Design, synthesis, molecular modeling, in vivo studies and anticancer evaluation of quinazolin-4(3H)-one derivatives as potential VEGFR-2 inhibitors and apoptosis inducers

Article abstract of DOI:10.1016/j.bioorg.2019.103422

Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer. Accordingly, new quinazoline-based derivatives having the structural features of VEGFR-2 inhibitors were designed and synthesized. Anti-proliferative activities 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. Compounds 26_b, 29_a, 29_b and 30 showed excellent anti-cancer activities against all cell lines. Moreover, compound 31 was the most active with IC ₅₀ values of 3.97 ± 0.2, 4.83 ± 0.2 and 4.58 ± 0.3 μM, respectively. The most active cytotoxic agents were further evaluated in vitro for their VEGFR-2 inhibitory activities, compound 31 showed a high activity against VEGFR-2 with an IC₅₀ value of 2.5 ± 0.04 μM, almost equal to that of sorafenib (IC₅₀ = 2.4 ± 0.05 μM). Further studies revealed the ability of this promising quinazoline derivative 31 to induce apoptosis and arrest cell cycle growth at G2/M phase. In vivo antitumor activities of the synthesized compounds revealed that compounds 30 and 31 possessed significant tumor growth inhibition effect. Molecular docking studies were also performed and finally we can say that VEGFR-2 inhibition confers the reported cytotoxic activities.

Full text of DOI:10.1016/j.bioorg.2019.103422

Journal Pre-proofs
Design, synthesis, molecular modeling, in vivo studies and anticancer evalua-
tion of quinazolin-4(3H)-one derivatives as potential VEGFR-2 inhibitors and
apoptosis inducers
Hazem A. Mahdy, Mohammed K. Ibrahim, Ahmed M. Metwaly, Amany
Belal, Ahmed B.M. Mehany, Abdou El-Sharkawy, Mostafa A. Elhendawy,
Mohamed M. Radwan, Mahmoud A. Elsohly, Ibrahim H. Eissa
PII:
S0045-2068(19)31098-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.103422
YBIOO 103422
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
10 July 2019
3 October 2019
3 November 2019
Please cite this article as: H.A. Mahdy, M.K. Ibrahim, A.M. Metwaly, A. Belal, A.B.M. Mehany, A. El-
Sharkawy, M.A. Elhendawy, M.M. Radwan, M.A. Elsohly, I.H. Eissa, Design, synthesis, molecular modeling, in
vivo studies and anticancer evaluation of quinazolin-4(3H)-one derivatives as potential VEGFR-2 inhibitors and
apoptosis inducers, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103422
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Design, synthesis, molecular modeling, in vivo studies and anticancer evaluation
of quinazolin-4(3H)-one derivatives as potential VEGFR-2 inhibitors and
apoptosis inducers
a
a
b
c,d
Hazem A. Mahdy Mohammed K. Ibrahim , Ahmed M. Metwaly , Amany Belal ,
e
f,g
h,i
Ahmed B. M. Mehany , Abdou El-Sharkawy* , Mostafa A. Elhendawy , Mohamed
M. Radwan and Mahmoud A. Elsohly* , Ibrahim H. Eissa*a
h,j
h,k
a
Pharmaceutical Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar
University, Cairo11884, Egypt.
b
Pharmacognosy Department, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo 11884, Egypt.
c
Medicinal Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni-
Suef 62514, Egypt.
d
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Taif University, Taif
2
1974, Saudi Arabia.
e
Zoology Department, Faculty of Science, Al-Azhar University, Cairo 11884, Egypt.
f
Department of Anatomy, Faculty of Medicine, Al-Azhar University, Cairo, Egypt.
Department of Anatomy, College of Medicine, Jouf University, Saudi Arabia
National Center for Natural Products Research, University of Mississippi, MS
g
h
3
8677, USA.
i
Department of Agriculture Chemistry, Damietta University, Damietta, Egypt
j
Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University,
Alexandria, Egypt.
k
Department of Pharmaceutics and Drug Delivery, University of Mississippi,
University, MS 38677, USA.
*
Corresponding authors:
Ibrahim H. Eissa
Pharmaceutical Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar
University, Cairo 11884, Egypt.
Email: Ibrahimeissa@azhar.edu.eg
1

M. A. ElSohly
National Center for Natural Products Research, University of Mississippi, University,
MS 38677, USA.
Email: melsohly@olemiss.edu
Abdou El-Sharkawy
Department of Anatomy, Faculty of Medicine, Al-Azhar University, Cairo, Egypt.
Email: abdouelsharkawy@gmail.com
2

Abstract:
Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of
cancer. Accordingly, new quinazoline-based derivatives having the structural features
of VEGFR-2 inhibitors were designed and synthesized. Anti-proliferative activities
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.
Compounds 26 , 29 , 29 and 30 showed excellent anti-cancer activities against all
b
a
b
cell lines. Moreover, compound 31 was the most active with IC 50 values of 3.97 ±
.2, 4.83 ± 0.2 and 4.58 ± 0.3 µM, respectively. The most active cytotoxic agents
0
were further evaluated in vitro for their VEGFR-2 inhibitory activities, compound 31
showed a high activity against VEGFR-2 with an IC value of 2.5 ± 0.04 µM, almost
5
0
equal to that of sorafenib (IC = 2.4 ± 0.05µM). Further studies revealed the ability
5
0
of this promising quinazoline derivative 31 to induce apoptosis and arrest cell cycle
growth at G2/M phase. In vivo antitumor activities of the synthesized compounds
revealed that compounds 30 and 31 possessed significant tumor growth inhibition
effect. Molecular docking studies were also performed and finally we can say that
VEGFR-2 inhibition confers the reported cytotoxic activities.
Key words: Anticancer; Apoptosis; Molecular docking; quinazolin-4(3H)-one;
VEGFR-2; in vivo studies.
3

