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

Article abstract of DOI:10.1016/j.bmc.2020.115872

Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer. Thus, nineteen new quinazoline-4(3H)-one derivatives were designed and synthesized. Preliminary cytotoxicity studies of the synthesized compounds were evaluated against three human cancer cell lines (HepG-2, MCF-7 and HCT-116) using MTT assay method. Doxorubicin and sorafenib were used as positive controls. Five compounds were found to have promising cytotoxic activities against all cell lines. Compound 16_f, containing a 2-chloro-5-nitrophenyl group, has emerged as the most active member. It was approximately 4.39-, 5.73- and 1.96-fold more active than doxorubicin and 3.88-, 5.59- and 1.84-fold more active than sorafenib against HepG2, HCT-116 and MCF-7 cells, respectively. The most active cytotoxic agents were further evaluated in vitro for their VEGFR-2 inhibitory activities. The results of in vitro VEGFR-2 inhibition were consistent with that of the cytotoxicity data. Molecular docking of these compounds into the kinase domain, moreover, supported the results.

Full text of DOI:10.1016/j.bmc.2020.115872

Journal Pre-proofs
Design, synthesis, and anti-proliferative evaluation of new quinazolin-4(3H)-
ones as potential VEGFR-2 inhibitors
Khaled. El-Adl, Abdel-Ghany A. El-Helby, Rezk R. Ayyad, 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, Ibrahim. H. Eissa
PII:
S0968-0896(20)30702-1
https://doi.org/10.1016/j.bmc.2020.115872
BMC 115872
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 July 2020
3 November 2020
6 November 2020
Please cite this article as: Khaled. El-Adl, A.A. El-Helby, R.R. Ayyad, H.A. Mahdy, M.M. Khalifa, H.A.
Elnagar, A.B.M. Mehany, d.M. Metwaly, a.A. Elhendawy, d.M. Radwan, d.A. ElSohly, m.H. Eissa, Design,
synthesis, and anti-proliferative evaluation of new quinazolin-4(3H)-ones as potential VEGFR-2 inhibitors,
Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115872
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Design, synthesis, and anti-proliferative evaluation of new quinazolin-4(3H)-ones
as potential VEGFR-2 inhibitors
Khaled. El-Adla,b,*, Abdel-Ghany A. El-Helbya , Rezk R. Ayyada, 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, Ibrahim. H. Eissa*a
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
1

Abstract
Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer.
Thus, nineteen new quinazoline-4(3H)-one derivatives were designed and synthesized.
Preliminary cytotoxicity studies of the synthesized compounds were evaluated against three
human cancer cell lines (HepG-2, MCF-7 and HCT-116) using MTT assay method.
Doxorubicin and sorafenib were used as positive controls. Five compounds were found to
have promising cytotoxic activities against all cell lines. Compound 16f, containing a 2-
chloro-5-nitrophenyl group, has emerged as the most active member. It was approximately
4.39-, 5.73- and 1.96-fold more active than doxorubicin and 3.88-, 5.59- and 1.84-fold more
active than sorafenib against HepG2, HCT-116 and MCF-7 cells, respectively. The most
active cytotoxic agents were further evaluated in vitro for their VEGFR-2 inhibitory activities.
The results of in vitro VEGFR-2 inhibition were consistent with that of the cytotoxicity data.
Molecular docking of these compounds into the kinase domain, moreover, supported the
results.
Keywords: Anticancer; Molecular docking; quinazolin-4(3H)-one; VEGFR-2.
2

1. Introduction
Tumor angiogenesis plays a vital role in cancer cell survival, tumor growth and the development
of distant metastasis [1-3]. Pathological angiogenesis has been associated with a diversity of
diseases, including diabetes retinopathy, psoriasis, rheumatoid arthritis and cancer [4]. Numerous
regulators of angiogenesis have been recognized and characterized [5, 6]. Among them Vascular
endothelial growth factor (VEGF) is considered as a key angiogenic factor [7].
VEGF is secreted by tumors [8]. It induces a mitogenic response via its binding to one of
three tyrosine kinase receptors (VEGFR-1–3) on the nearby endothelial cells [8]. Overexpression of
VEGF have been noticed in several types of cancer including colorectal cancer [11], breast cancer
[12] and hepatocellular carcinoma [13]. Accordingly, inhibition of this signaling pathway should
block angiogenesis and subsequent tumor growth [9, 10]. There is much evidence that direct
inhibition of the kinase activity of VEGFR-2 (also referred to as the kinase insert domain containing
receptor (KDR)) will result in the reduction of angiogenesis and the suppression of tumor growth
[11].
Recently, a lot of researches are focusing on the development of novel VEGFR-2 inhibitors
[12]. Many VEGFR-2 inhibitors have been reported with significant potency to inhibit tumor
angiogenesis (Fig. 1, 2) [13]. Several small molecule VEGFR-2 inhibitors, such as the birdlore
derivative sorafenib 1 (BAY-43-9006, Bayer) [14], the indol-2-one derivative sunitinib 2 (SU-
11248, Pfizer) [14], and quinolone derivative tivozanib 3 (FOTIVDA, Eusa Pharma UK) [15] have
been clinically approved for the treatment of several cancer diseases.
Numerous small VEGFR-2 inhibitors molecules have progressed to the clinical evaluation
stage [16]. Linifanib 4 (ABT-869, Abbott) is a novel, orally active VEGFR-2 inhibitor that exhibits
significant antitumor and antiangiogenic activities and is currently in phase III trials [13, 17].
Cediranib 5 (AZD2171, AstraZeneca) is an orally active angiogenesis inhibitor that works by
selectively inhibiting the tyrosine kinase activity of all VEGF receptor subtypes [18]. Cediranib 5, a
quinazoline derivative, is being tested in phase III clinical trials for treatment of ovarian cancer
[19]. However, the development of novel VEGFR-2 inhibitors analogs displayed a field of great
interest, as demonstrated by the number of patents filed and of a lot of papers published in the last
years [10].
VEGFR-2 was reported to be substantially upregulated in HepG-2 cells in a dose-dependent
manner with the stimulation of the hepatocyte growth factor (HGF), which is involved in cell
proliferation, invasion, and angiogenesis of hepatocellular carcinoma (HCC) [20-22]. Blockade of
VEGFR-2 signaling revealed a marked inhibition on both the growth and metastasis of HCC [22,
3

23]. As well, VEGFR-2 was found to be crucial to cell survival and regulates endothelial
differentiation in both the breast cancer cells (MCF-7) [23] and human colorectal carcinoma (HCT-
116) [24, 25]. Over-expression of VEGFR-2 receptors in breast cancer cells has documented a
contributor in resistance of such cancer type to the chemotherapeutic effect of tamoxifen [26].
VEGFR-2 degraders were documented to impair the in vitro endothelial differentiation, and to
promote angiogenesis, suggesting a VEGFR-2-mediated mechanism of antiproliferative activity in
human colorectal carcinoma [24].
Quinazolin-4(3H)-one is a promising class of heterocycles that is well-tolerated in humans
[27] and possesses antitumor activity [28-30]. Moreover, it is the backbone of many bioactive
compounds that show potential activities as VEGFR inhibitors [4, 31]. In addition, several new
sulfonamide [29] and thiosemicarbazone [32] moieties were reported to possess antitumor activities.
In view of this, our rationale of molecular design aimed to develop small quinazolin-4(3H)-
ones in order to act as VEGFR-2 inhibitors. They displayed potent antiproliferative effects and
could be considered as promising lead compounds for further optimization. A molecular docking
model and preliminary SAR based on enzymatic inhibitory activity were also discussed.
H
H
N
N
N
H
N
O
O
Cl
O
F
F
F
Sorafenib 1
H
N
H
O
N
N
N
H
O
N
O
O
Cl
F
N
H
O
O
O
N
H
N
Tivozanib 3
N
Sunitinib 2
F
H
N
H
N
O
O
NH2
N
N
N
HN
F
O
O
N
H
Linifanib 4
Cediranib 5
Fig. 1. Chemical structures of VEGFR-2 inhibitors
1.1. Rational of molecular design
4

