines. Doxorubicin and sorafenib were used as positive controls. Compound 18_d was observed to have promising cytotoxic activity (IC₅₀ = 3.74 ± 0.14, 5.00 ± 0.20 and 6.77 ± 0.27 μM) in comparison to the reference drug doxorubicin (IC₅₀ = 8.28, 9.63 and 7.67 μM) and sorafenib (IC₅₀ = 7.31, 9.40 and 7.21 μM). The most active compounds were tested for their in vitro VEGFR-2 inhibitory activities. Results of VEGFR-2 inhibition were consistent with that of the cytotoxicity data. Thus, compound 18_d showed VEGFR-2 inhibitory activity (IC₅₀ = 0.340 ± 0.04 μM) superior to that of the reference drug, sorafenib (IC₅₀ = 0.588 ± 0.06 μM). Furthermore, docking study was performed in order to understand the binding pattern of the new compounds into VEGFR-2 active site. Docking results attributed the potent VEGFR-2 inhibitory effect of the new compounds as they bound to the key amino acids in the active site, Glu883 and Asp1044, as well as their hydrophobic interaction with the receptor hydrophobic pocket. Results of cytotoxic activities, in vitro VEGFR-2 inhibition together with docking study argument the advantages of the synthesized analogues as promising anti-angiogenic agents.

Full text of DOI:10.1016/j.bioorg.2020.104380

Journal Pre-proofs
Discovery of new quinazolin-4(3H)-ones as VEGFR-2 inhibitors: Design,
synthesis, and anti-proliferative evaluation
Ibrahim.H. Eissa, Abdel-Ghany A. El-Helby, Hazem A. Mahdy, Mohamed M.
Khalifa, Hamdy A. Elnagar, Ahmed. B.M. Mehany, Ahmed.M. Metwaly,
Mostafa.A. Elhendawy, Mohamed.M. Radwan, Mahmoud.A. ElSohly,
Khaled. El-Adl
PII:
S0045-2068(20)31678-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.104380
YBIOO 104380
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 July 2020
14 September 2020
12 October 2020
Please cite this article as: Ibrahim.H. Eissa, A.A. El-Helby, H.A. Mahdy, M.M. Khalifa, H.A. Elnagar, A.B.M.
Mehany, Ahmed.M. Metwaly, Mostafa.A. Elhendawy, Mohamed.M. Radwan, Mahmoud.A. ElSohly, Khaled. El-
Adl, Discovery of new quinazolin-4(3H)-ones as VEGFR-2 inhibitors: Design, synthesis, and anti-proliferative
evaluation, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104380
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Discovery of new quinazolin-4(3H)-ones as VEGFR-2 inhibitors: Design,
synthesis, and anti-proliferative evaluation
Ibrahim. H. Eissa*a, Abdel-Ghany A. El-Helbya, Hazem A. Mahdya, Mohamed M.
Khalifaa, Hamdy A. Elnagara, Ahmed. B. M. Mehanyc, Ahmed. M. Metwalyd,
Mostafa. A. Elhendawye,f, Mohamed. M. Radwanf, g, Mahmoud. A. ElSohly*f,h,
Khaled. El-Adla,b,*
a Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of
Pharmacy (Boys), Al-Azhar University, Cairo11884, Egypt.
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University
for Sustainable Development, Cairo, Egypt.
c Zoology Department, Faculty of Science, Al-Azhar University, Cairo 11884, Egypt.
d Pharmacognosy Department, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo 11884, Egypt.
e Department of Agriculture Chemistry, Faculty of Agriculture, Damietta University,
Damietta, Egypt
f National Center for Natural Products Research, University of Mississippi,
MS 38677, USA.
g Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University,
Alexandria, Egypt.
h Department of Pharmaceutics and Drug Delivery, University of Mississippi,
University, MS 38677, USA.
* Corresponding authors:
Ibrahim H. Eissa
Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of
Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt.
Email: Ibrahimeissa@azhar.edu.eg
Khaled. El-Adl
Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of
Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt.
Email: eladlkhaled74@yahoo.com, eladlkhaled74@azhar.edu.eg,
khaled.eladl@hu.edu.eg
1

Abstract
Sixteen novel quinazoline-based derivatives were designed and synthesized via modification of
the VEGFR-2 reported inhibitor 7 in order to increase the binding affinity of the designed compounds
to the receptor active site. The designed compounds were evaluated for their VEGFR-2 inhibitory
effects. Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer. The
bioactivity of the new compounds was performed against HepG-2, MCF-7 and HCT-116 cell lines.
Doxorubicin and sorafenib were used as positive controls. Compound 18d was observed to have
promising cytotoxic activity (IC50 = 3.74 ± 0.14, 5.00 ± 0.20 and 6.77 ± 0.27 µM) in comparison to the
reference drug doxorubicin (IC50 = 8.28, 9.63 and 7.67 µM) and sorafenib (IC50 = 7.31, 9.40 and 7.21
µM). The most active compounds were tested for their in vitro VEGFR-2 inhibitory activities. Results
of VEGFR-2 inhibition were consistent with that of the cytotoxicity data. Thus, compound 18d showed
VEGFR-2 inhibitory activity (IC50 = 0.340± 0.04 µM) superior to that of the reference drug, sorafenib
(IC50 = 0.588 ± 0.06 µM). Furthermore, docking study was performed in order to understand the binding
pattern of the new compounds into VEGFR-2 active site. Docking results attributed the potent VEGFR-2
inhibitory effect of the new compounds as they bound to the key amino acids in the active site, Glu883
and Asp1044, as well as their hydrophobic interaction with the receptor hydrophobic pocket. Results of
cytotoxic activities, in vitro VEGFR-2 inhibition together with docking study argument the advantages
of the synthesized analogues as promising anti-angiogenic agents.
Keywords: Antiangiogenesis; Antiproliferative; Docking studies; Quinazolin-4(3H)-one; VEGFR-2.
2

1. Introduction
Cancer angiogenesis is a crucial process for the tumor growth [1]. It is the process of developing
new capillary blood vessels from preexisting ones that supplies oxygen and nutrients to the proliferating
cells, potentially promotes cancer growth, survival and metastasis [1-3]. Numerus factors are involved
in the stimulation of cancer angiogenesis [4]. Vascular endothelial growth factor (VEGF) family, which
includes VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF), is a group of vital
proteins that considered one of the most important angiogenic molecules [5]. VEGF and VEGV
receptors are highly expressed in tumors of almost all types of cancer analyzed. Members of the VEGF
family bind to three different tyrosine kinase (TK) receptors namely; VEGFR-1 (Flt-1), VEGFR-2 (Flk-
1- KDR), and VEGFR-3, which is expressed on the lymphatic and vascular endothelium (Fig. 1) [5].
Fig. 1. Natural ligands, target receptors and antiangiogenic compounds of the VEGF family
(based on Ref. [5].
VEGFR-2, a type II transmembrane TK receptor expressed on endothelial cells and on
circulating bone marrow-derived endothelial progenitor cells, is the key mediator of the VEGF-induced
angiogenic signaling [6]. This receptor contains three different parts, including an Ig-like domain
extracellular region, a hydrophobic transmembrane region containing the TK domain and the carboxyl
terminal tail [6]. Binding of VEGF to VEGFR-2 produces a cascade of different signaling pathways [7].
3

These pathways lead to dimerization of the receptor followed by the autophosphorylation of intracellular
TK domains, that results in great mitogenic and chemotactic effects on endothelial cells [8]. Approaches
for hindering these pathways include the use of definite inhibitors (antibodies or small molecules), which
may either bind VEGF or interfere with the different domains of VEGFR [9].
Several inhibitors have been clinically approved and are marketed as novel drugs targeting
cancer angiogenesis such as multikinase inhibitors; sorafenib 1 and sunitinib 2 and the monoclonal
antibody bevacizumab [10]. These agents were proved to have significant efficacy against many cancers
[11]. Putting this in consideration, researchers were encouraged to move forward in this area (Fig. 2).
Now, great efforts are being devoted to discover new anticancer agents that hopefully control different
pathological conditions [12-18].
O
H
H
N
N
N
N
N
H
H
N
O
F
O
Cl
N
H
O
O
F
F
N
F
H
2
Sunitinib
1
Sorafenib
Fig. 2. The reported VEGFR-2 inhibitors
Quinazoline nucleus is of well-known promising biological activity [19, 20]. A series of
substituted quinazoline derivatives was discovered in literature as effective VEGF/VEGFR inhibitors
for antiangiogenic cancer therapy [21, 22]. Fig. 3. illustrates some selected quinazoline derivatives
structures that were proved to be angiogenesis inhibitors. AZD2171 3 was reported as a novel highly
potent inhibitor of KDR tyrosine kinase [23]. It prevents the VEGF-stimulated proliferation in a
subnanomolar IC50. AZD2932 4 was discovered as multi-target VEGFR-2 and PDGFR kinases
inhibitors [24]. Vandetanib 5, introduced by AstraZeneca under a name of Caprelsa®, is an
anilinoquinazoline derivative with unique antiangiogenic characteristics as it inhibits vascular
endothelial growth factor receptor 2 (VEGFR-2), epidermal growth factor receptor (EGFR) in addition
to Ret tyrosine kinases (RET-TS) [25]. It became the first drug to be approved by the FDA for treatment
of late-stage (metastatic) medullary thyroid cancer in adult patients who are ineligible for surgery [25].
A series of novel 2-chloro-4-anilino-quinazolines 6 was developed as potent EGFR and VEGFR-2 dual
inhibitors an attractive therapeutic and pharmacokinetic properties [22].
4