1
. Introduction
Cancer is a major cause of mortality all over the world, it approximately
causes 13 % of all deaths, annually [1]. According to WHO fact sheet published on
September 2018, there are 9.6 million deaths occurred worldwide. Globally, about 1
in 6 deaths is due to cancer [2]. The most common causes of cancer death are cancers
of lung (1.76 million deaths), colorectal (862 000 deaths), stomach (783 000 deaths),
liver (782 000 deaths), and breast (627 000 deaths) [2]. It was predicted that global
cancer incidence would reach 22 million cases per year by 2030 [3]. The acquired
chemotherapeutic resistance of different cancer types was increased noticeably in the
past few years. [4]. In addition, the poor selectivity of chemotherapeutics causes
cytotoxicity for dividing cells leading to serious side effects, such as
immunosuppression, anemia, diarrhea, nausea, and alopecia [5]. These factors
necessitate the discovery of new more active, and more selective anticancer drugs.
Angiogenesis, the formation of new blood vessels from pre-existing ones, is a
normal physiological process responsible for most, if not all, blood vessel formation
in growth and development as well as in inflammation and wound healing [6].
Angiogenesis is a key step in the conversion of a benign tumor to a malignant one,
where new blood vessels infiltrate tumor masses supplying them with oxygen and
nutrients to enhance tumor growth and metastasis [7-9]. Hence, blocking angiogenesis
could be a successful strategy to hinder tumor growth.
Vascular endothelial growth factor (VEGF) is a pivotal contributor to
angiogenesis, increasing blood vessels number in a given network, stimulating both
physiological and pathological angiogenesis [9]. Cancer cells which cannot express
VEGF are not capable of growing nor metastasizing [10]. Overexpression of VEGF
have been noticed in several types of cancer including colorectal cancer [11], breast
cancer [12] and hepatocellular carcinoma [13]
VEGF ligands bind in an overlapping pattern to three different, but structurally
related VEGF-receptor tyrosine kinases(VEGFR-TK) including VEGFR-1, VEGFR-2
and VEGFR-3 [14]. Both VEGFR-1 and VEGFR-2 promote angiogenesis, while
VEGFR-3 stimulation leads to lymphangiogenesis [15]. While VEGFR-1 was found
to modulate VEGFR-2 effect, VEGFR-2 has proven to mediate almost all of the
known VEGF cellular responses [16].
4

VEGFR-2 is a tyrosine kinase receptor expressed in endothelial cells. Binding
of VEGF to VEGFR induces a conformational change in VEGFR, followed by
receptor dimerization and phosphorylation of tyrosine residues. VEGF signaling
through VEGFR-2 has been shown to play a major role in the regulation of tumor
angiogenesis [17, 18]. Therefore, VEGF/VEGFR-2 signaling is an attractive
therapeutic target in the treatment of cancer [19]. Hence, blocking of VEGFR-2 or
down-regulation of its signaling is a main approach for the discovery of new drugs
for many human angiogenesis-dependent malignancies [20].
Several potent VEGFR-2 inhibitors have been developed and achieved clinical
success in the treatment of cancer [21]. Considering the difference between structures,
these agents are mainly divided into two classes (type I and type II inhibitors) [22].
Type I inhibitors mainly recognize the active conformation of VEGFR-2, just binding
in and around the region which originally occupied by the adenine ring of ATP.
Sunitinib 1 is an example of this category (Fig. 1) [23]. The second type of VEGFR-2
inhibitors (type II) stabilizes the DFG-out (inactive) conformation of the enzyme by
movement of the DFG motif. This enables them to occupy a hydrophobic site, usually
called allosteric site, created by the new rearrangement and directly adjacent to the
ATP binding pocket. This allosteric site is only revealed in the inactive DFG-out
confirmation of the kinase[24]. Type-II inhibitors have several advantages over type-I
inhibitors, including improved kinase selectivity and slower off-rates[25]. Sorafenib 2
and tivozanib3 are classical type II VEGFR-2 inhibitors (Fig. 1). However, some
adverse effects as bleeding complications have been observed during clinical use,
indicating that the development of saferVEGFR-2 inhibitors is still an active field of
research [20].
A literature survey revealed that there are different scaffolds having VEGFR-2
inhibitory activities such as quinoline (e.g. lucitanib 4 and lenvatinib 5) [26, 27],
quinazolines (e.g. AZD-2932 6) [28, 29] and sulfonamides (e. g. pazopanib 7) [30].
Quinazolin-4(3H)-one is a privileged scaffold possesses promising antitumor activity
with a very good human tolerance [31, 32]. On other hand, several new 6-aryl-5-
cyanothiouracil [33], thiazolidine-2,4-dione [34], thiazol-4(5H)-one [35], benzylidene
[36], sulfonamide [32], sulfonylurea [37], sulfonylthiourea [38], semicarbazone [39]
and thiosemicarbazone [40] moieties were reported to possess antitumor activities.
5