In continuation of our previous works that proved potential anticancer activities of novel
chemical agents [33-46], herein we present the synthesis and the biological evaluation of some
novel derivatives carrying modifications in the quinazoline-4(3H)-one scaffolds.
Analyzing the binding interactions of sorafenib 1 and different VEGFR-2 inhibitors and the
common pharmacophoric features shared by them [47-49] revealed that VEGFR-2 inhibitors,
though being highly diverse in structure, share four common feature pharmacophores for binding as
illustrated in Fig. 2 and Fig. 3. (i) A flat aromatic ring system (the left cyan color) of the main
scaffold that adopts the active site via formation of an essential hydrogen bond with the backbone
NH of Cys917 residue, the key amino acid residue in the catalytic ATP-binding domain [48]. And
therefore, this flat system should contain at least one H-bond acceptor (N atom is preferred,
followed by the O atom). (ii) A central aryl ring or spacer (presented by the brown color),
occupying the linker region between the ATP-binding domain and the DFG domain of the enzyme
[50]. (iii) A functional group (presented by the yellow color) acting as pharmacophore (e.g., amide
or urea) that forming two hydrogen bonds with the enzyme allosteric site residues: one hydrogen
bond with the side chain of a conserved glutamic acid in the C-helix (Glu 883) and the other with
the backbone amide of aspartic acid in the DFG motif (Asp 1044) [51]. (iv) All VEGFR-2 inhibitors
also have a hydrophobic moiety (the right pink color) that is located just after the hydrogen bond
donor–acceptor pair forming van der Waals interactions with the allosteric site [52, 53].
5

H
N
H
N
N
H
N
O
O
Cl
O
F
F
F
1
Sorafenib
O
N
N
H
F
N
H
O
N
H
2
Sunitinib
H
N
H
N
O
N
O
O
Cl
O
O
N
3
Tivozanib
Fig. 2. Basic pharmacophoric features of VEGFR-2 inhibitors
Our design was based on targeting VEGFR-2 in its DFG-out conformation. Inspection of
known type-II inhibitors, especially sorafenib, revealed that the conserved hydrogen bonds between
the ligand and the allosteric site residues were done using urea or amide moieties. In this respect,
we designed novel compounds based on quinazoline core molecularly hybridized with effective
antitumor moieties containing urea, amide and sulfonamide pharmacophores with extended alkyl or
aryl groups. The main core of our molecular design comprised bio-isosteric modification
approaches of VEGFR-2 inhibitors at four different positions (Fig. 3).
6

Hetero
aromatic
ring
Hydrophobic
tail
Central Aryl
Ring
HBA-HBD
Effective
antitumor
moieties
Quinazolin-4(3H)-one
(different site of
attachemet;
Cyclic / Open chain
linkers (spacers)
Substituted aromatic
or aliphatic groups
containing
amide, urea or
sulfonamide
position 2 or 3)
Fig. 3. Summary for the possible modifications of VEGFR-2 inhibitors
The first position was the heterocyclic aromatic ring in which the 2-mercapto-3-(4-
methylphenyl)quinazolin-4(3H)-one moiety was used. The choice of this quinazoline moiety was
based on some bio-isoeteric considerations, i) the bicyclic structure of quinazoline core is
convenient to the large size space of the ATP binding region [53], ii) the heterocyclic nitrogen
atoms serve as hydrogen-bond acceptors conferring excellent VEGFR-2 potency.
The second position was the linker (spacer) region. The length of the linker was modified to
be four atom bridges in addition to a phenyl moiety. The third position was the HBA/HBD region
(pharmacophore). We used many different effective anticancer moieties comprises the essential
HBA/HBD features to play the role of the pharmacophore. The pharmacophores were designed to
be open forms. The used open chain pharmacophores were hydrazone, sulfonamide,
acetamidobenzamide, 2-hydroxybenzolhydrazine, and thiosemicarbazone moieties.
Finally, the terminal hydrophobic moieties were selected to be aromatic heterocyclic,
aromatic non-heterocyclic or aliphatic structures. The wide variety of modifications enabled us to
study the SAR of these compounds as effective anti-cancer agents with potential VEGFR-2
inhibitory activities which is considered as a crucial objective of our work. All modification
pathways and molecular design rationale were demonstrated and summarized in Fig 4.
7

O
N
N
H
N
S
O
12
O
O
N
O
N
N
N
H
H
N
N
S
R1
H
S
S
N
O
N
O
N
N
H
NH2
O
CH3
CH3
18
16a-f
O
N
O
N
N
H
N
H
N
N
S
H
N
S
H
N
O
O
O
O
27
26
O
N
H
O
N
N
S
H
N
O
N
R
N
SH
O
28a-d
8
O
N
H
N
O
N
N
S
H
N
O
S
N
N
H
H
N
O
S
29
O
O
R1
O
N
S
N
H
O
N
34a-c
H
N
O
H
N
O
N
H
31
O
HO
Fig. 4. Design of the newly proposed quinazolinone derivatives as VEGFR-2 inhibitors.
8

2. Results and discussion
2.1. Chemistry
In our research to discover new anticancer compounds, we designed and evaluated
pharmacologically nineteen quinazolinone derivatives as potent VEGFR-2 inhibitors. These
compounds were prepared via a generalized route outlined in Schemes 1-6.
The commercial available anthranilic acid 6 was primary reacted in alkaline media with p-
toluidine 7 in the presence of carbon disulfide to furnish 2-mercapto-3-(4-methylphenyl)quinazolin-
4(3H)-one 8 [54] which was then heated with alcoholic potassium hydroxide to afford the
corresponding potassium salt 9 as the reported procedure [55]. On the other side, 4-
aminoacetophenone 10 was stirred with chloroacetyl chloride in dry DMF at room temperature to
produce N-(4-acetylphenyl)-2-chloroacetamide 11 [56]. The previously prepared potassium salt 9
was refluxed, however, with compound 11 according to the method reported by El-Helby et. al. to
afford compound 12 (Scheme 1) [57].
IR spectrum of compound 12 confirmed the presence of carbonyl absorption bands at 1670,
1685 and 1700 cm−1 and NH band at 3234 cm−1. Its 1H NMR spectra showed singlet signals of the
aliphatic protons at δ 2.55 and 4.11 ppm in addition to another one at δ 2.42 corresponding to
COCH3, SCH2 and Ar-CH3 protons, respectively (Scheme 1).
Scheme 2 was initiated via synthesis of some benzoic acid hydrazide intermediates 15a-f.
These intermediates were prepared by esterification of (un)substituted benzoic acids 13a-f with
absolute ethanol in the presence of sulfuric acid as a catalyst [58] followed by refluxing with
hydrazine hydrate [59, 60]. The later intermediates were allowed to react with compound 12 in the
presence of a catalytic amount of glacial acetic acid to give the target compounds 16a-f [61]. The
1
formation of compounds 16a-f were confirmed by H NMR spectra which showed shifting of the
CH3 protons to a range of δ 2.41-2.43 ppm than the CH3 protons of the corresponding ketone
derivative 12. Moreover, D2O exchangeable singlet signals appeared at a range of δ 10.74-11.33
ppm that corresponds to the NH protons.
Similarly, compound 12 was treated with thiosemicarbazide 17 to afford the corresponding
derivative 18 [61] which was approved by its spectral data (Scheme 2).
Scheme 3 was started primarily with stirring of 4-aminobenzoic acid 19 with chloroacetyl
chloride in DMF at room temperature to give 4-(3-chloroacetamido) benzoic acid 20 [62].
Different carboxylic acid amide intermediates were prepared from 4-(3-chloroacetamido)
benzoic acid 20 following the mixed anhydride procedure. This procedure involves the activation of
the acid as a mixed carboxylic-carbonic acid anhydride [63, 64]. The anhydride 21 was generated
by the action of ethyl chloroformate in the presence of TEA, and is not isolated before the
9

