Design and synthesis of new 1,6-dihydropyrimidin-2-thio derivatives targeting VEGFR-2: Molecular docking and antiproliferative evaluation

Article abstract of DOI:10.1016/j.bioorg.2020.104090

A series of new 1,6-dihydropyrimidin-2-thiol derivatives (scaffold A) as VEGFR-2 inhibitors has been designed and synthesized. Compounds 3a, 3b, 3e and 4b have been selected for in vitro anticancer screening by the National Cancer Institute. Compound 3e showed remarkable anticancer activity against most of the cell lines tested, where a complete cell death against leukemia, non-small cell lung cancer, colon, CNS, melanoma, and breast cancer cell lines was observed. In vitro five dose tests showed that compound 3e had high activity against most of the tested cell lines with GI₅₀ ranging from 19 to 100 μM and selectivity ratios ranging between 0.75 and 1.71 at the GI₅₀ level. VEGFR-2-kinase was tested against 3a, 3b, 3e, 4b and sorafenib was used as a reference. Compounds 3a and 3e were the most potent analogues with IC₅₀ values of 386.4 nM and 198.7 nM against VEGFR-2, respectively, in comparison to sorafenib (IC₅₀ = 0.17 nM). The results of the docking study showed a good fitting of the new compounds to the active site of VEGFR-2 with binding free energies in the range of ?9.80 to ?11.25 kcal/mol compared to ?12.12 kcal/mol for sorafenib. Compounds 4a-e with the hydroxyimino group had a higher affinity to VEGFR-2 than their parent derivatives 3a-e.

Full text of DOI:10.1016/j.bioorg.2020.104090

Journal Pre-proofs
Design and synthesis of new 1,6-dihydropyrimidin-2-thio derivatives target-
ing VEGFR-2: molecular docking and antiproliferative evaluation
Adel A. Marzouk, Salah A. Abdel-Aziz, Kamal S. Abdelrahman, Amira S.
Wanas, Ahmed M. Gouda, Bahaa G.M. Youssif, Mohamed Abdel-Aziz
PII:
S0045-2068(20)31387-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.104090
YBIOO 104090
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
6 April 2020
16 June 2020
7 July 2020
Please cite this article as: A.A. Marzouk, S.A. Abdel-Aziz, K.S. Abdelrahman, A.S. Wanas, A.M. Gouda, B.G.M.
Youssif, M. Abdel-Aziz, Design and synthesis of new 1,6-dihydropyrimidin-2-thio derivatives targeting
VEGFR-2: molecular docking and antiproliferative evaluation, Bioorganic Chemistry (2020), doi: https://doi.org/
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Design and synthesis of new 1,6-dihydropyrimidin-2-thio derivatives
targeting VEGFR-2: molecular docking and antiproliferative evaluation
a
a,b
a
Adel A. Marzouk , Salah A. Abdel-Aziz , Kamal S. Abdelrahman , Amira S.
Wanas c,d, Ahmed M. Gouda , Bahaa G. M. Youssif , Mohamed Abdel-Aziz
e
f
g
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University,
b
Assiut Branch, Assiut-71524, Egypt; Department of Pharmaceutical Chemistry, Faculty
c
of Pharmacy, Deraya University, Minia, Egypt; National Center for Natural Products
Research, University of Mississippi, MS 38677, USA; d Pharmacognosy Department,
Faculty of Pharmacy, Minia University, 61519-Minia, Egypt; e Department of Medicinal
f
Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt;
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt; g Department of Medicinal Chemistry, Faculty of Pharmacy, Minia
University, 61519-Minia, Egypt.
Short running title: Novel dihydropyrimidin derivatives as VEGFR-2 inhibitors
*
To whom correspondence should be addressed:
Mohamed Abdel-Aziz, Ph.D. Department of Medicinal Chemistry, Faculty of
Pharmacy, Minia University, 61519-Minia, Egypt.
Tel.:(002)-01003311327
E-mail address: Abulnil@hotmail.com
Bahaa G. M. Youssif, Ph.D. Pharmaceutical Organic Chemistry Department, Faculty
of Pharmacy, Assiut University, Assiut 71526, Egypt.
Tel.: (002)-01098294419
E-mail address: bgyoussif@ju.edu.sa, bahaa.youssif@pharm.aun.edu.eg

Abstract
A series of new 1,6-dihydropyrimidin-2-thiol derivatives (scaffold A) as VEGFR-2
inhibitors has been designed and synthesized. Compounds 3a, 3b, 3e and 4b have
been selected for in vitro anticancer screening by the National Cancer Institute.
Compound 3e showed remarkable anticancer activity against most of the cell lines
tested, where a complete cell death against leukemia, non-small cell lung cancer,
colon, CNS, melanoma, and breast cancer cell lines was observed. In vitro five dose
tests showed that compound 3e had high activity against most of the tested cell lines with
GI ranging from 19 to 100 μM and selectivity ratios ranging between 0.75 and 1.71
5
0
at the GI level. VEGFR-2-kinase was tested against 3a, 3b, 3e, 4b and sorafenib was
5
0
used as a reference. Compounds 3a and 3e were the most potent analogues with IC50
values of 386.4 nM and 198.7 nM against VEGFR-2, respectively, in comparison to
sorafenib (IC = 0.17 nM). The results of the docking study showed a good fitting of
5
0
the new compounds to the active site of VEGFR-2 with binding free energies in the
range of -9.80 to -11.25 kcal/mol compared to -12.12 kcal/mol for sorafenib.
Compounds 4a-e with the hydroxyimino group had a higher affinity to VEGFR-2 than
their parent derivatives 3a-e.
Keywords: VEGFR-2, Dihydropyrimidine, Docking, Antiproliferative