Depending on ligand-based drug design [41-44] particularly the molecular
hybridization approach that involves the coupling of two or more groups with relevant
biological properties [45, 46], a molecular hybridization of quinazolin-4(3H)-one and
other effective antitumor moieties was carried out in an attempt to get new effective
antitumor molecules, having the main pharmacophoric features of VEGFR-2
inhibitors.
1
.1 Rationale of molecular design
In continuation of our previous works [47-54], that proved potential anticancer
activities of novel chemical agents, a new series of quinazolin-4(3H)-one derivatives
were designed and synthesized. Study of the structure-activity relationships (SAR)
and common pharmacophoric features shared by various VEGFR-2 antagonists
revealed that most of them have four main features as shown in Fig. 1 and Fig. 2 [19,
5
5, 56]. These features are; (i) a flat hetero aromatic ring system containing at least
one H-bond acceptor (N atom is preferred, followed by the O atom). This H-bond
acceptor forms hydrogen bonding interactions with the key amino acid residue
(Cys917) in the catalytic ATP-binding domain [19] (ii) A central aryl ring (spacer),
which occupies the linker region between the ATP-binding domain and the DFG
domain [57], (iii) a pharmacophore moiety comprises two groups of an H-bond
acceptor and an H-bond donor (e.g., amide or urea). This pharmacophore binds with
two crucial residues (Glu883 and Asp1044) in the DFG (Asp-Phe-Gly) motif. The NH
motifs of the urea or amide moieties usually form two hydrogen bonds with Glu883,
whereas the C=O motif forms another hydrogen bond with Asp1044 [58], (iv) a
terminal hydrophobic group, which occupies the created allosteric hydrophobic
pocket via different hydrophobic interactions [59]. Moreover, the X-ray structure
analysis of many VEGFR-2 inhibitors confirmed the sufficient space available for
various substituents around the terminal hetero aromatic ring [60, 61].
6

F
H
H
N
N
O
N
F
O
N
O
N
Cl
H
HN
CF3
O
N
O
H
NH
Sorafenib 2
Sunitinib 1
H
O
N
CH3
H
H
N
N
O
N
O
O
Cl
O
N
O
O
O
O
H N
2
N
Tivozanib 3
Lucitanib 4
H
H
H
N
N
N
N
O
N
O
O
O
O
N
Cl
O
O
N
H N
2
N
O
Lenvatinib 5
AZD-2932
6
O
H
NH2
N
N
N
S
N
N
O
N
Pazopanib 7
Fig. 1. Basic pharmacophoric features of VEGFR-2 inhibitors.
7

Hetero
aromatic
ring
Hydrophobic
tail
Central Aryl
Ring
HBA-HBD
H
H
N
N
CF3
Cl
N
H
O
N
H C
3
O
O
Sorafenib
Interact with
Glu883& Asp1044
in DFG domain
Occupy
linker
region
Occupy the
allosteric
binding region
ATP binding
domain
Fig. 2. The basic structural requirements for sorafenib as reported VEGFR-2
inhibitor.
The goal of our work is to synthesize new agents with the same essential
pharmacophoric features of the reported and clinically used VEGFR-2 inhibitors, in
addition, to being molecularly hybridized with effective antitumor moieties in an
attempt to get more potent antitumor molecules. The main core of our molecular
design rationale comprised bio-isosteric modification strategies of VEGFR-2
inhibitors at four different positions (Fig. 3).
Hetero
aromatic
ring
Central Aryl
Ring
Hydrophobic
tail
HBA-HBD
Open chain
linkers (spacers)
Effective antitumor Substituted aromatic
moieties
or aliphatic groups
Quinazolin-4(3H)-one
Fig. 3. Summary for the possible modifications of VEGFR-2 inhibitors.
The first position was the heterocyclic aromatic ring; three different
quinazoline moieties were used; i) 2-phenylquinazolin-4(3H)-one (compounds 13a-c,
1
7a-d and 21 ), ii) 2-mercapto-3-phenylquinazolin-4(3H)-one (compounds 25a,b,d,f
a-c
and 26 ), and iii) 3-ethyl-2-mercaptoquinazolin-4(3H)-one (compounds 25 26 ,
a,b
c,e,g
c,d
8

2
9a,b, 30 and 31). The choice of these quinazoline moieties was based on some bio-
isosteric considerations; i) the bicyclic structure of quinazoline core is convenient to
the large size space of the ATP binding region [62], ii) the 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 one (compounds 21 ), three (compounds 17 ), or four atom
a-c
a-d
bridges (compounds 13 ). In some cases, it was four atoms in addition to phenyl
a-c,
moiety (compounds 25a-g, 26 , 29 , 30 and 31).
a-d
a,b
The third position was the HBA/HBD region (pharmacophore). We used many
different effective anticancer moieties having the essential HBA/HBD features to play
the role of the pharmacophore. The pharmacophores were designed to be either open
chains (as compounds 25a-g, 26 , 29 , 30 and 31), or cyclized (compounds 13 17a-
a-d
a,b
a-c,
and 21 ). Different cyclized forms were used including six-membered rings
d
a-c
(
(
(
compounds 13 ) and five membered rings such as; thiazolidine2,4-dione
a-c
compounds 17 ), thiazolidin-4-one (compounds 21 ) and pyranothiazoles
a-d
a-c
compounds 21 ). On the other hand, the open chain pharmacophores were
a-c
sulfonamide (compounds 25 ), sulfonylurea (compounds 26 ), semicarbazone
a-g
a-d
(compound 29 ), thiosemicarbazone (compound 29 ), N-ethylideneformohydrazide
a b
(compound 30) or oxime moieties (compound 31).
Finally, the terminal hydrophobic moieties were selected to be aromatic
heterocyclic (compounds 25 ), aromatic non-heterocyclic (compounds 13 17a-d,
d-g
a-c,
2
1a-c, 26 ), or aliphatic structures (compounds 26 29 , 30 and 31). The wide variety
d a-c, a,b
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 a
crucial objective of our work. All modification pathways and molecular design
rationale were illustrated and summarized in Fig. 4 & 5.
In addition, to explore and emphasize the mechanism of action of the
synthesized compounds, molecular docking studies were conducted to understand the
expected binding interactions of the target compounds with VEGFR-2 active sites.
For the same purpose, other studies regarding the ability of the most active
compounds to produce in vivo tumor growth inhibition, induce apoptosis and arrest
cell cycle growth at G2/M phase have been done.
9