subsequent reaction. The formed anhydride was then treated with different aromatic or aliphatic
amines at room temperature to give the desired intermediates 22, 23, 24a-d and 25 (Scheme 3).
With respect to the previously prepared intermediates, they were allowed to interact with the
potassium salt 9 in DMF affording compounds 26, 27, 28a-d and 29 (Scheme 4) [65]. IR spectra of
this series confirmed presence of the carbonyl absorption band at a range of 1654-1751 cm-1 and
presence of NH band at a range of 3179 - 3462 cm-1. 1H NMR spectra showed singlet signals of the
aliphatic -SCH2 protons at a δ range of 4.09 - 4.15 ppm and presence of D2O exchangeable NH-
amide protons at a δ range of 8.32- 10.74 ppm.
The potassium salt 9 reacted also with compound 30, prepared by mixed anhydride procedure
via the reaction of 4-(2-chloroacetamido) benzoic acid 20 with 2-hydroxybenzohydrazide 15e, to
give the target compound 31 (Scheme 5) [64]. The formation of compound 31 was confirmed by 1H
NMR spectra which showed the appearance of a singlet signal of the aliphatic -SCH2 protons at δ
4.12 ppm in addition to D2O exchangeable signals of the NH-amide protons at δ 10.47 and 10.68
ppm.
The intermediates 33a-c were prepared according to the reported methods [66, 67]. These
intermediates were used in the present work to introduce the N-(4-(N-
(substituted)sulfamoylphenyl)acetamide moieties to the previously prepared potassium salt 9 to
afford compounds 34a-c (Scheme 6) [65]. IR spectra of this series confirmed the presence of the
carbonyl absorption bands at a range of 1710-1671cm-1 and NH bands at a range of 3447-3117 cm-
1. 1H NMR spectra showed singlet signals of the aliphatic -SCH2 protons at a δ range of 4.09 - 4.12
ppm and D2O exchangeable NH-amide protons at a δ range of 9.67-10.90 ppm.
10

O
N
1) KOH/CS2
CH3OH/Reflux /10h
COOH
NH2
N
2) HCl
H2N
(7)
SH
(6)
(8)
O
H2N
KOH/
EtOH
Reflux /0.5h
(10)
O
DMF/ K2CO3
Stirring / 1.5 h
Cl
Cl
O
O
N
O
N
S K
Cl
N
H
(9)
(11)
DMF/KI/
/ 6 h
O
N
N
H
N
S
O
(12)
O
Scheme 1. General procedure for synthesis of target compound 12.
11

HO
O
R
H2N NH
O
C2H5O
R
EtOH
R
R
NH2NH2.H2O
O
Conc H2SO4
Reflux /3 h
(13a-f
)
(14a-f
)
(15a-f
)
H2N NH
O
O
N
H
N
(15a-f
)
R
H
N
S
N
O
N
gl. Acetic acid
O
N
O
EtOH /Reflux /8 h
(16a-f
)
N
S
R
H
H
N
16a
16b
16c
16d
16e
16f
2-Cl
3-Cl
4-Cl
2-OH
2-Cl,5-NO2
O
(12)
O
S
H2N
O
N
N
NH2
H
N
(17)
H
N
gl. Acetic acid
S
S
EtOH /Reflux /8 h
O
N
N
NH2
(18)
H
Scheme 2. General procedure for synthesis of target compounds 16a-f and 18.
12

COOH
NH2
(19)
O
H
N
O
Cl
DMF /K2CO3
stirring / 2h
Cl
R1-NH2
COOH
DCM / stirring / 3h / rt
HN
Cl
O
(22)
HN
NH2
Cl
H
N
O
O
(20)
O
DCM / E3N
ice
DCM / stirring / 3h / rt
O
Cl
bath/stirring
/1h
HN
(23)
Cl
O
O
O
O
NH2
R
H
Cl
CH3
H
N
O
24a
24b
24c
24d
O
R
R
COOC2H5
HN
DCM / stirring / 3h / rt
Cl
O
HN
Cl
(21)
O
(24a-d
)
H
N
O
H2NHN
N
H
DCM / stirring / 3h / rt
HN
(25)
Cl
Scheme 3. General procedure for synthesis of the intermediates 22, 23, 24a-d and 25.
13

O
O
N
O
N
H
Cl
N
N
(22)
H
H
N
S
H
DMF/ KI/ Reflux/ 8h
O
O
N
(26)
O
O
N
H
O
Cl
N
(23)
N
H
H
N
N
S
DMF/ KI/ Reflux/ 8h
H
O
N
(27)
O
O
N
N
SK
R
(9)
O
28
a H
R
O
N
H
28
28
b 4-Cl
c4-CH3
O
N
N
Cl
N
(24a-d
)
28
d4-COOC2H5
H
H
N
DMF/ KI/ Reflux/ 8h
S
H
N
R
O
O
(28a-d
)
H
O
N
O
N
H
O
N
N
Cl
N
(25)
H
H
N
DMF/ KI/ Reflux/ 8h
S
H
N
O
N
(29)
H
O
Scheme 4. General procedure for synthesis of target compounds 26, 27, 28a-d and 29.
14

O
HO
O
1)
Cl
-5oC/Et3N
DCM/stirring1h
O
H
N
COOH
O
N
O
H
Cl
O
Cl
OH
N
N
H
H
O
(20)
(30)
2)
HN NH2
(15e)
O
DMF /KI //6 h
stirring/3 h / room temp.
N
N
SK
(9)
O
N
H
N
N
S
O
H
O
N
N
H
O
(31)
HO
Scheme 5. General procedure for synthesis of target compound 31.
15