H
H
N
N
N
O
N
O
N
O
N
O
O
F
O
O
N
N
N
3
AZD2171
4
AZD2932
R3
F
Br
HN
N
HN
N
R1
R2
O
N
O
N
Cl
N
5
Vandetanib
6
Fig. 3. Anti-angiogenic quinazoline based compounds
Throughout the recent few years, quinazolin-4(3H)-ones were reported as promising anti-
angiogenic agents through VEGFR-2 inhibition [26]. It has been identified as an excellent hinge binding
moiety for multi-target inhibitors of angiogenic VEGFR-2, epidermal growth factor homology domain-2
(Tie-2) and erythropoietin-producing hepatoma receptor B4 (EphB4) [27]. Therefore, looking for
derivatives of quinazolin-4(3H)-ones through modification and functionalization of the structure is
a significant point of research targeting to find analogs having strong angiogenesis inhibition.
The current work is a continuation of our effort to design novel anti-angiogenic quinazolin-
4(3H)-ones [26]. This pursue is inspired by our introduction of the effective N-(4-
(1(hydroxylimino)ethyl)phenyl)-2-[(3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio] acetamide 7 which
was reported as a potent anti-angiogenic agent [26]. It was discovered to reduce the angiogenesis in vivo
in diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) model. Compound 7 was also
found to induce apoptosis in HepG-2 cells by more than 14 folds over the control when evaluated using
Annexin V and PI double staining assay. Moreover, compound 7 arrested cell growth in G2-M phase
when evaluated using HepG-2 cells according to the procedure described by Wand et al [26].
The VEGFR-2 inhibitory effect of this series is assumed to be mediated through
accommodation of the large size bicyclic quinazoline core into the space of the ATP binding region.
Further interactions may also occur with the side chain carboxylate of Glu885 of the αC helix and/or
5

with Asp1046 in the conserved DFG motif through hydrogen bonding with the appropriate moieties in
the inhibitors.
In the present work a modification was carried out to the quinazoline ring through the
replacement of the ethyl group at position 3 of compound 7 by a phenyl ring that will increase the
hydrophobic nature of the designed candidates aiming to magnify their binding affinity to VEGFR-2 via
increasing the hydrophobic interaction with its allosteric hydrophobic back pocket. Another
modification took place through extension or replacement of the hydrogen bonding moiety of the
inhibitor 7 with various moieties that may lead to increasing the binding interaction with Glu885 of the
αC helix and/or with Asp1046 in the conserved DFG motif of the receptor. Fig. 4.
O
N
H
N
N
S
O
N
OH
7
CH3
H
N
O
R
R = 2-Cl, 3-Cl, 4-Cl, 2-OH, 2-Cl 5-NO2
N
N
N
NH2
O
H
CH3
CH3
H
H
N
N
R = -C3H7, -Cyc C6H11
R1
O
O
O
H
N
H
N
N
R = H, 4-Cl, 4-CH3, 4-COOC2H5
R
H
O
O
H
N
N
H
O
HO
Fig. 4. Schematic representation showing the designing strategy of quinazolin-4(3H)-ones.
6

2. Results and discussion
2.1. Chemistry
The target compounds 14-33 were synthesized according to the reaction sequences outlined in
Schemes 1-5. 2-Mercapto-3-phenylquinazolin-4(3H)-one 10 and its salt 11 were prepared according to
the literature procedure via reaction of anthranilic acid 8 with aniline 9 in the presence of carbon disulfide
followed by heating of the obtained compound 10 with alcoholic potassium hydroxide [28, 29]. On the
other side, stirring of 4-aminoacetophenone 12 with chloroacetyl chloride in dry DMF at room
temperature produced N-(4-acetylphenyl)-2-chloroacetamide 13 [30]. According to the reported method
[31-33] , the previously synthesized potassium salt 11 was refluxed with compound 13 to afford
compound 14 (Scheme 1) The formation of compound 14 was confirmed by the appearance of carbonyl
bands at 1733 and 1671 cm-1 and NH absorption band at 3318 cm-1 in its IR spectrum. 1H NMR spectra
showed singlet signals of the aliphatic protons at δ 2.51 ppm in addition to another one at 4.13 ppm
corresponding to COCH3 and SCH2 protons, respectively (Scheme 1).
The reported intermediates 17a-e were obtained by esterification of substituted benzoic acid
derivatives 15a-e with absolute ethanol in the presence of sulfuric acid as a catalyst followed by refluxing
with hydrazine hydrate [34, 35]. Then, these intermediates reacted with compound 14 in the presence of
a catalytic amount of glacial acetic acid to give the target compounds 18a-e . The suggested reaction
mechanism involved nucleophilic attack of the hydrazide moieties of the intermediates 17a-e on the α-
carbon atom of compound 14. Synthesis of compound 20 was also achieved by the same mechanism
through reaction of compound 14 with semicarbazide 19 in ethanol (Scheme 2).
Following the mixed anhydride synthesis procedure, different intermediates 24a,b, 25, 26a-d and
27 were successfully prepared [36, 37]. This procedure started with stirring of 4-aminobenzoic acid 21
with chloroacetyl chloride in DMF at room temperature to give 4-(3-chloroacetamido) benzoic acid 22
[38]. The later compound was then allowed to react with ethyl chloroformate in the presence of TEA to
afford the anhydride 23 which is not isolated before the subsequent reaction. The formed anhydride was
then treated with different aromatic or aliphatic amines at room temperature to give the desired
intermediates 24a,b, 25, 26a-d and 27 (Scheme 3).
Regarding the previously prepared intermediates, they were then treated with the prepared salt
11 in DMF to afford compounds 28a,b, 29, 30a-d and 31 adopting methods described in literature (Scheme
4) [39]. IR spectra of this series revealed presence of the carbonyl absorption band at a range of 1659-
1
1751 cm-1 and presence of NH band at a range of 3192 - 3385 cm-1. H NMR spectra showed singlet
7

signals of the aliphatic -SCH2 protons at a δ range of 4.10 - 4.16 ppm and presence of D2O exchangeable
NH-amide protons at a δ range of 8.32 - 10.74 ppm.
Treatment of the potassium salt 11 with compound 32, synthesized following mixed anhydride
procedure via the reaction of 4-(2-chloroacetamido) benzoic acid 22 with 2-hydroxybenzohydrazide 17d,
1
produce the target candidate 33 (Scheme 5) [37]. The formation of compound 33 was proved by H
NMR spectra which showed the appearance of a singlet signal of the aliphatic -SCH2 protons at δ 4.10
ppm in addition to D2O exchangeable signals of the NH-amide protons at δ 10.47 and 10.67 ppm.
Moreover, the methylene group found to be resonating at 37.80 ppm in both of 13C NMR and DEPT 135
NMR spectra.
O
COOH
/
1) KOH/CS2 CH3OH / reflux /10h
N
H2N
2) HCl
NH2
N
SH
(10)
(9)
(8)
O
H2N
KOH/EtOH Reflux /0.5h
(12)
O
DMF/ K2CO3
Stirring / 1.5 h
Cl
Cl
O
N
O
O
N
S K
Cl
N
H
(11)
(13)
O
O
O
N
Cl
O
N
N
H
N
(13)
DMF/KI/
N
H
N
/ 6 h
S K
S
O
(11)
(14)
O
Scheme 1: General procedure for synthesis of target compound 14.
8

HO
O
R
H2N NH
O
C2H5O
O
R
EtOH
R
NH2NH2.H2O
Conc H2SO4
reflux / 3 h
(15a-e
)
(16a-e
)
(17a-e
)
O
gl. acetic acid/EtOH / reflux / 8 h
N
H
N
R
H2N NH
O
R
H
N
S
N
O
N
O
(17a-e
)
O
N
N
(18a-e
)
R
H
18 )
N
2-Cl
3-Cl
4-Cl
a
S
18 )
b
O
18 )
c
(14)
18d) 2-OH
O
2-Cl,5-NO
18e)
2
O
H2N
O
N
N
NH2
(19)
gl. acetic acid/EtOH / reflux / 8 h
H
N
H
N
S
O
O
N
N
H
NH2
(20)
Scheme 2: General procedure for synthesis of target compounds 18a-e and 20.
9