O
CN
HN
O
N
S
O
N
R
NH
N
13a-c
N
N
O
N
?
N
11
O
O
NH
S
N
S
O
R2
O
O
N
O
O
N
R1
O
N
H
S
S
N
N
NH
N
N
O
17a-d
19a-c
Fig. 4. The rationale of molecular design of the new proposed VEGFR-2 inhibitors
having substitutions at position-3 of quinazoline nucleus.
1
0

O
N
O
N
R4
S
R4
O
N
O
S
N
O
S
H
N
H
H
N
R6
R5
N
N
N
S
O
H
H
O
O
O
25a-g
2
6a-d
O
N
R4
S
N
?
22a-c
O
N
X
O
O
CN
NH2
N
N
N NH
CH3
N NH
CH3
H
N
H
N
S
N
S
O
O
2
9a,b
30
Fig. 5. The rationale of molecular design of the new proposed VEGFR-2 inhibitors
having substitutions at position-2 of quinazoline nucleus.
2
2
. Results and discussion
.1. Chemistry
The synthetic employed pathway to prepare the new quinazoline derivatives is
outlined in Schemes 1-5. The chemical synthesis was initiated by stirring anthranilic
acid with benzoyl chloride in DMF at room temperature to furnish N-benzoyl
anthranilic acid 8 [63], which was heated with acetic anhydride using Bogert and
Seil method [64] to provide 2-phenyl-4H-3,1-benzoxazin-4-one 9 [63], which was
condensed with hydrazine hydrate in refluxing n-butanol to yield, 3-amino-2-
phenylquinazolin-4(3H)-one 10 [65],. Acylation of 10 with 2-chloroacetyl chloride in
DMF provided 2-chloro-N-(4-oxo-2-phenyl-quinazolin-3(4H)-yl)-acetamide 11 [66],
which is the key intermediate in scheme 1, 2 and 3. On the other hand, one pot
1
1

reaction of ethyl cyanoacetate, thiourea and substituted benzaldehydes, afforded the
other intermediates 2-mercapto-6-oxo-4-(substituted) phenyl-1,6-dihydropyrimidine-
5
-carbonitriles 12a-c. The target compounds 13a-c were obtained by reaction of the key
intermediate 11 with the appropriate 2-mercapto-6-oxo-4-(substituted) phenyl-1,6-
dihydropyrimidine-5-carbonitriles 12a-c in refluxing dry acetone using anhydrous
potassium carbonate in presence of catalytic amount of potassium iodide (Scheme 1).
The IR spectra of compounds 13a-c revealed the presence of C=O absorption
-1
band at a range of 1677 - 1697 cm , C≡N stretching band at a range of 2206 - 2228
-1
-1 1
cm and NH band at a range of 3132 - 3264 cm . H NMR spectra showed two up-
field doublet signals of the aliphatic methylene protons at a δ range of 3.93 - 4.12
ppm, methylene protons resonated differently because they are diastereomers [67-69].
1
Moreover, their H NMR spectra revealed the presence of D O exchangeable 2NH
2
1
3
protons at δ range of 11.43 - 11.64 ppm and 13.74 - 13.91 ppm. In addition, C NMR
and DEPT 135 NMR showed the presence of one methylene carbon resonating at a δ
1
3
range of 32.4 – 32.5 ppm. Three amide carbonyls have been detected in C NMR
spectrum at a δ range of 159.6– 167.5 ppm and they were missed at DEPT 135 NMR
spectrum.
Refluxing the thiourea with chloroacetic acid in water with the presence of 4N
HCl, furnished thiazolidine-2, 4-dione 14 [70], subsequent treatment of 14 with
alcoholic potassium hydroxide afforded the corresponding potassium salt 15. Heating
the potassium salt of TZD 15 with compound 11 in dry DMF afforded the
intermediate,
2-(2,4-dioxothiazolidin-3-yl)-N-(4-oxo-2-phenylquinazolin-3(4H)-yl)
acetamide 16 which was condensed with various substituted benzaldehydes via
Knoevenagel condensation [71, 72], in dry toluene in the presence of piperidine to
give the target benzylidene analogues 17a-d (Scheme 2).
The reaction of 2-chloroacetamide derivative 11 with ammonium thiocyanate
in refluxing ethanol yielded 2-{[4-oxo-2-phenylquinazolin-3(4H)yl]amino}
thiazolidin-4-one 18, via intermolecular cyclization and the Dimroth-like
rearrangements [73] (Scheme 3). The final compounds 19a-c were obtained in good
yields by condensation of compound 18 with appropriate aromatic aldehydes in dry
toluene in the presence of catalytic amounts of piperidine. The formation of
1
benzylidene derivatives 17a-d and 19a-c were confirmed by H NMR spectra, which
showed the disappearance of the singlet signal of methylene protons of TZD ring and
1
2