O
O
NHR
NHR
O
S
S
DMF/K2CO3
Stirring /2 h
O
O
Cl
O
Cl
Cl
H2N
N
H
(32)
(33a-c
)
O
NHR
S
O
O
Cl
O
O
N
H
(32a-c
)
N
N
H
DMF /KI
N
N
S K
N
S
Reflux / 12 h
O
O
(34a-c
(9)
S
)
NHR
O
R
O
C
34a
34b
34c
N
N
N
Scheme 6. General procedure for synthesis of target compounds 34a-c.
2.2. Biological evaluation
2.2.1. In vitro anti-proliferative activity
The synthesized quinazolin-4(3H)-one derivatives were evaluated for their in vitro anti-
proliferative effects against a panel of three human tumor cell lines namely; hepatocellular
carcinoma (HepG-2), colorectal carcinoma (HCT-116) and breast cancer (MCF-7). In such cell
lines, VEGF has been overexpressed [68-70]. Sorafenib and doxorubicin, two of the most effective
anti-proliferative drugs, were used as standard drugs. The inhibitory effects on cell proliferation
were determined by MTT assay [71-73] and the results were expressed as growth inhibitory
concentration (IC50) values and summarized in Table 1.
From the obtained results, it was obvious that the tested compounds displayed
excellent, marked, moderate or weak anti-proliferative activities against the three
16

tested cell lines with a clear observation that almost all the tested compounds showed better
activities against HepG-2 and HCT-116 than against MCF-7.
In general, compounds 16f, 18, 28b, 34a and 34c were found to be the most potent derivatives.
However, they were found to be mostly more active than the standard and sometimes a little lower
or almost the same. In particular, compound 16f (IC50 = 1.88 ± 0.07, 1.68 ± 0.06 and 3.91 ± 0.15
µM) was found to be the most potent counterpart as it was 4.39, 5.73 and 1.96 times more active
than doxorubicin (IC50 = 8.28 ± 0.33, 9.63 ± 0.38 and 7.67 ± 0.30 µM) and 3.88, 5.59 and 1.84
times more active than sorafenib (IC50 = 7.31 ± 0.29, 9.40 ± 0.37and 7.21 ± 0.28 µM) against
HepG2, HCT-116 and MCF-7 cells, respectively. In addition, compound 28b possessed excellent
anti-proliferative activities against the three cell lines; however, its IC50 values against HepG2,
HCT-116 and MCF-7 were 4.12 ± 0.12, 3.67 ± 0.14 and 5.99 ± 0.23 µM, respectively.
On the other hand, the rest of compounds exhibited mild to moderate activity according to the
type of cancer cell line with IC50 ranging from 8.27 ± 0.33 to 43.96 ± 1.75 µM.
Table 1. Anti-proliferative activities of the tested compounds toward HepG-2, HCT-116 and
MCF-7 cell lines
Comp.
In vitro anti-proliferative IC50 ( µM )a
HepG-2
Adjusted
P Valueb
HCT-116
Adjusted
P Valueb
0.3784
MCF-7
Adjusted
P Valueb
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
12
16a
16b
16c
16d
16e
16f
18
12.33 ± 0.49 <0.0001
8.27 ± 0.33
13.48 ± 0.53
30.49 ± 1.21 <0.0001 28.98 ± 1.15
22.56 ± 0.90 <0.0001 17.06 ± 0.68
33.81 ± 1.35 <0.0001 30.41 ± 1.21
41.55 ± 1.66 <0.0001 37.14 ± 1.48
11.92 ± 0.47 <0.0001 12.37 ± 0.49
<0.0001 35.92 ± 1.43
<0.0001 23.04 ± 0.92
<0.0001 40.86 ± 1.63
<0.0001 43.96 ± 1.75
<0.0001 15.44 ± 0.61
1.88 ± 0.07
8.38 ± 0.33
<0.0001
0.6308
1.68 ± 0.06
8.32 ± 0.33
<0.0001
0.4363
3.91 ± 0.15
9.89 ± 0.39
0.0009
0.0101
26
16.48 ± 0.65 <0.0001 14.08 ± 0.56
17.43 ± 0.69 <0.0001 13.42 ± 0.53
21.76 ± 0.87 <0.0001 16.55 ± 0.66
<0.0001 24.89 ± 0.99
<0.0001 18.31 ± 0.73
<0.0001 26.85 ± 1.07
<0.0001
27
28a
28b
<0.0001
<0.0001
0.6504
4.12 ± 0.12
0.0002
3.67 ± 0.14
<0.0001
5.99 ± 0.23
17

28c
28d
29
19.02 ± 0.76 <0.0001 17.43 ± 0.69
21.51 ± 0.86 <0.0001 17.02 ± 0.68
<0.0001 28.35 ± 1.13
<0.0001 23.74 ± 0.94
>0.9999 16.07 ± 0.64
<0.0001
<0.0001
<0.0001
<0.0001
0.9992
12.14 ± 0.48 <0.0001
11.69 ± 0.46 <0.0001 10.76 ± 0.43
4.19 ± 0.16 0.0003 6.14 ± 0.24
29.98 ± 1.19 <0.0001 24.11 ± 0.96
9.40 ± 0.37
31
0.1773
14.38 ± 0.57
6.79 ± 0.27
34a
34b
34c
<0.0001
<0.0001 30.64 ± 1.22
<0.0001
0.9998
6.76 ± 0.27
0.9912
0.7475
5.24 ± 0.20
9.63 ± 0.38
9.40 ± 0.37
<0.0001
0.9994
7.10 ± 0.28
7.67 ± 0.30
7.21 ± 0.28
Doxorubicin 8.28 ± 0.33
0.9991
Sorafenib
7.31 ± 0.29
a
All experiments were repeated three times. The results were expressed as the mean ±
standard deviation (mean ± SD) calculated from three independent experiments (n = 3). P value <
0.05 was considered significant.
b Adjusted P Value compared to standard sorafenib
2.2.2. In vitro VEGFR-2 enzyme assay inhibition
The most active cytotoxic compounds (12, 16e, 16f, 18, 27, 28a, 28b, 29, 31, 34a and 34c) were
further assessed to determine their inhibitory activities against VEGFR-2 with sorafenib as positive
control. The results were summarized in Table 2. As shown in Table 2, the majority of the tested
compounds displayed superior to moderate enzyme inhibitory activity with IC50 values ranging
from 0.290 ± 0.05 to 0.790 ± 0.11 µg/mL. Among them, five compounds, 16f, 18, 28b, 34a and 34c,
out of eleven potently inhibited VEGFR-2 with IC50 values (0.290 ± 0.05, 0.517 ± 0.07, 0.380 ±
0.04, 0.377 ± 0.04, and 0.415 ± 0.03 µg/mL, respectively) than the reference drug; Sorafinib (0.588
µg/mL).
18