COOH
NH2
(21)
H
N
O
O
R
Cl
DMF /K2CO3
stirring / 2h
Cl
R1-NH2
R
24a) Propyl
DCM / stirring / 3h / rt.
COOH
24 )
Cyclohexyl
b
HN
Cl
O
(24a,b
)
NH2
HN
Cl
H
N
O
O
(22)
O
DCM / E3N
ice bath/stirring /1h
DCM / stirring / 3h / rt.
O
Cl
HN
Cl
(25)
O
O
O
O
NH2
R1
H
N
O
24a)
24b)
24c)
24d)
O
H
Cl
R
R1
CH3
COOC2H5
DCM / stirring / 3h / rt.
HN
Cl
O
HN
Cl
(23)
O
(26a-d
)
H
N
O
H2NHN
N
H
DCM / stirring / 3h / rt.
HN
(27)
Cl
O
Scheme 3: General procedure for synthesis of the intermediates 24a,b, 25, 26a-d and 27.
10

O
R
O
N
O
N
28a)
28b) Cyclohexyl
Propyl
H
Cl
N
N
H
(24a,b
)
H
N
S
H
N
R
DMF/ KI/ reflux/ 8h
O
(28a,b
)
O
O
O
N
O
H
Cl
N
H
(25)
N
H
N
N
S
H
N
DMF/ KI/ reflux/ 8h
O
(29)
O
O
N
N
SK
Cl
R1
(11)
O
R1
30a) H
30b)
30c)
Cl
CH3
O
N
H
O
N
N
N
(26a-d
)
30d)
COOC2H5
H
H
N
DMF/ KI/ reflux/ 8h
O
S
H
N
O
(30a-d
)
H
O
N
R1
O
N
H
O
N
N
Cl
N
(27)
H
H
N
S
DMF/ KI/ reflux/ 8h
H
N
N
H
O
(31)
O
Scheme 4: General procedure for synthesis of target compounds 28a,b, 29, 30a-d and 31.
11

O
HO
O
1)
Cl
O
H
-5oC/Et3N
N
COOH
O
N
O
DCM/stirring1h
H
Cl
O
Cl
OH
O
N
N
H
H
2)
(32)
(22)
HN NH2
(17d)
O
stirring/3 h / rt
N
DMF /KI / /6 h
N
SK
(11)
O
N
N
H
N
S
O
H
N
O
N
H
O
HO
(33)
Scheme 5: General procedure for synthesis of target compound 33.
2.2. Biological evaluation
2.2.1. In vitro anti-proliferative activity
In vitro cytotoxicity of the synthesized compounds 14-33 was tested using MTT assay against
a panel of three human tumor cell lines namely; hepatocellular carcinoma (HepG-2), colorectal
carcinoma (HCT-116) and breast cancer (MCF-7) [40-42]. The cytotoxicity results were compared to
that of sorafenib, a well-defined VEGFR-2 inhibitor, and doxorubicin, one of the most effective
anticancer agents. The observed cytotoxic activity of the target compounds was recorded in Table 1.
Compound 18d exhibited the highest cytotoxic activity among the tested compounds with IC50
values of 3.74, 5.00 and 6.77 µM. It was 1.88, 1.92 and 1.13 folds more active than doxorubicin (IC50 =
8.28, 9.63 and 7.67 µM) and 1.95, 1.88 and 1.06 times more active than sorafenib (IC50 = 7.31, 9.40 and
7.21 µM) against HepG-2, HCT-116 and MCF-7 cells, respectively. Furthermore, it is obvious from the
obtained data that compounds 14, 18b, 18e, 20, 30d and 33 showed promising activities with IC50 almost
lower than or equal to that of the reference drugs.
12

Table 1. Anti-proliferative activities of the tested compounds toward HepG-2, HCT-116 and
MCF-7 cell lines
In vitro cytotoxicity IC50 ( µM )a
Comp.
HepG-2
HCT-116
MCF-7
14
18b
18c
7.33 ± 0.29
5.65 ± 0.22
10.73 ± 0.42
8.86 ± 0.35
21.88 ± 0.87
9.87 ± 0.39
10.32 ± 0.41
15.65 ± 0.62
17.78 ± 0.71
18d
18e
3.74 ± 0.14
8.90 ± 0.35
5.00 ± 0.20
8.4 ± 0.33
6.77 ± 0.27
11.5 ± 0.46
20
7.89 ± 0.31
6.39 ± 0.25
9.78 ± 0.39
28a
19.40 ± 0.77
16.90 ± 0.67
20.29 ± 0.81
28b
27.97 ± 1.11
19.41 ± 0.77
14.76 ± 0.59
14.26 ± 0.57
13.71 ± 0.54
6.87 ± 0.27
29.40 ± 1.17
8.17 ± 0.52
8.28 ± 0.33
7.31 ± 0.29
22.92 ± 0.91
17.22 ± 0.68
16.36 ± 0.65
12.42 ± 0.49
9.29 ± 0.37
5.56 ± 0.22
25.05 ± 1
29.63 ± 1.18
15.11 ± 0.60
12.87 ± 0.51
16.17 ± 0.64
15.44 ± 0.61
7.06 ± 0.28
31.07 ± 1.24
7.53 ± 0.58
7.67 ± 0.30
7.21 ± 0.28
29
30a
30b
30c
30d
31
33
9.42 ± 0.45
9.63 ± 0.38
9.40 ± 0.37
Doxorubicin
Sorafenib
a IC50 values are the mean ± S.D. of three separate experiments.
13

2.2.2. In vitro VEGFR-2 enzyme assay inhibition
The most active cytotoxic compounds (14, 18b, 18d, 18e, 20, 29, 30a, 30b, 30c, 30d and 33) were
further assessed to determine their inhibitory activities against VEGFR-2 with sorafenib as positive
control. Several published articles reported the IC50 inhibitory activity of sorafenib. However, Lin Zhang
et al [27] reported the IC50 of sorafenib to be 0.55 µM which was consistent with our experimental
results. The results were summarized in Table 2. As shown in Table 2, most of the tested compounds
exhibited superior to moderate enzyme inhibitory activity with IC50 values ranging from 0.340 to 0.751
µM in a sub micromolar level. Among them, five compounds, 14, 18d, 18e, 20 and 30d, out of eleven
potently inhibited VEGFR-2 with IC50 values (0.578, 0.340, 0.495, 0.546, and 0.446 µM, respectively)
than the reference drug; sorafenib (0.588 µM).
Table 2: IC50 (µM) for the tested compounds as inhibitors for VEGFR-2
Comp.
VEGFR-2 inhibition
IC50 (µM) a
14
20
0.578 ± 0.05
0.546 ± 0.03
0.619 ± 0.06
0.340± 0.04
0.495 ± 0.03
0.751 ± 0.05
0.721 ± 0.05
0.671 ± 0.07
0.713 ± 0.05
0.446 ± 0.06
0.590 ± 0.08
0.588 ± 0.06
18b
18d
18e
29
30a
30b
30c
30d
33
Sorafenib
a IC50 values are the mean ± S.D. of three separate experiments.
14

2.3. Molecular docking
Molecular docking study was performed aiming to get further insight into the binding modes
and orientations of the newly synthesized compounds into the ATP binding site of VEGFR-2 kinase
enzyme. The study was used to evaluate how the small molecules and the target macromolecule
(receptor) fit together. Docking study was carried out using C-Docker protocol in Discovery Studio 2.5
Software. Validation of the docking protocol was achieved through redocking of the co-crystallized
ligand in the VEGFR-2 active site using C-Docker algorithm. The validation step proved the suitability
of the used protocol for the intended docking study as demonstrated by the small RMSD (0.58Å)
between the docked pose and the co-crystallized ligand. Moreover, the ability of the docking algorithm
to retrieve the reported binding mode of co-crystallized ligand also indicated the validity of the selected
docking algorithm Fig. 5.
Fig. 5. Superimposition of the co-crystallized pose (green) and the docking pose (maroon) of the same
ligand
The newly synthesized candidates were docked into the active site of VEGFR-2 to understand
their binding mode. Sorafenib was used as a reference compound. The distinctive binding pattern of
sorafenib to VEGFR-2 kinase active site has been discussed and compared to the tested compounds.
The two N-H groups of the urea moiety of sorafenib were involved in the formation of two
hydrogen bonds with the side chain carboxylate of Glu883 with distances of 1.71 and 1.70 Å. Besides,
the urea carbonyl group was also observed to be involved in a hydrogen bond interaction with the
backbone NH of Asp1044 with distance of 1.75 Å. Moreover, the spacer between urea and oxygen
15