the appearance of a new singlet signal for benzylidene methine proton in the range of
1
3
δ 7.81 – 8.06 ppm. This methine proton was detected also in both of C NMR and
DEPT 135 NMR spectra resonating around 127.0 ppm.
In addition, the reaction of malononitrile with different aldehydes gave 2-
(substituted benzylidene) malononitrile derivatives 20a-c. Subsequent reaction of 20a-c
with 18 in refluxing ethanol with the presence of a catalytic amount of piperidine
[74], afforded the corresponding pyranothiazole derivatives 21a-c (Scheme 3).
The IR spectra of compounds 21a-c showed the disappearance of the carbonyl
band of thiazolidin-4-one ring, and the appearance of C≡N band at a range of 2197 -
-1
-1 1
2
270 cm , in addition to a forked NH band at a range of 3320 - 3335 cm . H NMR
2
spectra showed the presence of two singlet signals at ranges of δ 12.77 - 13.10 ppm
and δ 7.58 - 7.74 ppm corresponding to NH and NH protons, respectively
2
(disappeared upon deuteration) in addition to the presence of singlet signal at a range
of δ 7.63 - 7.74 ppm corresponding to CH proton of the heterocyclic pyran ring. In
addition, 13C NMR and DEPT135 NMR spectra showed the disappearance of the
methylene group and the appearance of a characteristic signal at ranges of δ 40.4 –
4
4.21 ppm corresponding to the methine proton of the heterocyclic pyran ring.
Anthranilic acid was refluxed with isothiocyanates in absolute ethanol
containing a catalytic amount of TEA, as in reported procedure [75, 76], where the
corresponding 3-(substituted)-2-sulfanylquinazolin-4(3H)-ones 22a-c were obtained.
Potassium salt of 3-(substituted)-2-sulfanylquinazolin-4(3H)-ones 23a-c were prepared
by heating of compound 22a-c with potassium hydroxide in absolute ethanol with
continuous
stirring.
The
reaction
of
23a-c
and
2-chloro-N-[4-(N-
(substituted)sulfamoylphenyl]acetamide 24a-g in dry DMF over a water bath in the
presence of catalytic amount of KI yielded the target compounds, 25a-g (Scheme 4).
The IR spectra of 25a-g illustrated the presence of the carbonyl absorption band
-1
at a range of 1671-1679 cm and the presence of NH band at a range of 3390 - 3393
-1
cm . In addition; compounds 25
were characterized by the appearance of
a-c
-1
1
characteristic bands of NH at a range of 3284 - 3196 cm . Additionally, the H NMR
2
spectra showed singlet signals of the aliphatic -SCH protons at a δ range of 4.09 -
2
4
.12 ppm and presence of D O exchangeable NH-amide protons at a δ range of 10.63
2
-
10.81 ppm. Moreover, NH protons of compounds 25 appeared as a singlet at a
2 a-c
1
3
range of δ 7.19 - 7.25 ppm and disappeared with D O. The spectra of C NMR and
2
1
3

DEPT 135 NMR revealed the presence of SCH carbons resonating at a δ range of
2
3
1
7.2-37.8 ppm. Also, two different amide carbonyls have been detected at δ range of
1
3
60.6-161.1 and 166.8-166.9 ppm in C NMR spectrum.
The reaction of compounds 25 with substituted isocyanates in dry acetone in
a-c
presence of anhydrous K CO [77, 78], afforded the corresponding sulfonylurea
2
3
derivatives 26a-d (Scheme 4).
IR charts of compounds 26a-d revealed disappearance of NH bands and
2
appearance of different NH bands of sulfonylureas. IR spectra of cyclohexyl
1
derivatives exhibited the appearance of intense peaks at CH aliphatic region. H NMR
spectra exhibited the appearance of two new NH signals and disappearance of NH2
signals. Cyclohexyl, ethyl and propyl protons appeared upfield, between δ 1.04 and
1
13
4
.10 ppm in H NMR spectrum and between δ 24.21 and 49.75 ppm in the C NMR
spectrum.
Meanwhile, the reaction of the potassium salt of 3-ethyl-2-sulfanylquinazolin-
(3H)-one 23 with N-(4-acetylphenyl)-2-chlroacetamide 27 in dry DMF, yielded the
4
c
desired intermediate compound 28. Condensation of 28 with various derivatives
namely, semicarbazide, thiosemicarbazide, 2-cyanoacetohydrazide and
hydroxylamine HCl in ethanol furnished the corresponding compounds 29a, b, 30 and,
3
1 respectively (Scheme 5).
1H NMR spectrum of 28 showed singlet signals of the aliphatic protons at δ
1
3
2
.51 and 4.29 ppm and at δ 13.48 and 37.23 ppm in C NMR spectrum corresponding
1
to COCH and SCH groups, respectively. Moreover, H NMR spectrum showed the
3
2
presence of D O exchangeable singlet signal of NH group at about δ 10.99 ppm. IR
2
-1
spectrum of 29 showed absorption bands at 3312, 3280 and 1672 cm corrosponding
a
to NH, NH and C=O groups, respectively. Furthermore, the signals of the 2 NH and
2
NH protons appeared as D O exchangeable singlet signals. Moreover, IR spectrum of
2
2
compound 30 revealed characteristic strong intensity bands at 3340, 3219 and 2259
cm -1 for the introduced 2NH and C≡N groups, respectively confirming the formation
1
of cyanoacetylhydrazono derivative. In addition, H NMR spectrum displayed up-
field singlet signal at δ 3.2 ppm for the introduced CH group and downfield shifted
2
singlet signals appeared at δ 10.32 and 10.52 ppm for the two NH groups. The
1
3
introduced methylene group found to be resonating at 26.88 ppm in both of C NMR
1
4

1
and DEPT 135 NMR spectra. Furthermore, the H NMR of 31 revealed the presence
of D O exchangeable singlet signal of OH group at about δ 11.06 ppm.
2
COOH
O
Cl
O
ii
COOH
NH2
i
NH
O
+
O
N
9
8
O
iii
NC
O
O
+
H
O
N
NH2
N
R
10
+
S
H N
NH2
2
iv
v
O
O
N
O
Cl
CN
HN
NH
HS
N
N
R
11
(12a-c)
vi
O
CN
HN
O
N
O
N
S
N
NH
R
13a ; R = H
(13a-c
)
13 ; R = OCH3
b
13c ; R = Cl
.
Scheme 1. General procedure for preparation of target compounds 13 ; Reagents
a-c
and conditions: (i) DMF / stirring / 2h, (ii) acetic anhydride / reflux / 2-3h, (iii)
NH NH · H O / n-butanol/ reflux 8 h, (iv) chloroacetyl chloride / DMF / stirring /
2
2
2
1
2h, (v) K CO / ethanol/ reflux/6h, (vi) acetone / K CO / reflux / 8h.
2 3 2 3
1
5