Graphical abstract

Highlights




A series of novel 1,6-dihydropyrimidin-2-thio (3a-e and 4a-e) was designed
and synthesized.
Compounds 3a, 3b, 3e and 4b were selected for in vitro anticancer activity by
National Cancer Institute.
Compound 3e exhibited a remarkable anticancer activity against most of the
cell lines tested.
In vitro five dose assay results showed that 3e have GI ranging from 19 to
5
0
1
00 μM with selectivity ratios from 0.75 to 1.71.

Compounds 3a and 3e, the most potent analogues against VEGFR-2, showed a
good fitting to the active site of VEGFR-2.

1
. INTRODUCTION
Cancer is a major global health issue. While progress has been made in the treatment
and prevention of cancer, it remains the second biggest cause of death in the world.
st
However, efficacy in cancer treatment remains a concern in the 21 century, and
innovative and safer anticancer agents with a wider range of cytotoxicity to tumor
cells need to be investigated [1]. Biginelli first reported dihydropyrimidinones
(DHPMs) a century ago when the multicomponent reaction was discovered [2].
Dihydropyrimidinone scaffold is a class of highly pharmacologically effective
heterocyclic compounds that attract considerable interest. They have a diverse drug
profile such as antimicrobials [3], antimalarial [4], antitrypanosomal [5] and
anticancer activity [6-8]. Monastrol I (Figure 1) is one of the tetrahydropyrimidine
derivatives that has been established as a new cell permeable molecule that results in
mitotic mammalian cell arrest by blocking the bipolar mitotic spindle. [9]. In addition,
several diverse dihydropyrimidinones have been synthesized, which show promising
antitumor activity, including ethanehydrazonoyl-pyrimidinone II [10] (Figure 1).
O
NH
O
OH
N
N
O
Cl
NH
O
NH
N
S
H
COCH3
Monastrol (I)
II
Figure 1: Structure of monastrol I and compound II

Abnormal angiogenesis plays an important role in cancer and tumor metastases and
represents the necessary pathway through which the tumor passes from benign to
malignant. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the main and
most critical regulator of angiogenic factors associated with tumor angiogenesis [11].
VEGFR-2 can lead to angiogenesis by binding to VEGF leading to stimulation of
downstream signaling cascades and certain endothelial reactions, such as increased
vessel permeability and increased endothelial cell proliferation. VEGF/ VEGFR-2
blockage therefore offers a promising strategy for anti-angiogenic therapy in the
treatment of carcinoma, especially metastatic and progressive solid tumors. [12].
Oxime moiety is distinguished by its simple coordination with metal ions and may
participate in hydrogen bonding to the residues of amino acids in the active site of
different enzymes. By doing so, the oxime moiety can improve the binding site of the
entire molecule [13]. Oximes may also release nitric oxide (NO) free radicals that
have a cytostatic and cytotoxic effect on tumor cells. NO can prevent tumor cells from
metastasizing and help the macrophage kill tumor cells [14].
Based on the aspects described above and in the continuation of our efforts to
discover small molecules with potential anticancer activity [15-19], the objective of
this work is to synthesize a hybrid series of dihydropyrimidine derivatives containing
oxime as NO release moiety (Scaffold A). This hybridization was intended for
synergistic effect, enhancement of anticancer activity and/or decrease of side effects,
if any.

1
.1.
Rational Design
Activation of VEGFR-2 plays an important role in tumor survival, angiogenesis, and
migration. Over the last two decades, several potent VEGFR-2 inhibitors have been
identified and approved by the FDA for the treatment of different types of cancers.
[
20-22]. To identify the essential binding interactions involved in inhibition of
VEGFR-2, five crystal structures of the enzyme (4ASD, 4AC [22], 3WZD, 3WZD,
WZE [23], 2RL5 [24]) bound to different inhibitors (sorafenib, lenvatinib, 2RL, and
3
axitinib) were analyzed, Fig. 2. Investigation of the binding modes of these inhibitors
using discovery studio Visualizer [25] and LigPlot+ [26] revealed the importance of
hydrogen bonding interaction with Lys868, Glu885, Cys919, and Asp1046. In
addition, the four inhibitors exhibited similar hydrophobic interactions with Val848,
Ala866, Leu889, Val916, Leu1035, Cys1045, and Phe1047 amino acids in VEGFR-2
(Supplementary data, Fig. S1-4).
The general structure of VEGFR-2 inhibitors consists of four distinct moieties [27].
These moieties include hinge-binding moiety, linker (3-5 atoms), hydrogen bonding
moiety (amide/urea) and a hydrophobic tail that occupy the allosteric hydrophobic
pocket of the enzyme, Fig. 2.