Table 2: IC50 values of the tested compounds against VEGFR-2
VEGFR-2 inhibition IC50
Comp.
(µg/mL)a
12
18
0.790 ± 0.11
0.517 ± 0.07
0.610 ± 0.09
0.290 ± 0.05
0.751 ± 0.10
0.713 ± 0.07
0.380 ± 0.04
0.648 ± 0.06
0.619 ± 0.05
0.377 ± 0.04
0.415 ± 0.03
0.588 ± 0.04
16e
16f
27
28a
28b
29
31
34a
34c
Sorafenib
a All experiments were repeated three times. The results were expressed as the mean ± stander
deviation (mean ± SD) calculated from three independent experiments (n = 3). P value < 0.05 was
considered significant. IC50 values are the mean ± S.D. of three separate experiments.
2.2.3. Structure-Activity Relationship (SAR)
Investigation of results of the different biological tests (in vitro anti-proliferative activity and
in vitro VEGFR-2 enzyme assay inhibition) revealed a range of potent VEGFR-2 inhibition activity.
Five series of new quinazolines differentiated by incorporating a diverse of donor/acceptor groups
such as a hydrazone, sulfonamide, acetamidobenzamide, 2-hydroxybenzolhydrazine, and
thiosemicarbazone moieties have been designed using structure-activity relationships.
Generally, our results showed that some derivatives have high activity on either cell lines or
VEGFR-2 compared to the lead compound Sorafenib. Among these derivatives, it was noticed that
introduction of a hydrazone group (16f) led to an increase in VEGFR-2 enzymatic activity (IC50 =
0.290 µg/mL) and anti-proliferative effect (IC50 = 1.88, 1.68, and 3.91 µM against HepG-2, HCT-
116 and MCF-7 cell lines, respectively) Also, replacement of the urea entity of sorafenib by
acetamidobenzamide (28b) presented a higher VEGFR-2 inhibitory effect and an increased anti-
tumor activity (IC50 = 0.380 µg/mL against VEGFR-2 and 4.12, 3.67, and 5.99 µM against HepG-2,
HCT-116 and MCF-7 cell lines, respectively). Moreover, and according to our investigations, the
19

insertion of sulfonamide (34a and 34c) instead of urea moiety increased enzymatic activity and
appeared to be a new opportunity to develop VEGFR-2 inhibitors. On the other hand, when the
sorafenib urea was replaced by thiosemicarbazone motif (18) it afforded a compound with almost
equal activity compared to sorafenib (IC50 = 0.517 µg/mL against VEGFR-2 and 8.38, 8.32, and
9.89 µM against HepG-2, HCT-116 and MCF-7 cell lines, respectively). Unfortunately, neither the
enzymatic inhibition nor the anti-tumor activities of the newly synthesized compound with
hydroxybenzoyl hydrazine moiety (31) were enhanced (IC50 = 0.619 µg/mL against VEGFR-2 and
11.69, 10.76, and 14.38 µM against HepG-2, HCT-116 and MCF-7 cell lines, respectively).
Regarding hydrazone containing compounds, the most active inhibitor was 16f bearing a 2-
chloro-5-nitrophenyl group as a hydrophobic tail (IC50 = 0.290 µg/mL against VEGFR-2 and 1.88,
1.68, and 3.91 µM against HepG-2, HCT-116 and MCF-7 cell lines, respectively). However,
insertion of 2-hydroxyphenyl moiety as a hydrophobic tail produced compound 16e showed a
decreased anti-proliferative effect (IC50 = 11.92, 12.37, and 15.44 µM against HepG-2, HCT-116
and MCF-7 cell lines, respectively). These results indicate that grafting a large electron
withdrawing substituents at the terminal hydrophobic tail is beneficial for the activity.
Comparing the anticancer activities of acetamido benzamide derivatives of 2-mercapto-3-(4-
methylphenyl)quinazolin-4(3H)-one showed that the activity of 4-chloro 28b (IC50 = 0.380 µg/mL
against VEGFR-2 and 4.12, 3.67, and 5.99 µM against HepG-2, HCT-116 and MCF-7 cell lines,
respectively) is superior than that expressed by sorafenib and better than 4-methyl 28c (IC50 = 19.02,
17.43, and 28.35 µM against HepG-2, HCT-116 and MCF-7 cell lines, respectively), and ester 28d
(IC50 = 21.51, 17.02, and 23.74 µM against HepG-2, HCT-116 and MCF-7 cell lines, respectively),
indicating that grafting electron withdrawing substituents at positions 4 is beneficial for activity.
For N-substituted benzene sulfonamide derivatives, it was found that compound
incorporating N-acetyl derivative 34a (IC50 = 0.377 µg/mL against VEGFR-2 and 4.19, 6.14, and
6.79 µM against HepG-2, HCT-116 and MCF-7 cell lines, respectively) was more preferred
biologically than that those having aromatic heterocyclic rings (34b and 34c).
2.3. Molecular docking
The target compounds were docked into the active site of VEGFR-2 (PDB ID: 2OH4) to
understand their binding mode. The reason we chose 2OH4 as receptor is that 2OH4 complex
contains a urea containing ligand. Molecular insights based on molecular docking indicated
favorable binding interactions of the newly synthesized compounds with the active site of VEGFR-
2. 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.
20

1N–H of the urea unit of sorafenib formed a hydrogen bond with Glu883 with distance of 1.68
3
Å. N–H could also bind to the side chain carboxylate of Glu883 with distance of 1.59 Å.
Moreover, carbonyl group was identified to be involved in a hydrogen bond interaction with the
backbone NH of Asp1044 with distance of 1.74 Å. Besides, the phenyl group (spacer) between urea
and oxygen 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 [74] Fig. 5.
Fig. 5. The Predicted binding mode for sorafenib with the active site of VEGFR-2
Results of the docking study indicated that most of the target compounds showed a similar
position and orientation inside the putative binding sites of VEGFR-2. The calculated ∆G (binding
free energies) of the synthesized compounds and the reference drug against VEGFR-2 were
summarized in Table 3.
The binding mode of compound 16f (affinity value of -58.02 kcal/mol), revealed that the N-H
group of the hydrazono moiety formed hydrogen bond with Glu883 in the active site (2.45Å) while
oxygen atom of the carbonyl group formed one hydrogen bond with Asp1044 with a distance of
2.52 Å. Additionally, the oxygen atom of carbonyl group of the quinazoline nucleus formed
hydrogen bond with Asn921 (2.10 Å). Additionally, this compound formed three arene-arene
interactions, one of which occurred between the central phenyl moiety (spacer) and Lys866, the
other two cationic pi bonds were formed between quinazoline moiety and Arg1049 Fig. 6 (A). In
order to compare their spatial orientations, superimposition of Sorafenib and 16f was performed and
illustrated in Fig. 6 (B).
21

Fig. 6. (A). Predicted binding mode of compound 16f with the active site of VEGFR-
2. (B). Superimposing Sorafenib and 16f (Sorafenib in green color, 16f in maroon
color).
Investigation of the top docking poses of compound 28b (affinity value of -51.47 kcal/mol),
showed three hydrogen bond interactions in the active site of VEGFR-2. The oxygen atom of
carbonyl group of the spacer formed hydrogen bond with Asp1044(1.97Å), while the N-H of the
amide (spacer) formed two hydrogen bonds of 2.18, 2.47 Å with Glu883. This compound formed
22

tow arene-arene interaction, one of them is a cationic pi interaction with Phe916 and the other one
formed between the phenyl of spacer and (Lys866) Fig. 7.
Fig. 7. Predicted binding mode of compound 28b with the active site of VEGFR-2.
The binding mode of compound 34a (affinity value of -51.79 kcal/mol), revealed that the N-
H groups of the spacer moiety formed two hydrogen bonds with Glu883 in the active site (2.16 Å,
2.27 Å) while oxygen atom of the carbonyl group formed one hydrogen bond with Asp1044 with a
distance of 2.31 Å. This compound formed one cationic pi bond with (Lys866) Fig. 8.
23