O
N
O
N
Cl
S
O
NH
Cl
H N
NH2
+
2
OH
1
1
i
O
O
ii
KN
iii
S
HN
S
O
O
1
5
14
O
O
O
N
O
N
O
N
N
O
N
iv
NH
NH
S
S
O
O
H
O
N
R1
1
6
(17a-d)
R1
1
1
1
1
7a ; R = H
7b ; R = OCH3
7c ; R = Cl
7d ; R = OH
Scheme 2. General procedure for preparation of target compounds 17 ; Reagents
a-d
o
and conditions: (i) a) water 0-5 C / stirring / 15min, b) Conc. HCl / reflux 10 h, (ii)
KOH / ethanol / reflux 1h, (iii) DMF / heating / 20 h, (iv) dry toluene / piperidine /
reflux 8 h.
1
6

O
O
R2
O
N
HN
S
HN
S
O
N
Cl
O
O
NH
ii
N
i
N
N
N
N
N
O
H
11
18
(19a-c)
R2
19 ; R = OH
2
a
2
1
1
9 ; R = CH
b
3
O
2
9 ; R = NO
c
3
H
H
CN
CN
R3
iii
iv
+
CN
CN
R3
(20a-c)
H N
CN
2
O
R3
HN
O
S
N
N
N
(21a-c)
3
2
2
2
1 ; R = H
a
3
1 ; R = Cl
b
3
1 ; R =OCH
c
3
Scheme 3. General procedure for preparation of target compounds 19a-c & 21a-c.
Reagents and conditions: (i) NH SCN / ethanol / reflux / 6h, (ii) dry toluene /
4
piperidine / reflux 8h, (iii) TEA / ethanol / stirring / 30 min, (iv) dry toluene /
piperidine / reflux 8 h,
1
7

NH2
COOH
NH2
S
O S O
NHR5
R4
i
C
N
O
N
iii
R4
N
SH
Cl
(22a-c)
HN
O
ii
O
N
R4
O S O
NHR5
N
SK
(24a-g)
(
23a-c)
R4
C H
R5
iv
25a-g
a
O
N
H
6
5
R4
S
N
b
c
H
4-Br-C H4
6
H
N
H
C H
2
5
O
d
e
pyridine-2-yl
O
C H
6
5
S
NHR5
pyridine-2-yl
C H
2 5
(
25a-g
)
O
f
C H 4,6-dimethylpyrimidine-2-yl
6
5
v
g
C H
2
5 4,6-dimethylpyrimidine-2-yl
O
R4
S
26a-d
R4
C H
R6
N
H
a
Cyclohexyl
6
5
N
N
b
c
4-Br-C H4 Cyclohexyl
6
O
O
O
S
R6
C H
2
Cyclohexyl
5
N
N
O
(
26a-d
)
H
H
d
C H
2
C H
6 5
5
Scheme 4. General procedure for synthesis of target compounds 25a-g & 26a-d.
Reagents and conditions: (i) ethanol / TEA / reflux / 2h, (ii) KOH / ethanol / reflux
h, (iii) chloroacetyl chloride / DMF /stirring /1 h, (iv) DMF / KI / heating 12 h, (v)
2
substituted isocyanate / dry acetone / K CO / reflux 24 h.
2
3
1
8

O
O
O
N
NH2
Cl
NH
N
i
SK
2
3c
H C
O
3
CH3
27
ii
O
N
N
H
N
iii
S
v
O
CH3
28
O
O
N
O
N
iv
N
H
N
N
H
N
S
X
S
O
N
N
NH2
O
N
H
OH
CH3
2
9a,b
CH3
31
2
2
9a
; X = O
; X = S
9b
O
N
H
N
N
S
O
O
N
CN
N
H
CH3
30
Scheme 5. General procedure for the synthesis of target compounds 29 , 30 & 31.
a,b
Reagents and conditions: (i) p-amino acetophenone / chloro acetylchloride / DMF /
stirring / 2h, (ii) DMF / KI / heating / 8 h, (iii) semicarbazide, thiosemicarbazide /
ethanol / gl. acetic acid /reflux /1 h, (iv) cyanoacetohydrazide / ethanol/ reflux3 h, (v)
hydroxylamine HCl / dry acetone / K CO / reflux / 42 h
2
3
2
.2. Biological evaluation
.2.1. In vitro anti-proliferative activity
All final synthesized compounds were evaluated for their in vitro anticancer
activities via standard MTT method [79-81], against a panel of three human tumor
2
1
9

cell lines namely; hepatocellular carcinoma (HepG-2), colorectal carcinoma (HCT-
16) and breast cancer (MCF-7). Doxorubicin and sorafenib were used as standard
anticancer drugs. The results were expressed as growth inhibitory concentration (IC )
1
5
0
values and summarized in Table 1.
The tested compounds displayed excellent, good, moderate or weak anti-
proliferative activities against the three tested cell lines as shown in Table 1.
In general, compounds 26 , 29 , 29 , 30 and 31 were found to be the most
b
a
b
potent derivatives against the three cell lines. In particular, compound 31 exhibited
excellent activities against hepatocellular carcinoma (HepG-2), colorectal carcinoma
(
HCT-116) and breast cancer (MCF-7) with IC values of 3.97 ± 0.2, 4.83 ± 0.2 and
50
4
.58 ± 0.3 µg/ml, respectively. These results were found to be mostly more active
than the standard and sometimes a little lower or almost the same.
Moreover, several compounds such as 26 , 29 , 29 and 30 showed excellent
b
a
b
anti-proliferative activities with IC values ranging from 4.91 to 9.23 µg/ml. Also,
5
0
compounds 13 , 17 , 25g and 26 displayed remarkable activities against HepG-2 and
c
a
d
HCT-116 cell lines with IC values ranging from 11.13 to 16.93 µg/ml.
5
0
On the other hand, several compounds showed weak anti-proliferative
activities against the tested cell lines. Finally, compounds 17 , 19 , 21 and 25 were
d
b
c
c
appeared to be inactive.
Table 1. Anti-proliferative activities of the tested compounds toward HepG-2, HCT-
1
16 and MCF-7cell lines.
a, b
In vitro cytotoxicity IC (µg/ml)
5
0
Comp.
HepG-2
HCT-116
MCF-7
1
3a
3b
66.46 ± 3.8
58.43 ± 3.4
68.21 ± 3.7
13.16 ± 1.2
14.88 ± 1.4
45.63 ± 2.7
23.96 ± 1.7
NA c
80.42 ± 4.3
89.88 ± 4.8
15.74 ± 1.5
31.35 ± 1.9
63.81 ± 3.8
51.78 ± 2.9
NA c
1
79.51 ± 4.4
11.13 ± 1.0
16.93 ± 1.4
55.71 ± 3.4
35.60 ± 2.3
NA c
1
3c
7a
1
1
7b
7c
7d
1
1
2
0