through its phenyl group acts as a hydrophobic moiety and interacts with the hydrophobic site (Lys866)
in the hinge region. These results were the same as the reported data (Fig. 6 a) [43].
Analysis of the docking results revealed the existence of noticeable correlation between the
biological results and the binding free energies (the calculated ∆G) of the synthesized compounds. The
calculated ∆G of the synthesized compounds and the reference drug against VEGFR-2 were cited in
Table 3.
As shown in Fig. 6 b. and illustrated in Table 3, compound 18d has a promising antiproliferative
activity through its high PTK binding affinity. It bound into VEGFR-2 kinase via formation of six
hydrogen bonds. Two hydrogen bonds were formed between the hydrazino N-H group and the Glu883
carboxylate group with distance 1.43 and 2.30 Å. The hydrazino carbonyl group as well as the terminal
OH group were also involved in hydrogen bonding with DFT motif Asp1046 in the active site. The
former binding mode allowed compound 18d to accommodate its quinazolinone moiety in the vicinity
of hydrophobic pocket of Leu1033, Val914, Lys918, Phe1045, Phe916 and Val846 amino acids forming
a hydrophobic interaction with Val914 in addition to two hydrogen bonding between the quinazolinone
carbonyl and Asn912.
As illustrated in Fig. 6 c., compound 20 possessed a significant potential binding affinity (∆G
= - 55.31) into the VEGFR-2 active site. This high binding affinity presumably attributed to the
formation of five hydrogen bond interaction. the terminal NH2 in addition to the N-H were involved in
hydrogen bonding with the carboxylate side chain of Glu883. While the side chain carbonyl interacted
with the DFT motif Asp1046 of the active site through formation of hydrogen bond with 1.82 Å. Two
more hydrogen bonds were formed between the quinazolinone carbonyl and Asn921 in the active site.
Furthermore, compound 20 interacted hydrophobically with the surrounding Arg1049 amino acid.
16

17

Fig. 6. (a) 3D representation of sorafenib with VEGFR-2 active site. (b) the predicted binding
pattern of 18d with the active site of VEGFR-2. (c) the predicted binding pattern of 20 with the active
site of VEGFR-2
Table 3: The calculated ∆G (binding free energies) of the synthesized compounds and reference drug
against VEGFR-2 (∆G in Kcal/mol).
Comp.
14
∆G [Kcal/mol
- 45.21
- 40.12
- 45.73
- 31.01
- 56.21
- 39.61
- 55.31
- 37.33
- 31.21
]
Comp.
29
∆G [Kcal/mol
- 41.44
]
18a
18b
18c
30a
- 33.47
30b
- 43.58
30c
- 43.54
18d
18e
30d
- 51.05
31
- 37.44
20
33
- 32.03
28a
28b
Sorafenib
- 52.20
2.4. Structure-Activity Relationship (SAR)
The evaluation of the biological efficacy along with the molecular docking affinities and
interactions of the designed compounds provided more insight into the structure activity relationship of
quinazolin-4(3H)-ones derivatives as VEGFR-2 kinase inhibitors. It was clear that the hydrazone
containing compounds 18a-e exhibited a significant increase in VEGFR-2 enzymatic activity and anti-
proliferative effect. Substitution of the phenyl group attached to hydrazone moiety with hydroxyl group
as in 18d resulted in better biological activity and binding affinity with VEGFR-2 than members
substituted with Cl group 18a, 18b, 18c and 18e. This emphasizes the important role of the hydroxyl group
in increasing the antiangiogenic activity of 18d. The former observation was also confirmed by the strong
activity of the hydroxyl containing derivative 33.
On the other hand, the high activity of compound 20 could be attributed to its strong binding to
the active site through the role exerted by the phenyl group in position 3 of quinazoline moiety that cause
non covalent pi-pi interaction with the receptor in addition to the presence of NH2 and NH groups that
magnified the interaction via hydrogen bonding with the receptor. Comparing the anticancer activities
18

of acetamido benzamide derivatives of 2-mercapto-3-(p-tolyl)quinazolin-4(3H)-one showed that the
activity of ester (30d) is superior than that expressed by sorafenib and better than 4-chloro (30b) and 4-
methyl (28c) analogue, indicating that grafting electron withdrawing substituents at positions 4 is
beneficial for activity.
3. Conclusion
In a continuation of our previous work in the discovery of potent VEGFR-2 inhibitors, we
designed and synthesized sixteen novel quinazoline-based derivatives via modification of the VEGFR-
2 reported inhibitor 7. Modification of compound 7 was performed in two positions in order to increase
the binding affinity of the designed compounds to the receptor active site. The bioactivity of the new
compounds was carried out against HepG-2, MCF-7 and HCT-116 cell lines. Compound 18d was
observed to have promising cytotoxic activity (IC50 = 3.74 ± 0.14, 5.00 ± 0.20 and 6.77 ± 0.27 µM) in
comparison to the reference drug doxorubicin (IC50 = 8.28, 9.63 and 7.67 µM) and sorafenib (IC50 =
7.31, 9.40 and 7.21 µM). Additionally, member 14, 18b, 18e, 20 and 30d showed potent inhibitory
activities with IC50 values ranging from 5.56 ± 0.22 to 11.5 ± 0.46 which were almost lower than or
equal to that of the reference drugs. Moreover, the highly active compounds were tested for their in vitro
VEGFR-2 inhibitory activities. Thus, compound 18d showed VEGFR-2 inhibitory activity (IC50 =
0.340± 0.04 µM) superior to that of the reference drug, sorafenib (IC50 = 0.588 ± 0.06 µM). Interestingly,
the results of VEGFR-2 inhibition were consistent with that of the cytotoxicity data. Furthermore,
docking study was performed in order to understand the binding pattern of the new compounds into
VEGFR-2 active site. Docking results attributed the potent VEGFR-2 inhibitory effect of the new
compounds as they bound to the key amino acids in the active site, Glu883 and Asp1044, as well as their
hydrophobic interaction with the receptor hydrophobic pocket. Results of cytotoxic activities, in vitro
VEGFR-2 inhibition together with docking study argument the advantages of the synthesized analogues
as promising anti-angiogenic agents.
4. Experimental
4.1. Chemistry
Determination of melting points were carried out using a Gallen lamp melting point apparatus
and were uncorrected. Silica gel Merck 60 F254 commercial plates were used for analytical TLC as
well as UV light and/or with iodine to follow the course of the reaction. The structure of each
compound was confirmed by IR (pye Unicam SP 1000 IR spectrophotometer), 1H NMR (400 MHz,
Bruker Biospin GmbH spectrophotometer) and 13C NMR spectra (100 MHz, Bruker Biospin GmbH
19

spectrophotometer). Chemicals shifts (δ) are reported in parts per million downfield from TMS, J
values are in hertz, and the splitting patterns are abbreviated as follows: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet. The mass spectra were recorded on Varian MAT 311-A (70 e.v.).
2-Mercapto-3-phenyl)quinazolin-4(3H)-one 10 [44], and its corresponding salt 11, N-(4-
acetylphenyl)-2-chloroacetamide 13 [30], ethyl-substitutedbenzoate 16a-e [45], substituted-benzoic acid
hydrazides 17a-e [34, 35], 4-(2-chloroacetamido)benzoic acid 22 [38], 4-(2-chloroacetamido)-N-
(substituted)benzamide
24-27
[36,
37],
N-(4-(2-benzoylhydrazine-1-carbonyl)phenyl)-2-
chloroacetamide 32 [36] were synthesized according to the reported procedures.
4.1.1. N-(4-Acetylphenyl)-2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio) acetamide 14.
A mixture of the potassium salt of 2-mercapto-3-phenylquinazolin-4(3H)-one 11 (0.01 mol) and N-
(4-acetylphenyl)-2-chloroacetamide 13 (2.15 g, 0.01 mol) in DMF (50 ml) was heated on a water bath
for 8 h. After cooling to room temperature, the reaction mixture was poured on crushed ice. The
precipitate was collected by filtration, dried and crystalized from ethanol to give the corresponding target
compound 14.
o
Buff crystals (yield 83%); m.p.: 158-159 C; IR (KBr, ν cm-1): 3318 (NH), 1733, 1671 (C=O);
1H NMR (400 MHz, DMSO-d6) δ ppm; 2,51 (s, 3H, COCH3), 4.13 (s, 2H, S-CH2), 7.42 (t, 1H, J =
7.6 Hz, Ar-H), 7.48 (m, 3H, Ar-H), 7.58 (m, 3H, Ar-H), 7.74 (d, 2H, J = 8.8 Hz, Ar-H), 7.79 (d, 1H, J
= 7.6 Hz, Ar-H), 7.92 (d, 2H, J = 8.8 Hz, Ar-H), 8.06 (d, 1H, J = 8.0 Hz, Ar-H), 10.72 (s, 1H, NH, D2O
13
exchangeable).; C NMR: 26.86, 37.84 , 118.8(2C) , 118.87, 120, 124.1, 126.31, 126.4 , 127.08 ,
128.87, 129.87, 129.99(2C) , 130.47, 132.29, 135.37 , 136.23 , 143.72 , 147.55 , 157.34, 161.07, 166.92,
196.92 DEPT 135 NMR; 26.86 (1CH3), 37.84 (1CH2), 118.8, 118.87, 124.1, 126.31,126.4 ,127.08,
129.87, 129.90, 129.99 (2CH), 130.02, 130.47, 135.37; MS (m/z): 429.99 (M+, 100 %, base peak),
429.09 (30.36 %) 411.89 (12.95%), 383.94 (12.98%), 294.95 (27.17 %). Anal. Calcd. for C24H19N3O3S
(429.49): C, 67.12; H, 4.46; N, 9.78; Found: C, 67.34; H, 4.53; N, 9.6%.
4.1.2. General procedure for preparation of compounds 18a-e.
N-(4-Acetylphenyl)-2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetami-de 14 (0.001
mol) was treated with the appropriate prepared benzohydrazide derivatives namely,
2-
chlorobenzohydrazide, 3-chlorobenzohydrazide, 4-chlorobenzohydrazide, 2-hydroxybenzohydrazide
and 2-chloro-5-nitrobenzohydrazide 17a-e (0.001 mol) in ethanol (50 ml) in the presence of catalytic
amount of glacial acetic acid and refluxed for 8 h. Resulting solids were filtered, washed with water,
dried and recrystallized from ethanol to afford the corresponding target compounds 18a-e respectively.
20