Fig. 8. Predicted binding mode of compound 34a 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.
12
∆G [Kcal/mol]
- 36.81
- 39.00
- 35.26
- 36.09
- 36.35
-43.28
Comp.
28a
∆G [Kcal/mol]
-34.42
16a
16b
16c
16d
16e
16f
18
28b
- 51.47
-39.00
28c
28d
-42.18
29
- 28.81
-26.27
31
-58.02
34a
-51.79
-47.72
34b
-32.48
26
-36.51
34c
-51.13
27
-40.42
Sorafenib
-52.20
3. Conclusion
In summary, we have designed and synthesized nineteen final novel quinazoline-based
derivatives incorporating, sulfonamide, hydrazono, acetamidobenzamide, 2-hydroxybenzoyl
hydrazine and thiosemicarbazone moieties. The newly synthesized compounds were evaluated for
their anti-proliferative activities against panel of three human cancer cell lines namely;
hepatocellular carcinoma (HepG2), breast cancer (MCF-7) and colorectal carcinoma (HCT-116)
using MTT assay. Compound 16f has emerged as the most active member with IC50 values of 1.88
± 0.07, 1.68 ± 0.06, 3.91 ± 0.15 µM as it was 4.39, 5.73 and 1.96 times more active than
doxorubicin (IC50 = 8.28 ± 0.33, 9.63 ± 0.38 and 7.67 ± 0.30 µM) and 3.88, 5.59 and 1.84 times
more active than sorafenib (IC50 = 7.31 ± 0.29, 9.40 ± 0.37and 7.21 ± 0.28 µM) against HepG2,
HCT-116 and MCF-7 cells, respectively. Also, compounds 18, 28b, 34a and 34c displayed excellent
anti-proliferative activities against HepG2 and HCT-116 cell lines. In addition, the most potent
compounds were examined for their VEGFR-2 inhibitory activities. Interestingly, the results of
VEGFR-2 inhibition were consistent with that of the cytotoxicity data. Most of tested compounds
24

displayed superior to moderate inhibitory activity compared to the reference drug, sorafenib.
Docking studies supported the previous results via prediction of the possible binding interactions of
the designed compounds with VEGFR-2 active sites. The most active candidates may serve as
useful lead compounds in the search for powerful and selective antineoplastic agents and deserve
further investigations.
4. Experimental
4.1. Chemistry
Melting points were determined with a Gallen lamp melting point apparatus and are
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
1
was confirmed by IR (pye Unicam SP 1000 IR spectrophotometer), H NMR (400 MHz, Bruker
Biospin GmbH spectrophotometer) and 13C NMR spectra (100 MHz, Bruker Biospin GmbH
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.).
Compounds 2-Mercapto-3-(4-methylphenyl)quinazolin-4(3H)-one
8
[75, 76], N-(4-
Acetylphenyl)-2-chloroacetamide 11 [56], ethyl (un)substitutedbenzoate 14a-f [58], (un)substituted-
benzoic acid hydrazides 15a-f [59, 60], 4-(2-chloroacetamido)benzoic acid 20 [62], 4-(2-
chloroacetamido)-N-(substituted)benzamide 22-25 [63, 64, 77], N-(4-(2-Benzoylhydrazine-1-
carbonyl)phenyl)-2-chloroacetamide 30 [63] and 2-chloro-N-(4-(N-(substituted) sulfamoyl)
phenyl)acetamide 33a-c [66, 67] were obtained according to the reported procedures.
4.1.1. Potassium salts of 2-mercapto-3-(4methylphenyl) quinazolin-4(3H)-one (9)
To a mixture of 2-mercapto-3-(4-methylphenyl) quinazolin-4(3H)-one 8 (0.05 mol) potassium
hydroxide (2.8 g, 0.05 mol) was added. The mixture was refluxed in absolute ethanol (50 ml) for
1.5 h. Upon cooling to room temperature, precipitated product was obtained, collected washed with
diethyl ether (30 mL) and dried. Yield 92%; mp > 350 C.
4.1.2. N-(4-acetylphenyl)-2-((4-oxo-3-(4-methylphenyl)-3,4-dihydroquinazolin-2-yl)thio)aceta-
mide (12)
A solution of the potassium salt 9 (0.01 mol) and N-(4-acetylphenyl)-2-chloroacetamide 11
(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 12.
25

o
Buff crystals (yield 85 %); m.p.: 168-170 C; IR (KBr, ν cm−1): 3234 (NH), 1670, 1685
1
(3C=O).; H NMR (400 MHz, DMSO-d6) δ ppm; 2.42 (s, 3H, CH3-C6H5), 2.55 (s, 3H, COCH3),
4.11 (s, 2H, S-CH2), 7.37-7.42 (m, 4H, Ar-H), 7.44 (t, 1H, Ar-H), 7.50 (d, 1H, J = 8.8 Hz, Ar-H),
7.74 (d, 2H, J = 7.6 Ar-H), 7.80 (t, 1H, Ar-H), 7.92 (d, 2H, J = 7.6 Hz, Ar-H), 8.06 (d, 1H, J = 8.8
Hz, Ar-H), 10.74 (s, 1H, NH, D2O exchangeable).; MS (m/z): 443.17 (M+, 17.95 %), 309.14 (100
%),; Anal. Calcd. For C25H21N3O3S (443.13): C, 67.70; H, 4.77; N, 9.47; Found: C, 67.98; H, 4.83;
N, 9.68%.
4.1.3. General procedure for synthesis of compounds (16a-f)
A solution of N-(4-acetylphenyl)-2-((4-oxo-3-(4-methylphenyl)-3,4-dihydroquinazolin-2-
yl)thio)acetamide 12 (0.001 mol) was treated with the appropriate prepared benzohydrazide
derivatives namely, benzohydrazide, 2-chlorobenzohydrazide, 3-chlorobenzohydrazide, 4-
chlorobenzohydrazide, 2-hydroxybenzohydrazide and 2-chloro-5-nitrobenzohydrazide 15a-f (0.001
mol) in ethanol (50 ml) in the presence of catalytic amount of glacial acetic acid and refluxed for 8
h. The resulting solids were filtered, washed with water (15 mL), dried and recrystallized from
ethanol to afford the corresponding target compounds 16a-f respectively.
4.1.3.1.
N-(4-(1-(2-Benzoylhydrazono)ethyl)phenyl)-2-((4-oxo-3-(4-methylphenyl)-3,4-dihyd-
roquinazolin-2-yl)thio)acetamide 16a
Pale yellow crystals (yield 83 %); m.p.: 188-190 oC; IR (KBr) cm-1: 3421, 3310 (2NH), 1716,
1658 (3C=O).; 1H NMR (DMSO.d6,  ppm): 2.34 (s, 3H, CH3–C6H5), 2.42 (s, 3H, CH3-C=N), 4.10
(s, 2H, -SCH2), 7.35 (d, 2H, J = 6.4 Hz, Ar-H), 7.39 (d, 2H, J = 6.4 Hz, Ar-H), 7.47 (t, 1H, J = 6.0
Hz, Ar-H), 7.49-7.58 (m, 4H, Ar-H), 7.68 (d, 2H, J = 6.8 Ar-H), 7.80 -7.85 (m, 3H, Ar-H), 7.89 (d,
2H, J = 6.8 Hz, Ar-H), 8.07 (d, 1H, J = 6.4 Hz, Ar-H), 10.55 (s, 1H, CONH; D2O exchangeable),
10.74 (s, 1H, NHCO; D2O exchangeable); 13C NMR: 19.03, 21.35, 37.83, 56.51, 119.07 (2C),
119.15, 120 (2C), 126.35, 126.46, 127.08, 127.40, 127.63, 128.30, 128.79, 129.59 (2C), 130.52
(2C), 131.90, 133.41, 134.58, 135.38 (2C), 140.19, 140.62, 147.59, 155.75, 157.69, 161.13, 166.47;
DEPT135 NMR: 19.03 (2CH3), 21.35 (2CH3), 37.83 (2CH2), 119.07 (2CH), 119.15, 126.35,
126.46, 127.09, 127.39, 127.63, 128.29, 128.79, 129.59 (2CH), 130.52 (2CH), 131.92, 135.38
(2CH); Anal. Calcd. for C32H27N5O3S (561.66): C, 68.43; H, 4.85; N, 12.47, Found: C, 68.61; H,
4.97; N,12.73 %.
4.1.3.2. N-(4-(1-(2-(2-Chlorobenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-(4-methylphenyl)-
3,4-dihydroquinazolin-2-yl)thio)acetamide 16b
White crystals (yield 65 %); m.p.: 217-220 oC; IR (KBr) cm-1: 3308, 3259 (2NH), 1712, 1701,
1
1656 (3C=O); H NMR (DMSO.d6,  ppm): 2.26, 2.29 (2s, 3H, CH3 –C6H5), 2.41, 2.42 (2s, 3H,
26