1
9a
9b
76.81 ± 4.2
NA c
27.42 ± 1.8
NA c
21.65 ± 1.7
NA c
1
19c
21a
21c
85.57 ± 4.6
96.52 ± 5.3
NA c
78.08 ± 4.2
92.16 ± 4.9
NA c
94.50 ± 5.1
NA c
NA c
2
2
2
1b
5a
5b
91.42 ± 4.7
60.26 ± 3.7
69.14 ± 3.9
NA c
74.87 ± 4.1
85.94 ± 4.7
61.42 ± 3.4
NA c
92.48 ± 4.9
NA
83.74 ± 4.4
NA c
2
5c
5d
5e
5f
2
82.81 ± 4.5
23.06 ± 1.9
73.65 ± 4.1
14.67 ± 1.3
42.13 ± 2.6
7.81 ± 0.5
48.93 ± 2.9
13.78 ± 1.2
6.86 ± 0.4
6.42 ± 0.3
71.55 ± 3.8
42.51 ± 2.5
51.67 ± 3.0
10.41 ± 1.0
40.16 ± 2.3
9.23 ± 1.0
30.58 ± 1.9
14.76 ± 1.4
8.28 ± 0.8
8.11 ± 0.7
91.71 ± 4.9
87.41 ± 4.6
74.99 ± 3.9
12.69 ± 1.2
60.21 ± 3.3
13.34 ± 1.2
56.91 ± 3.2
25.74 ± 1.8
9.48 ± 0.7
7.83 ± 0.5
2
2
25g
26a
26b
2
6c
26d
29a
29b
3
0
1
8.35 ±0.7
3.97 ± 0.2
4.50 ± 0.2
3.40 ± 0.2
11.87±1.1
4.83 ± 0.2
5.23 ± 0.2
5.30 ± 0.2
13.79 ±1.3
4.58 ± 0.3
4.17 ± 0.2
4.21 ± 0.6
3
Doxorubicin
Sorafenib
a
b
c
IC values are the mean ± S.D. of three separate experiments.
5
0
IC (µg/ml): 1-10 (very strong), 11-25 (strong), 26-50 (moderate), 51-100 (weak).
5
0
NA: Compounds having IC value > 100µg/ml.
5
0
2
1

2
.2.2. In vitro VEGFR-2 enzyme assay inhibition
The most active cytotoxic compounds (13 , 17 , 17 , 25 , 26 , 26 , 29 , 29 , 30
c
a
c
g
b
d
a
b
and 31) were subjected to further investigation for their ability to inhibit VEGFR-2
using sorafenib as a reference drug. The results were expressed as growth inhibitory
concentration (IC ) values and summarized in Table 2.
5
0
Compound 31 was the most potent VEGFR-2 inhibitor with an IC value of
5
0
2
.5 ± 0.04 μM, nearly equal to that of sorafenib (IC = 2.4 ± 0.05 μM). Additionally,
50
compounds 29 , 29 and 30 showed promising IC values less than 5.2 μM. Also,
a
b
50
Compounds 13 , 25 , 26 and 26 showed moderate IC values less than 10 μM.
c
g
b
d
50
Finally, Compounds 17 and 17 exhibited the lowest anti VEGFR-2 activities with
a
a
IC values of 11.4 ± 0.8 and 21.8 ± 0.42 μM, respectively.
5
0
Table 2: IC (µM) for the tested compounds as inhibitors for VEGFR-2
5
0
IC (µM)
5
0
Comp.
VEGFR-2
7.9 ± 0.6
13c
17a
17c
25g
11.4 ± 0.8
21.8 ± 0.42
9.7 ± 0.9
2
2
2
2
6b
6d
9a
9b
5.3 ± 0.41
8.4 ± 0.78
4.3 ± 0.06
4.1 ± 0.06
5.2 ± 0.07
2.5 ± 0.04
2.4 ± 0.05
3
0
1
3
Sorafenib
2
.2.3. Effect on cell cycle progression
To get a better insight regarding the effect of 31 on growth inhibition of cancer
cells, its effect on the cell cycle distribution and apoptosis induction was evaluated
using HepG-2 cells according to the procedure described by Wand et al [82]. The
2
2

cells were treated with compound 31 at a concentration equals its IC50 value on
VEGFR-2 (2.5 µM) for 24 h.
The results (Table 3 and Fig. 6) showed that compound 31 arrested cell
growth in G2-M phase, accumulation of cells at that phase became 33.37% after
treatment of control HepG-2 cells with compound 31. It was noticeable also that
treatment of HepG-2 cells with 31 induced apoptosis at pri-G1 phase, percent of
apoptosis became 12.17 after being 2.04 in control cells.
Table 3: Effect of compound 31 on cell cycle progression in HepG-2 cells.
Cell cycle distribution (%)
Sample
%
G0-G1
%S
%G2-M
33.37
%Pre-G1
12.17
3
1 / HepG-2
49.25
52.31
17.38
31.38
Cont. HepG-2
16.31
2.04
Fig. 6. Cell cycle analysis and apoptosis effect in HepG-2 cell line when treated with
compound 31
2
.2.4. Induction of apoptosis
Induced apoptosis in HepG-2 cells by compound 31 was evaluated using Annexin
V and PI double staining assay [83] to explore the mode of induced cell death. HepG-
cells were incubated with 31 at 2.5 µM concentration for 24 h. The obtained results
are represented in Table 4 and Fig. 7 & 8.
2
2
3