4.1.2.1.
N-(4-(1-(2-(2-Chlorobenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-phenyl-3,4-dihydro
quinazolin-2-yl)thio)acetamide 18a.
Green crystals (yield 65%); m.p.: 173-176 oC; IR (KBr) cm-1: 3280 (NH) and 1648 (C═O).; 1H
NMR (DMSO.d6,  ppm): 2.50, 2.52 (s, s, 3H, CH3), 4.08, 4.12 (s, s, 2H, S-CH2), 7.3-97.52 (m, 6H,
Ar-H), 7.57-7.61 (m, 4H, Ar-H), 7.69 (d, 2H, J = 6.8 Hz, Ar-H), 7.83 (d, 2H, J = 6.8 Hz, Ar-H), 7.93 (d,
2H, J = 6.8 Hz, Ar-H), 8.06 (t, 1H, J = 6.8 Hz, Ar-H), 10.45, 10.57 (s, s, 1H, CONH, D2O exchangeable),
13
10.96, 11.20 (s, s, 1H, NHCO, D2O exchangeable).; C NMR; 14.88, 37.80, 118.97, 119.06, 120.02,
126.36, 126.51, 126.93, 127.08, 127.68 (2C), 129.26, 129.90 (2C), 130.03 (2C), 130.49, 130.93, 131.55,
133.28, 133.38, 135.43, 136.25 (2C), 137.21, 147.59, 157.42, 161.10, 163.38, 166.47, 169.98; Mass
(m/z): 582.05 (M+, 26.02%), 389.69 (100 %, base peak), 380.67 (85.55%), 233.31 (46.54%), 171.23
(38.56%). Anal. Calcd. for C31H24ClN5O3S (582.08): C, 63.97; H, 4.16; N, 12.03; Found: C, 64.16; H,
4.38; N, 12.29 %.
4.1.2.2.
N-(4-(1-(2-(3-Chlorobenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-phenyl-3,4-dihydro
quinazolin-2-yl)thio)acetamide (18b ).
o
Green crystals (yield 73 %); m.p.: 181-182 C; IR (KBr) cm-1: 3447, 3283 (2NH) and 1692
1
(C═O).; HNMR (DMSO.d6, ppm): 2.36 (s, 3H, CH3), 4.13 (s, 2H, SCH2), 7.44 (t, 2H, J = 8.0 Hz,
Ar-H), 7.50-7.55 (m, 4H, Ar-H), 7.57-7.63 (m, 4H, Ar-H), 7.70 (d, 2H, J = 8.0 Hz, Ar-H), 7.79-7.87 (m,
2H, Ar-H) ,7.94 (s, 2H, Ar-H), 8.08 (d, 1H, J = 8.0 Hz, Ar-H), 10.56 (s, 1H, CONH, D2O exchangeable),
10.86 (s, 1H, NHCO, D2O exchangeable).; 13C NMR; 14.98, 37.84, 119.14 (2C), 120.03, 126.37, 126.48,
127.09 (2C), 127.69, 128.08, 129.89 (2C), 130.02 (2C), 130.46, 130.74, 131.70, 133.31, 133.59, 135.39,
136.27, 136.58, 140.74, 147.60, 156.48, 157.39, 161.10, 162.93, 166.46, 168.20. DEPT135 NMR; 15
(1CH3), 37.84 (1CH2), 119.13 (2CH), 126.37 (2CH), 126.48, 127.08 (2CH), 127.68, 128.06, 130.02,
130.46, 130.74, 131.70, 135.38.; Mass (m/z): 581.77 (M+, 66.65 %), 254.17 (100 %, base peak), 236.24
(54.38%), 76.19 (49.09%) Anal. Calcd. for C31H24ClN5O3S (582.08): C, 63.97; H, 4.16; N, 12.03;
Found: C, 64.21; H, 4.39; N, 12.34 %.
4.1.2.3.
N-(4-(1-(2-(4-Chlorobenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-phenyl-3,4-dihydro
quinazolin-2-yl)thio)acetamide 18c.
o
Green crystals (yield 77 %); m.p.: 188-189 C; IR (KBr) cm-1: 3299, 3189 (2NH) and 1692
(C═O).; 1H NMR (DMSO.d6,  ppm): 2.5 (s, 3H, CH3), 4.11 (s, 2H, S-CH2), 7.35 (dd, 2H, J = 8.4 Hz,
Ar-H), 7.39 (dd, 2H, J = 8.4 Hz, Ar-H), 7.44 (t, 2H, Ar-H), 7.54 (dd, 2H, J = 8.0 Hz, Ar-H), 7.59 (dd,
2H, J = 8.0 Hz, Ar-H), 7.67 (d, 1H, J = 8.4 Hz, Ar-H), 7.79 (t, 3H, J = 8.0 Hz, Ar-H), 7.92-7.95 (m, 2H,
21

ArH), 8.06 (d, 1H, J = 8.0 Hz, Ar-H), 10.34 (s, 1H, CONH; D2O exchangeable), 10.79 (s, 1H, NHCO,
13
D2O exchangeable).; C NMR; 14.93, 36.93, 119.24(C), 120.01, 126.35, 126.55, 127.09(C), 127.64,
128.83(C), 128.87, 129.89, 130.03, 130.11, 130.27(C), 133.29(C), 135.48, 136.28, 136.81, 147.98,
156.22, 157.41, 160.16, 161.23, 163.17, 166.46, 168.17; DEPT135 NMR; 14.95, 36.60, 119.07, 119.15
(2CH), 126.35, 126.51, 127.08, 127.61 (2CH), 128.84 (2CH), 129.89 (2CH), 130.03 (2CH), 130.25
(2CH), 130.49, 135.44 Anal.Calcd. for C31H24ClN5O3S (582.08): C, 63.97; H, 4.16; N, 12.03; Found: C,
64.15; H, 4.32; N, 12.17 %.
4.1.2.4.
N-(4-(1-(2-(2-Hydroxybenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-phenyl-3,4-dihydro
quinazolin-2-yl)thio)acetamide 18d.
Buff crystals (yield 68 %); m.p.: 155-158 oC; IR (KBr) cm-1: 3631 (OH), 3284 (NH) and 1658
(C═O).; 1H NMR (DMSO.d6,  ppm): 2.32 (s, 3H, CH3), 4.14 (s, 2H, S-CH2), 6.96 (t, 1H, J = 7.6 Hz,
Ar-H), 7.05 (d, 1H, J = 8.0 Hz, Ar-H), 7.40-7.46 (m, 2H, Ar-H), 7.49 (d, 2H, J = 6.8 Hz, Ar-H), 7.55-
7.60 (m, 4H, Ar-H), 7.72 (d, 2H, J = 8.4 Hz, Ar-H), 7.78 (t, 1H, J = 7.2 Hz, Ar-H), 7.85 (d, 2H, J =
8.4 Hz, Ar-H), 8.04 (t, 2H, J = 7.6 Hz, Ar-H), 10.48 (s, 1H, CONH; D2O exchangeable), 10.58 (s, 1H,
13
NHCO, D2O exchangeable), 11.39 (s, 1H, OH; D2O exchangeable); C NMR; 14.14, 37.86, 117.41,
118.31, 119.20 (2C), 119.99, 120.07, 126.39, 126.46, 127.07, 127.62 (2C), 129.88 (2C), 130.02 (2C),
130.46, 131, 133.27, 133.79, 135.36, 136.26, 140.56, 147.59, 152.59, 157.20, 157.35, 161.12, 162.67,
166.48 DEPT135 NMR; 14.14 (1CH3), 37.87 (1CH2), 117.41, 119.20 (2CH), 120.07, 126.38, 126.46,
127.07, 127.62 (2CH), 129.88 (2CH), 130.02 (2CH), 130.46, 131, 133.79, 135.36; Anal. Calcd. for
C31H25N5O4S (563.63): C, 66.06; H, 4.47; N, 12.43; Found: C, 65.89; H, 4.56; N, 12.60 %.
4.1.2.5. N-(4-(1-(2-(2-Chloro-5-nitrobenzoyl)hydrazono)ethyl)phenyl) -2-((4-oxo -3-phenyl-3,4-
dihydroquinazolin-2-yl)thio)acetamide 18e.
Yellow crystals (yield 89 %); m.p.: 157-159 oC; IR (KBr) cm-1: 3457 (NH) and 1685 (C═O).;
1H NMR (DMSO.d6,  ppm): 2.30 (s, 3H, CH3), 4.11 (s, 2H, S-CH2), 7.47-7.56 (m, 5H, Ar-H),7.60 (s,
2H, Ar-H), 7.68 (s, 2H, Ar-H), 7.82-7.85 (m, 4H, Ar-H), 8.07 (s, 1H, Ar-H), 8.29 (s, 1H, Ar-H), 8.62 (s,
1H, Ar-H), 10.56 (s, 1H, CONH, D2O exchangeable), 11.36 (s, 1H, NHCO, D2O exchangeable).; Mass
(m/z): 627.92 (M+, 25.50%), 90.28 (100 %, base peak), 334.88 (81.52%), 239.11 (74.21%); Anal. Calcd.
for C31H23ClN6O5S (627.07): C, 59.38; H, 3.70; N, 13.40; Found: C, 59.21; H, 3.87; N, 13.68 %.
22