CH3-C=N) 4.07, 4.12 (2s, 2H, S-CH2), 7.35-7.48 (m, 6H, Ar-H), 7.50-7.53 (m, 4H, Ar-H), 7.54
(dd, 1H, J = 6 Hz, Ar-H), 7.69 (d, 1H, J = 8.8 Hz, Ar-H), 7.77 (s, 1H, Ar-H), 7.85 (dd, 2H, J = 8.8,
8.0 Hz, Ar-H), 8.06 (dt, 1H, J = 6.4 Hz, Ar-H), 10.44, 10.56 (2s, 1H, CONH; D2O exchangeable),
13
10.96, 11.20 (2s, 1H, NHCO, D2O exchangeable).; C NMR: 14.88, 19.04, 21.34, 37.83, 56.53,
119.01 (2C), 120.01, 126.35, 126.46, 127.08, 127.73 (2C), 129.59 (2C), 130.53 (2C), 132.99,
133.63, 135.37, 140.19 (2C), 140.39, 147.57, 147.60, 157.65, 157.69, 161.13, 161.15, 166.38,
166.50, 179.99; DEPT135 NMR: 14.01 (1CH3), 14.889 (1CH3), 19.04 (1CH3), 21.35 (1CH3), 37.82
(1CH2), 56.53 (1CH2), 118.99, 119.08 (2CH), 126.34, 126.46, 126.93, 127.08, 127.25, 127.56,
127.67 (2CH), 129.01, 129.26, 129.58 (2CH), 130.02, 130.52 (2CH), 130.70, 131.53, 135.37; Anal.
Calcd. for C32H26ClN5O3S (596.10): C, 64.48; H, 4.40; N, 11.75 Found: C, 64.60; H, 4.37; N,
12.01%.
4.1.3.3. N-(4-(1-(2-(3-Chlorobenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-(4-methylphenyl)-3,4-
dihydroquinazolin-2-yl)thio)acetamide 16c
Beige crystals (yield 58 %); m.p.: 221-223 oC; IR (KBr) cm-1 :3337, 3266 (2 NH), 1718, 1685
1
(3C═O).; H NMR (DMSO.d6,  ppm): 2.34 (s, 3H, CH3-C6H5), 2.43 (s, 3H, CH3-C=N) 4.10 (s,
2H, S-CH2), 7.34 (d, 2H, J = 8.4, 8.0 Hz, Ar-H), 7.39 (d, 2H, J = 8.4 Hz, Ar-H), 7.44 (t, 1H, J = 8.0,
8.4 Hz, Ar-H), 7.53 (d, 2H, J = 8.4 Hz, Ar-H), 7.64 (d, 2H, J = 9.2 Hz, Ar-H), 7.68 (d, 1H, Ar-H),
7.79-7.84 (m, 4H, Ar-H), 7.92 (s, 1H, Ar-H), 8.08 (d, 1H, J = 9.2 Hz, Ar-H), 10.53 (s, 1H, CONH;
D2O exchangeable), 10.83 (s, 1H, NHCO, D2O exchangeable).; 13C NMR; 21.34, 37.80, 119.1 (4C),
120.00, 126.34, 126.47 (2C), 127.09 (2C), 127.71, 129.59 (4C), 130.51 (4C), 130.79, 133.29,
133.62 (2C), 135.38, 140.18 (2C), 147.59, 157.68, 161.13, 166.46; DEPT135 NMR; 14.99, 21.34,
37.80, 119.09 (2CH), 126.34, 126.47, 127.08 (2CH), 127.69, 129.58 (2CH), 130.51 (2CH), 135.38
Anal. Calcd. for C32H26ClN5O3S (596.10): C, 64.48; H, 4.40; N, 11.75; Found: C, 64.67; H, 4.53;
N, 11.88 %.
4.1.3.4. N-(4-(1-(2-(4-Chlorobenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-(4-methylphenyl)-3,4-
dihydroquinazolin-2-yl)thio)acetamide 16d
o
White crystals (yield 79 %); m.p.: 247 C; IR (KBr) cm-1: 3336, 3256 (2 NH), 1751, 1715,
1651 (3C=O).; 1H NMR (DMSO.d6,  ppm): 2.33 (s, 3H, CH3-C6H5), 2.43 (s, 3H, CH3-C=N) 4.11
(s, 2H, S-CH2), 7.35 (dd, 2H, J = 8.4, 8.0 Hz, Ar-H), 7.39 (dd, 2H, J = 8.4, 8.0 Hz, Ar-H), 7.44 (t,
1H, J = 7.6 Hz, Ar-H), 7.54 (d, 2H, J = 8.0 Hz, Ar-H), 7.59 (d, 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, Ar-H), 8.06 (d, 1H, J = 8.0 Hz,
Ar-H), 10.56 (s, 1H, CONH; D2O exchangeable), 10.81 (s, 1H, NHCO; D2O exchangeable).;13C
NMR; 21.34, 37.84, 119.12 (2C), 120.01 (2C), 126.35, 126.45 (2C), 127.08 (2C), 127.64, 128.81
27