The obtained results showed that compound 31 induced apoptosis (10.96%) by
more than 14 folds over the control (0.76%). Compound 31 revealed to induce early
apoptosis (7.51%) and enhanced late apoptosis (3.45%) when compared with the
untreated control HepG-2 cells.
Table 4: Apoptosis and necrosis percent induced by compound 31 on HepG-2 cells.
Apoptosis
Sample
Necrosis
Total
13.5
2.04
Early
7.51
0.51
Late
3.45
0.25
3
1 / HepG-2
2.54
1.28
Cont. HepG-2
Fig. 7. Percentage of induced cell death by compound 31 on HepG-2 cells.
2
4

A
-
Control
Compound 31
B
-
Control
Compound 31
Fig. 8. A) HepG-2 cells distribution upon treatment with compound 31.
B) Induced apoptosis on HepG-2 cells by compound 31
2
.2.5. In vivo antitumor activity of the synthesized compounds
The in vivo anticancer efficiencies of compounds 30 and 31 were further evaluated
using diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) model [84,
st
8
5]. Thirty rats were randomized into five groups, the 1 group was used as a negative
nd
rd
control, the 2 group was injected with DEN, the 3 group was injected with DEN
th
then with doxorubicin, the 4 group was injected with DEN then compound 30, the
2
5

5th group was injected with DEN then compound 31 as described in experimental
section.
st
As shown in Fig. 9, the 1 group (negative control) showed hepatic polygonal
cells (HC) of normal liver structure with the round nucleus, normal central vein (CV),
blood sinusoids (BS) and normal portal vein (PV). Fig. 10 showed the effect of DEN
nd
on the 2 group producing a loss of liver tissue architecture, displaying accumulation
of collagen fiber, necrosis, apoptotic changes and degeneration of portal tract. The 3rd
group (doxorubicin treated one) revealed widespread preserving the normal hepatic
architecture. However, degenerative and apoptotic changes were encountered in
th
hepatocytes with dilatation in central vein (Fig. 11). The 4 group (compound 30
treated one) revealed mild to moderate congestion of hepatic blood vessels, portal
edema, biliary proliferation and round cells infiltration. Moreover, there were some
th
degenerative changes in a variable number of hepatocytes (Fig. 12). The 5 group
(compound 31 treated one) showed apoptotic changes in some hepatocytes, increased
Kupffer cells and the hepatic sinusoids were dilated in some areas. In addition, it
showed lymphocytic infiltration (Fig. 13). This confirmed that compounds 30 and 31
possessed significant tumor growth inhibition in vivo.
Fig. 9. Photomicrograph of normal liver structure in adult albino rat showing hepatic
polygonal cells (HC) with round nucleus, normal central vein (CV), blood sinusoids
(BS) and normal portal vein (PV) (H&E X 100, 400).
2
6

Fig. 10. Photomicrograph of DEN Liver showing, liver tissue loss architecture,
displaying accumulated collagen fiber (black arrow), area of necrosis (yellow arrow),
degenerated of portal tract (red arrow) and apoptotic changes (blue arrow) (H&E X
1
00, 400).
C
C
V
Fig. 11. Photomicrograph of liver for DEN-doxorubicin treated group showing,
widespread preserving the normal hepatic architecture. However, degenerative and
apoptotic changes were encountered in a few hepatocytes with dilated central vein
(H&E X 100, 400).
2
7

Fig. 12. Compound 30 after DEN revealed mild to moderate congestion of hepatic
blood vessels, portal edema, biliary proliferation and round cells infiltration and
degenerative changes in a variable number of hepatocytes (H&E X 100, 400).
Fig. 13. Compound 31 after DEN showing apoptotic changes in some hepatocyte,
mild hypertrophy in kupffur cells, dilatation of hepatic sinusoids, and lymphocytic
infiltration in some areas (H&E X 100, 400).
2
.3. Molecular docking
Molecular docking studies for the target compounds were performed to
rationalize the obtained biological results as well as to help us to understand how they
can inhibit the activation of VEGFR-2 kinase and its downstream signaling pathways.
Sorafenib was used as a reference compound. The distinctive binding pattern of
2
8

sorafenib to VEGFR-2 kinase active site has been discussed and compared to the
tested compounds. Sorafenib interactions with the amino acids of the pocket have
been studied and displayed in 3D style in Fig. 14. The proposed binding mode of
sorafenib, revealed an affinity value of -52.20 kcal/mol. It demonstrated the important
interactions with the residues at the active site of VEGFR-2. The urea side chain
possessed both an H-bond acceptor and donor in order to form three important
hydrogen bonds with two crucial residues (Glu883 and Asp1044) of the receptor,
where the two NH motifs of the urea moiety usually formed two hydrogen bonds with
the carboxylate moiety of Glu883 with a distance of 1.68 and 1.59 Å. Whereas the
C=O motif formed another hydrogen bond with the backbone NH of Asp1044 with a
distance of 1.73 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 [59, 86].
Fig. 14: The Predicted binding mode for sorafenib with the active site of
VEGFR-2
The obtained results 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 5.
Docking simulation of compound 29 showed that it has a good fitting into the
a
enzyme active sites almost at the same position of sorafenib with comparable docking
2
9