4.1.3.
2-(1-(4-(2-((4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamido)phenyl)ethylidene)
hydrazine-1-carboxamide 20.
N-(4-Acetylphenyl)-2-((4-oxo-3-phenyl-3,4-dihydroquina-zoline-2-yl)thio)acet-amide
14
(0.001 mol) was treated with semicarbazide 19 (0.001 mol) in ethanol (50 ml) in the presence of catalytic
amount of glacial acetic acid and refluxed for 8 h. Resulting solid were filtered, washed with water, dried
and crystallized from ethanol.
o
Yellowish green crystals (yield 63 %); m.p.:187-189 C; IR (KBr) cm-1: 3484 (NH2), 3290,
1
3196 (2 NH) and 1718, 1686 (C═O).; H NMR (DMSO.d6,  ppm): 2.38 (s, 3H, CH3-C=O), 4.11 (s,
2H, S-CH2), 6.56 (s, 2H, NHCONH2, D2O exchangeable), 7.34 (d, 2H, J = 8.0, Ar-H), 7.39 (d, 2H, J =
8.4 Hz, ArH), 7.46 (t, 1H, J = 8.0 Hz, Ar-H), 7.53 (d, 1H, J = 8.8 Hz, Ar-H), 7.58 (m, 1H, Ar-H), 7.63
(d, 2H, J = 8.0 Hz, Ar-H), 7.81 (t, 1H, J = 8.8 Hz, Ar-H), 7.89 (d, 1H, J = 8.8 Hz, Ar-H), 8.06 (d, 1H, J
= 8.0 Hz, Ar-H), 8.25 (d, 1H, J = 8.0 Hz, Ar-H), 10.14 (s, 1H, CONH, D2O exchangeable), 10.56 (s, 1H,
13
NHCONH2, D2O exchangeable); C NMR; 26.35, 36.64, 113.40, 119.09 (2C), 121.62 (2C), 126.90
(2C), 127.63 (2C), 129.88, 130.02 (2C), 130.97, 133.64, 136.01, 139.72, 143.97, 144.20, 157.74, 157.83,
158.25, 168.17, 195.5 DEPT135 NMR; 26.35 (1CH3), 36.64 (1CH2), 113.40, 119.08 (2CH), 121.62
(2CH), 126.90 (2CH), 127.63 (2CH), 129.89, 130.03 (2CH), 130.97; Anal. Calcd. for C25H22N6O3S
(486.55): C, 61.72; H, 4.56; N, 17.27; Found: C, 61.98; H, 4.70; N, 17.51) %.
4.1.4. General procedure for preparation of compounds 28-31
A mixture of potassium salt of 2-mercapto-3-phenylquinazolin-4(3H)-one 11 (0.01mol) and 4-
(2-chloroacetamido)-N-(substituted)benzamide 24a,b, 25, 26a-d and 27 (0.01 mol) in DMF (50 ml) was
heated on a water bath for 8 h. After cooling to room temperature, the reaction mixture was poured onto
crushed ice. The precipitate was collected by filtration, dried and crystalized from ethanol to give the
corresponding target compound 28a,b, 29, 30a-d and 31 respectively.
4.1.4.1. 4-(2-((4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamido)-N- propylbenzamide 28a.
o
White crystals (yield 68 %); m.p.: 153-155 C; IR (KBr) cm-1: 3385, 3263 (2NH), and 1697,
1
1664 (C═O).; H NMR (DMSO.d6,  ppm): 0.86 (t, 3H, J = 7.2 Hz, CH2CH3), 1.47 (m, 2H, CH2CH3),
3.17 (m, 2H, J = 6.8 Hz NHCH2CH2), 4.11 (s, 2H, S-CH2), 7.44 (d, 2H, J = 7.6 Hz, Ar-H), 7.49-7.54
(m, 2H, Ar-H), 7.59-7.62 (m, 3H, Ar-H), 7.65 (d, 2H, J = 8.8 Hz, Ar-H), 7.79 (m, 1H, Ar-H), 7.80 (d,
2H, J = 8.8 Hz, Ar-H), 8.06 (d, 1H, J = 8.0 Hz, Ar-H), 8.32 (t, 1H, CONHCH2, D2O exchangeable),
23

10.58 (s, 1H, CONH; D2O exchangeable).; 13C NMR; 11.93, 22.91, 37.77, 41.40, 118.69 (2C), 120.01,
126.33, 126.51, 127.09, 128.51 (2C), 129.89 (2C), 130.02 (2C), 130.48, 135.41, 136.24, 141.79, 147.57,
157.39, 161.07, 166, 166.60 DEPT135 NMR; 11.94 (1CH3), 22.91 (1CH2), 37.77 (1CH2), 41.40 (1CH2),
118.68 (2CH), 126.33 (2CH), 126.51, 127.08 (2CH), 128.50, 129.88, 130.02 (2CH), 130.48 (2CH),
135.41; Anal. Calcd. for C26H24N4O3S (472.56): C, 66.08; H, 5.12; N, 11.86; Found: C, 66.31; H, 5.30;
N, 12.01 %.
4.1.4.2. N-Cyclohexyl-4-(2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio) acetamido)benzamide
28b.
Beige crystals (yield 61 %); m.p.: 235-237 oC; IR (KBr) cm-1: 3326 (NH), and 1695 (C═O).;
1H NMR (DMSO.d6,  ppm): 1.10 (m, 2H, CH2 of cyclohexyl), 1.27 (m, 2H, CH2 of cyclohexyl), 1.58
(m, 2H, CH2 of cyclohexyl), 1.72 (m, 2H, CH2 of cyclohexyl), 1.79 (m, 2H, CH2 of cyclohexyl), 3.73
(m, 1H, CH- cyclohexyl), 4.10 (s, 2H, S-CH2), 7.44–7.55 (m, 4H, Ar-H), 7.59 (s, 4H ,Ar-H), 7.67 (d,
1H, J = 8.4 Hz, Ar-H), 7.79-7.85 (m, 3H, Ar-H), 7.93 (d, 1H, CONH-cyclohexyl; D2O exchangeable),
8.07 (d, 1H, J = 8.0 Hz, Ar-H), 10.63 (s, 1H, CONH; D2O exchangeable).; 13C NMR; 25.45 (2C), 25.75,
32.95 (2C), 37.8, 48.75, 118.62 (2C), 120.01, 126.34, 126.49, 127.09, 128.66 (2C), 129.89 (2C), 130.03
(2C), 130.48, 135.39(2C), 136.25, 141.79, 147.58, 157.39, 161.09, 165.23, 166.61 DEPT135 NMR;
25.45 (2CH2), 25.75 (CH2), 32.95 (2CH2), 37.8 (CH2), 48.75 (CH), 118.61 (2CH), 126.33, 126.49,
127.08, 128.66 (2CH), 129.88 (2CH), 130.02 (2CH), 130.47, 135.38; Anal. Calcd. for C29H28N4O3S
(512.63) C, 67.95; H, 5.51; N, 10.93 Found: C, 68.13; H, 5.64; N,11.17 %.
4.1.4.3. N-Benzyl-4-(2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamido) benzamide 29.
o
White crystals (yield 64 %); m.p.: 228-230 C; IR (KBr) cm-1: 3316, 3280 (2NH) and 1659
(C═O); 1H NMR (DMSO.d6,  ppm): 4.13 (s, 2H, S-CH2), 4.47 (d, 2H, CH2–C6H5), 7.31 (s, 5H, Ar-H),
7.44-7.55 (m, 4H, Ar-H), 7.59 (m, 3H, Ar-H), 7.70 (d, 2H, J = 8.4 Hz, Ar-H), 7.79 (t, 1H, J = 8.0 Hz,
Ar-H), 7.88 (d, 2H, J = 8.4 Hz, Ar-H), 8.07 (d, 1H, J = 8.0 Hz, Ar-H), 8.92 (t, 1H, CONHCH2; D2O
exchangeable), 10.60 (s, 1H, CONH; D2O exchangeable).; 13C NMR; 37.80, 43.06, 118.79 (2C), 126.34,
126.5, 127.09, 127.15 (2C), 127.68 (2C), 128.67, 128.70 (2C), 129.53, 129.89 (2C), 130.02 (2C),
130.48(2C), 135.40, 136.25, 140.25, 142.03, 147.58, 157.39, 161.08, 166.12, 166.65 DEPT135 NMR;
37.80 (1CH2), 43.06 (1CH2), 118.79 (2CH), 126.34, 126.5, 127.09, 127.15 (2CH), 127.68 (2CH),
128.67, 128.70 (2CH), 129.89 (2CH), 130.02 (2CH), 130.48, 135.40; Anal. Calcd. for C30H24N4O3S
(520.61): C, 69.21; H, 4.65; N, 10.76; Found: C, 69.49; H, 4.78; N, 10.93 %.
4.1.4.4. 4-(2-((4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamido)-N-phenylbenzamide 30a.
24