(2C), 129.59 (2C), 130.25, 130.51 (2C), 133.34 (2C), 133.63 (2C), 135.36 (2C), 140.18 (2C),
147.60, 157.69, 161.13, 166.45; DEPT135 NMR; 14.90 (1CH3), 21.35 (1CH3), 37.84 (1CH2),
119.11 (2CH), 126.35, 126.46 (2CH), 127.09 (2CH), 127.64, 128.81, 129.59 (2CH), 130.25, 130.51
(2CH), 135.37 (2CH). Anal. Calcd. For: C32H26ClN5O3S (596.10): C, 64.48; H, 4.40; N, 11.75;
Found: C, 64.65; H, 4.51; N, 11.92 %.
4.1.3.5.
N-(4-(1-(2-(2-Hydroxybenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-(4-methylphenyl)-
3,4-dihydroquinazolin-2-yl)thio)acetamide 16e
o
Pale yellow crystals (yield 75 %); m.p.: 203-206 C; IR (KBr) cm-1: 3628 (OH), 3447, 3276
1
(2NH), 1716, 1686, (3C=O).; H NMR (DMSO.d6,  ppm): 2.31 (s, 3H, CH3 –C6H5), 2.42 (s, 3H,
CH3-C=N), 4.11 (s, 2H, S-CH2), 7.37 (d, 2H, J = 8.4 Hz, Ar-H), 7.39 (d, 2H, J = 8.4 Hz, Ar-H),
7.46 (t, 1H, J = 7.6 Hz, Ar-H), 7.54 (d, 1H, J = 8.0 Hz, Ar-H), 7.59 (d, 2H, J = 8.4 Hz, Ar-H), 7.67
(d, 2H, J = 8.4 Hz, Ar-H), 7.79 (d, 2H, J = 8.4 Hz, Ar-H), 7.84 (t, 1H, Ar-H), 7.92 (d, 2H, J = 8.4
Hz, Ar-H), 8.06 (dd, 1H, J = 8.0 Hz, Ar-H), 10.54 (s, 1H, CONH; D2O exchangeable), 11.33 (s,1H,
NHCO; D2O exchangeable), 11.84 (s, 1H, OH; D2O exchangeable); 13C NMR; 14.14, 21.34, 37.85,
117.37, 118.34, 119.15 (2C), 120.01(2C), 126.35, 126.44, 127.08, 127.60 (2C), 129.59 (2C),
130.51(2C), 130.96, 133.26, 133.63, 133.76, 135.35, 140.18, 140.55, 147.60, 152.55, 157.14,
157.68, 161.13, 162.56, 166.46; DEPT135 NMR: 14.14 (1CH3), 21.34 (1CH3), 37.85, 117.37,
119.14 (2CH), 120.05, 126.35, 126.44, 127.08, 127.60 (2CH), 129.59 (2CH), 130.51 (2CH),
130.96, 133.75, 135.35; Mass (m/z): 577.53 (M+, 11.01 %), 526.21 (100 %).; Anal. Calcd. for
C32H27N5O4S (577.66): C, 66.54; H, 4.71; N, 12.12; Found: C, 66.41; H, 4.89; N, 12.34%.
4.1.3.6.
N-(4-(1-(2-(2-Chloro-5-nitrobenzoyl)hydrazono)ethyl)phenyl)-2-((4-oxo-3-(4-
methylphenyl)-3,4-dihydroquinazolin-2-yl)thio)acetamide 16f
o
Orange crystals (yield 87 %); m.p.: 185-187 C; IR (KBr) cm-1: 3481 (NH), 1725, 1672
1
(3C═O).; H NMR (DMSO.d6,  ppm): 2.31 (s, 3H, CH3 –C6H5), 2.43 (s, 3H, CH3–C=N), 4.10 (s,
2H, S-CH2), 7.34 (d, 2H, J = 8.0 Hz, Ar-H), 7.39 (d, 2H, J = 8.0 Hz, Ar-H), 7.44 (t, 1H, J = 6.8, 8.0
Hz, Ar-H), 7.53 (d, 1H, J = 8.8 Hz, Ar-H), 7.66 (d, 2H, J = 8.0 Hz, Ar-H), 7.81 (t, 1H, J = 7.6, 8.8
Hz, Ar-H), 7.89 (d, 2H, J = 8.4 Hz, Ar-H), 8.03 (d, 1H, J = 8.0 Hz, Ar-H), 8.05 (d, 1H, J = 8.0 Hz,
Ar-H), 8.28 (s, 1H, OH; D2O exchangeable), 10.56 (s, 1H, CONH; D2O exchangeable), 10.83 (s,
1H, NHCO, D2O exchangeable).; Mass (m/z) : 641.12 (M+, 35.65 %), 367.70 (100 %).; Anal. Calcd.
for C32H25CLN6O5S (641.10): C, 59.95; H, 3.93; N, 13.11; Found: C, 60.13; H, 4.07; N, 13.37 %.
4.1.4.
N-(4-(1-(2-Carbamothioylhydrazono)ethyl)phenyl)-2-((4-oxo-3-(4-methylphenyl)-3,4-
dihydroquinazolin-2-yl)thio)acetamide 18
28

A solution of N-(4-acetylphenyl)-2-((4-oxo-3-(4-methylphenyl)-3,4-dihydroquinazolin-2-
yl)thio)acetamide 12 (0.001 mol) in ethanol (50 mL) was treated with thiosemicarbazide (0.001
mol) in the presence of catalytic amount of glacial acetic acid. The solution was refluxed for 8 h.
Resulting solid was filtered, washed with water (15 mL), dried and crystallized from ethanol.
Pale green solid (yield 71 %); m.p.: 221-223oC; IR (KBr) cm-1: 3421 (NH2), 3340, 3295 (2
1
NH), 1734, 1675 (2C═O).; H NMR (DMSO.d6,  ppm): 2.26 (s, 3H, CH3 –C6H5), 2.43 (s, 3H,
CH3-C=N), 4.09 (s, 2H, S-CH2), 7.34 (d, 2H, J = 8.0, Ar-H), 7.39 (d, 2H, J = 8.4 Hz, Ar-H), 7.46 (t,
1H, J = 8.0 Hz, Ar-H), 7.53 (d, 1H, J = 8.8 Hz, 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), 7.91 (s, 2H, NHCSNH2, D2O exchangeable),
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, NHCSNH2, D2O exchangeable); 13C NMR; 19.03, 21.34, 37.88,
119.04 (2C), 119.99, 126.31, 126.44, 127.08, 127.72 (2C), 129.57 (2C), 130.51 (2C), 132.98,
133.60, 135.33, 140.18, 140.38, 147.58, 147.91, 157.67, 161.13, 166.41, 179.22; DEPT135 NMR;
19.03 (1CH3), 21.34 (1CH3), 37.88 (CH2), 119.01 (2CH), 126.32, 126.44, 127.08, 127.72(2CH),
129.57 (2CH), 130.52 (2CH), 135.33; Mass (m/z): 516.86 (M+, 36.41 %), 266.96 (100 %).; Anal.
Calcd. for C26H24N6O2S2 (516.64); C, 60.45; H, 4.68; N, 16.27; Found: C, 60.71; H, 4.86; N, 16.49
%.
4.1.5. General procedure for preparation of target compounds 26-29
A mixture of the potassium salt of 2-mercapto-3-(4-methylphenyl)quinazolin-4(3H)-one 9
(0.01mol) and the appropriate 4-(2-chloroacetamido)-N-(substituted)benzamide derivatives 22, 23,
24a-d or 25 (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 26, 27, 28a-
d and 29 respectively.
4.1.5.1.
N-Cyclohexyl-4-(2-((4-oxo-3-(4-methylphenyl)-3,4-dihydroquinazolin-2-yl)thio)
acetamido)benzamide 26
o
White crystals (yield 63 %); m.p.: 215-217 C; IR (KBr) cm-1: 3321, 3179 (2NH), 1681
1
(C=O).; H 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), 2.48 (s, 3H, CH3 –C6H5), 3.73 (m, 1H, CH- cyclohexyl), 4.09 (s, 2H, S-CH2), 7.36-
7.52 (m, 6H, Ar-H), 7.66 (d, 2H, Ar-H), 7.67 (d, 1H, J = 8.4 Hz, Ar-H), 7.79 (d, 2H, Ar-H), 7.74 (s,
1H, S-CH2CONHcyclohexyl; D2O exchangeable), 8.07 (d, 1H, J = 8.0 Hz ,Ar-H), 10.57 (s, 1H,
29