White crystals (yield 72 %); m.p.: 208-209 oC; IR (KBr) cm-1: 3315, 3283 (2 NH) and 1659,1703
1
(C═O); H NMR (DMSO.d6,  ppm): 4.16 (s, 2H, S-CH2), 7.08 (s, 1H, Ar-H), 7.34 (s, 2H, Ar-H), 7.43-
7.59 (m, 6H, Ar-H), 7.79 (s, 6H, Ar-H), 7.98 (d, 2H, J = 8.0 Hz, Ar-H), 8.08 (d, 1H, J = 8.0 Hz, Ar-H),
10.16 (s, 1H, CH2CONH; D2O exchangeable), 10.70 (s, 1H, CONH; D2O exchangeable).;13C NMR;
37.86, 118.81, 120.03, 120.82 (2C), 126.35, 126.49, 127.10, 129.02 (2C), 129.13, 129.19 (2C), 129.89
(2C), 129.94, 130.03 (2C), 130.48, 135.39, 136.25, 139.72, 142.37, 147.59, 157.37, 161.1, 165.34,
166.78, 168.46 DEPT135 NMR; 37.86 (CH2), 118.81 (2CH), 120, 82 (2CH), 123,97, 126.35, 126.49,
127.10 (2CH), 129.13, 129.19 (2CH), 129.89 (2CH), 130.03 (2CH), 130.48, 135.39; Anal. Calcd. for
C29H22N4O3S (506.58): C, 68.76; H, 4.38; N, 11.06; Found: C, 68.93; H, 4.57; N, 11.21%.
4.1.4.5.
N-(4-Chlorophenyl)-4-(2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2yl)thio)acetamido)
benzamide 22b.
Grey crystals (yield 55 %); m.p.: 184-186 oC; IR (KBr) cm-1: 3299 (NH) and 1692 (C═O); 1H
NMR (DMSO.d6,  ppm): 4.15 (s, 2H, S-CH2), 7.38 (d, 2H, J = 8.4 Hz, Ar-H), 7.46 (t, 1H, J = 7.6 Hz,
Ar-H), 7.50 (d, 2H, J = 8.0 Hz, Ar-H), 7.47 (d, 1H, J = 8.0 Hz, Ar-H), 7.61 (m, 3H, Ar-H), 7.76 (d, 2H,
J = 8.4 Hz, Ar-H), 7.81 (d, 2H, J = 8.4 Hz, Ar-H), 7.95 (d, 2H, J = 8.0 Hz, Ar-H), 8.08 (d, 1H, J = 8.0
Hz, Ar-H), 10.26 (s, 1H, CONH; D2O exchangeable), 10.68 (s, 1H, CONHC6H5; D2O exchangeable).;
13C NMR; 37.84, 118.82 (2C), 120.04, 122.28 (2C), 126.34, 126.5, 127.11, 127.59, 128.93 (2C), 129.22
(2C), 129.62, 129.89 (2C), 130.02 (2C), 130.48, 135.39, 136.25, 138.71, 142.50, 147.59, 157.37, 161.09,
165.40, 166.77, 168.45 DEPT135 NMR; 37.83 (1CH2), 118.81 (2CH), 122.27 (2CH), 126.34,126.5,
127.11, 128.93 (2CH), 129.23 (2CH), 129.89 (2CH), 130.03 (2CH), 130.48, 135.39 Mass (m/z): 540.37
(M+, 10.75 %), 498.22 (100 %, base peak),420.94 (27.69%), 119.78 (13.38%), 76.89 (10.02%); Anal.
Calcd. for C29H21ClN4O3S (541.02): C, 64.38; H, 3.91; N, 10.36; Found: C, 64.56; H, 4.13; N, 10.59 %.
4.1.4.6. 4-(2-((4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamido)-N-(p-tolyl)benzamide 30c.
Beige crystals (yield 67 %); m.p.: 205-207 oC; IR (KBr) cm-1: 3301, 3192 (2 NH) and 1665 (C═O).; 1H
NMR (DMSO.d6,  ppm): 2.26 (s, 3H, CH3 –C6H5), 4.11 (s, 2H, S-CH2), 7.05 (d, 1H, J = 8.0 Hz, Ar-
H), 7.12 (d, 2H, J = 8.0 Hz, Ar-H), 7.19 (d, 1H, J = 8.0 Hz, Ar-H), 7.23 (d, 1H, J = 8.0 Hz, Ar-H), 7.35-
7.47 (m, 3H, Ar-H), 7.55-7.61 (m, 3H, Ar-H), 7.69-7.72 (m, 2H, Ar-H), 7.74-7.82 (m, 1H, Ar-H), 7.92
(d, 2H, J = 8.0 Hz, Ar-H), 8.08 (d, 1H, J = 8.0 Hz, Ar-H), 10.55 (s, 1H, S-CH2CONH; D2O
exchangeable), 10.74 (s, 1H, NHCO; D2O exchangeable).; 13C NMR; 20.86, 37.73, 116.23, 119.12 (2C),
121.12 (2C), 124.91, 126.61, 127.92, 128.98 (2C), 129.41 (2C), 129.76, 130.05, 130.56, 133.28, 135.47,
136.16, 136.98, 139.62, 140.06, 142.15, 147.56, 157.25, 160.38, 165.37, 166.97, 176.68 DEPT135
25

NMR; 20.86 (CH3), 37.68 (CH2), 116.05, 119.06 (2CH), 121.10 (2CH), 122.36, 124.88, 126.67,
127.13, 128.94 (2CH), 129.40 (2CH), 129.69, 130.42, 135.45, 136.21; Anal. Calcd. for C30H24N4O3S
(520.61): C, 69.21; H, 4.65; N, 10.76; Found: C, 69.47; H, 4.82; N, 11.01 %.
4.1.4.7. Ethyl 4-(4-(2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamide benzamido)
benzoate 30d.
Brown crystals (yield 88 %); m.p.: 162 oC; IR (KBr) cm-1: 3309 (NH) and 1654 (C═O amidic) and 1751
1
(C═O ester); H NMR (DMSO.d6,  ppm): 1.3 (t, 3H, CH2CH3), 4.14 (s, 2H, S-CH2), 4.27 (q, 2H,
CH2CH3), 7.44 (t, 1H, J = 8.0 Hz, Ar-H), 7.50 (d, 1H, J = 8.0 Hz, Ar-H), 7.54-7.61 (m, 3H, Ar-H), 7.75-
7.83 (m, 4H, Ar-H), 7.89-7.99 (m, 7H, Ar-H), 8.08 (d, 1H, J = 8.0 Hz, Ar-H), 10.43 (s, 1H, S-CH2CONH;
D2O exchangeable), 10.68 (s, 1H, CONH; D2O exchangeable).; 13C NMR; 14.67, 36.69, 37.84, 118.81,
118.89 (2C), 119.97 (2C), 120.03, 124.89, 127.23, 129.31 (2C), 129.37, 129.44, 129.89 (2C),
130.01(2C), 130.48 (2C), 135.30, 136.18, 142.66, 144.19, 147.63, 157.22, 161.04, 165.69, 165.83,
166.75, 168.46; DEPT135 NMR; 14.67 (CH3), 36.69 (CH2), 37.84 (CH2), 118.88 (2CH), 119.95 (2CH),
119.97, 126.33, 126.49, 127.10, 129.31 (2CH), 129.89 (2CH), 130.02 (2CH), 130.48 (2CH), 135.38;
Anal. Calcd. for C32H26N4O5S (578.64): C, 66.42; H, 4.53; N, 9.68; Found: C, 66.78; H, 4.66; N, 9.90
%.
4.1.4.8. 2-((4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)-N-(4-(2-phenyl hydrazine-carbonyl)
phenyl)acetamide 31.
o
White crystals (yield 57 %); m.p.: 208-211 C; IR (KBr) cm-1: 3262 (NH) and 1722, 1697
(C═O); 1H NMR (DMSO.d6,  ppm): 4.12 (s, 2H, S-CH2), 6.70 (t, 2H, J = 7.2 Hz, Ar-H), 6.77 (d, 2H,
J = 7.6 Hz, Ar-H), 7.13 (t, 2H, J = 7.6 Hz, Ar-H), 7.45-7.51 (m, 3H, Ar-H), 7.54 (d, 1H, J = 8.0 Hz,
Ar-H), 7.60 (d, 3H, Ar-H), 7.71 (d, 2H, J = 8.4 Hz, Ar-H), 7.80 (t, 2H, J = 8.0 Hz, Ar-H), 7.89 (d, 1H,
J = 8.0 Hz, -NHNH-C6H5; D2O exchangeable), 8.07 (d, 1H, J = 8.0 Hz, Ar-H), 10.25 (s, 1H, CONH;
13
D2O exchangeable), 10.63 (s, 1H, CONHNH; D2O exchangeable).; C NMR; 37.79, 112.80 (2C),
118.91 (2C), 119.06, 120.01, 126.34, 126.53, 127.1, 128.12, 128.75 (2C), 129.17 (2C), 129.88 (2C),
130.04 (2C), 130.5, 135.44, 136.24, 142.35, 147.57, 150.05, 157.38, 161.09, 166.26, 166.72 DEPT135
NMR; 37.79 (CH2), 112.79 (2CH), 118.9 (2CH), 119.06, 126.34, 126.53, 127.1, 128.75 (2CH), 129.17
(2CH), 129.88 (2CH), 130.04 (2CH), 130.5, 135.44; Anal. Calcd. for C29H23N5O3S (521.60): C, 66.78;
H, 4.44; N, 13.43 Found: C, 67.58; H, 4.62; N, 13.29 %.
26

4.1.5.
N-(4-(2-(2-Hydroxybenzoyl)hydrazine-1-carbonyl)phenyl)-2-((4-oxo-3-phenyl-3,4dihydro
quinazolin-2-yl)thio)acetamide 33.
A mixture of potassium salt of 2-mercapto-3-phenylquinazolin-4(3H)-one 11 (3.48g, 0.01 mol)
and N-(4-(2-benzoylhydrazine-1-carbonyl)phenyl)-2-chloroacetamide 32 (0.01 mol) in DMF (100 ml)
was heated on a water bath for 8 h. After cooling to room temperature, reaction mixture was poured onto
crushed ice. The formed precipitate was filtered, dried and crystalized from ethanol to give the
corresponding target compound 33.
o
Beige crystals (yield 57 %); m.p.: 194-197 C; IR (KBr) cm-1: 3627 (OH), 3285, 3184 (3 NH),
and 1734, 1667 (C═O).; 1H NMR (DMSO.d6,  ppm): 4.10 (s, 2H, S-CH2), 6.90 (m, 2H, Ar-H), 6.96
(m, 2H, Ar-H), 7.50 (d, 2H, J = 8.0 Ar-H), 7.54 (d, 1H, J = 8.0 Hz, Ar-H), 7.60 (m, 4H, Ar-H), 7.74 (d,
2H, J = 8.4 Hz, Ar-H), 7.80 (t, 1H, J = 7.6 Hz, Ar-H), 7.91 (d, 2H, J = 7.6 Hz, Ar-H), 8.08 (d, 1H, J =
8.0 Hz, Ar-H), 10.47 (s, 1H, CONH; D2O exchangeable), 10.59 (s, 1H, OH; D2O exchangeable), 10.67
(s, 2H, CONHNHCO; D2O exchangeable).; 13C NMR; 37.81, 118.91 (2C), 120.03 (2C), 126.35, 126.53
(2C), 127.10, 128.86, 128.98 (2C), 129.89 (4C), 130.04 (4C), 130.5 (2C), 135.44, 136.26 (2C), 142.59,
147.58, 157.38, 161.09, 166.79 DEPT135 NMR; 37.80 (1CH2), 117.98, 118.92 (2CH), 126.35, 126.53,
127.09, 128.86, 128.98 (2CH), 129.89 (2CH), 130.04, 130.49 (2CH), 134.29, 135.44; Anal. Calcd. for
C30H23N5O5S (565.60): C, 63.71; H, 4.10; N, 12.38 Found: C, 63.52; H, 4.27; N, 12.64 %.
4.2. Biological testing
4.2.1. In vitro anti-proliferative activity
The anti-proliferative activities of the synthesized compounds against HepG-2, MCF-7 and
HCT-116 cell lines were carried out through standard MTT method. Cell lines were cultured in RPMI-
1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml penicillin and
100µg/ml streptomycin at 37 oC in a 5% CO2 incubator. The cell lines were seeded in a 96-well plate at
a density of 1.0 x 104 cells / well at 37 oC for 48 h under 5% CO2. After incubation, the cells were treated
with different concentration of synthesized compounds and incubated for 24 h. After 24 h of drug
treatment, 20 µl of MTT solution at 5mg/ml was added and incubated for 4 h. Dimethyl sulfoxide
(DMSO) in volume of 100 µl was added into each well to dissolve the purple formazan formed. The
colorimetric assay was estimated and recorded at absorbance of 570 nm using a plate reader (EXL 800,
USA). The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of
untreated sample) X 100. Results for IC50 values of the active compounds are summarized in Table 1.
27

4.2.2. In vitro VEGFR-2 kinase assay
Inhibitory activity of the selected compounds 14, 18b, 18d, 18e, 20, 29, 30a, 30b, 30c, 30d and
33
against
VEGFR-2
was
evaluated
using
Human
VEGFR-2
ELISA
kit
(Enzyme-Linked Immunosorbent Assay).
A
specific antibody for VEGFR-2 was
seeded on a 96-well plate and 100 μL of the standard solution or the tested compound
was added, all were incubated at room temperature for 2.5 hours. Then washed, 100
μL of the prepared biotin antibody was added, then incubated at room temperature for
additional 1 hour. Washed, 100 μL of streptavidin solution was added then incubated
for 45 min. at room temperature. Washed again, 100 μL of TMB Substrate reagent
was added and incubated for 30 min. at room temperature. 50 μL of the stop solution
was added, then read at 450 nm immediately. The standard curve was drawn,
concentrations on the X-axis and the absorbance on the Y-axis.
4.3. Molecular docking
VEGFR-2 crystal structure of was downloaded from the Protein Data Bank,
http://www.rcsb.org/pdb (PDB ID: 2OH4, resolution: 2.05 Å) using Discovery
Studio 2.5 software. Water molecules were removed from the downloaded protein.
Alternate conformations and valence monitor options were used to correct crystallographic disorders
and unfilled valence atoms. The energy of the 2OH4 protein was minimized using CHARMm and
MMFF94 force fields for charge and partial charge, respectively. The active binding site of the protein
was identified and prepared for docking [15, 31, 46]. Structures of the designed compounds and
reference ligand, sorafenib, were sketched using ChemBioDraw Ultra 14.0. Structures were saved in
MDL‐SD file format. Next, the SD file was opened followed by protonation of the 3D structures Then,
and energy was minimized by applying CHARMm and MMFF94 force fields. Energy minimization was
carried out as described in the Supporting Information. The structures were then prepared for docking
by optimization of the parameters. Docking was carried out using CDOCKER-CHARMm-based
technique in the interface of Accelry's Discovery Studio 2.5. A maximum of 10 conformers was
measured for each molecule in the docking analysis. Subsequently, the docking scores (CDOCKER
interaction energy) of the most ideal pose of each of the docked molecules with the amino acids at the
VEGFR-2 binding pocket were recorded [47-51].
28

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