Fig. 2. A superimposition plot of sorafenib, lenvatinib, 2RL, axitinib bound to
VEGFR-2 (pdb codes: 4ASD, 4AC, 3WZD, 3WZD, 3WZE, 2RL5), sorafenib is
shown in full purple color and is overlaid on lenvatinib, 2RL and axitinib (grey color),
red circles and ellipses identify the equivalent residues in the five 3D structures,
hydrogen bonding interactions with Lys868, Glu885, Cys919, and Asp1046 shown as
olive green dotted line.
In the current study, scaffold A (Fig. 3) was designed bearing the four
pharmacophoric features of VEGFR-2 inhibitors. The acetyl/hydroxyamino head in
scaffold A was designed to form hydrogen bonding interactions with Cys919.
Compared to sorafenib, the linker in scaffold A was designed to be more flexible and
can interact with VEGFR-2 through both hydrophobic and hydrogen bonding
interactions. Moreover, the amide group (hydrogen bonding moiety) was cyclized into
a dihydropyrimidine ring (B) which can provide multiple hydrogen bonding
interactions with the enzyme. Ring C was also substituted with mono/di/tri-methoxy
groups to occupy the allosteric hydrophobic pocket and interact hydrophobically with
hydrophobic residues in the enzyme.

Fig. 3. Rational design of scaffold A.
2. RESULTS AND DISCUSSION
2
.1. Chemistry
Synthesis of compounds 4a-e is outlined in Scheme 1. Compounds 1a-e were
synthesized by a single pot reaction of benzaldehyde derivatives, ethyl cyanoacetate
and thiourea in presence of anhydrous potassium carbonate according to modified
Biginelli reaction [28] (Scheme 1).

R
R
S
O
NC
H
S
NC
(a)
N
R
N
N
H N
2
NH2
O +
+
O
O
N
H
O
N
H
SH
1a-e
O
O
(c)
O
(b)
Br
H N
2
N
H
p-Aminoacetophenone
2
O
O
NC
NC
NH
NH
H
H
N
N
(d)
N
S
N
S
O
O
N
R
R
OH
O
3a-e
4a-e
R = H, 3-OMe, 4-OMe, 3,4-di OMe, 3,4,5-tri OMe
Scheme 1. Synthesis of compounds 3a-e and 4a-e
Reagent and reaction conditions: a) K CO , absolute ethanol; b) bromoacetyl
2
3
bromide, K CO , CH Cl ; c) acetone, K CO ; d) Hydroxyl amine hydrochloride,
2
3
2
2
2
3
absolute ethanol.
The target compounds 3a-e were prepared by refluxing compounds 1a-e with N-(4-
acetylphenyl)-2-bromoacetamide 2 in the presence of potassium carbonate for 4-8 h.
The structure of 3a-e was confirmed by IR, H NMR, 13C NMR and HRMS (ESI)
spectroscopy. The IR spectra of 3a-e showed significant stretching bands at 3453-
1
-1
-1
3
307 cm assigned to NH, 2212-2193 cm related to (CN), two (C=O) stretching
-1
-1
bands appears at 1674-1699 cm and 1674-1653 cm related to (CO-NH) and (CO-
-1
CH ) respectively. A stretching band at 1653-1592 cm related to (C=N) and 1606-
3
-
1
1
1
592 cm related to (C=C). Compounds 3b-d showed significant stretching band at
-1
1
327-1303 cm related to methoxy groups. H NMR spectra for compounds 3a-e
showed three singlet peaks at δ 11.17-11.29 ppm related to amidic (NH) proton, at δ
.8-3.9 ppm related to (S-CH ) and at δ 2.40-2.50 ppm related to (CO-CH ).
3
2
3

Moreover, compounds 3b and 3c showed singlet peak appeared at δ 3.74-3.75 ppm
related to methoxy groups. Furthermore, compound 3d have tow methoxy groups
showed two singlet peaks at δ 3.74 and 3.76 ppm. Moreover, compound 3e have three
methoxy groups showed two singlets for the three methoxy groups at δ 3.76 ppm and
1
3
at δ 3.69 ppm. C NMR spectra of compounds 3a-e showed characteristic signals at δ
1
2
1
96.50-196.92 ppm (CO-CH ) and a signal related to methyl group appeared at δ
3
6.28-26.75 ppm, (CN) appeared at δ 120.0-120.6 ppm and two signals at δ 166.48-
67.60 ppm and at δ 168.25-168.66 ppm related to cyclic amide and aromatic amide,
respectively. HRMS (ESI) data for compounds 3a-e further confirmed their assigned
structures. The m/z value of molecular ion peak [M-H] for each compound was close
to the calculated one in all cases.
Oxime derivatives 4a-e was prepared by heating at reflux a mixture of 3a-e and
hydroxylamine hydrochloride in absolute ethanol. IR spectra of compounds 4a-e were
-1
characterized by the appearance of intense broad bands at 3588-3300 cm related to
OH and NH groups. In addition to (C=N) and (CO-NH) groups which exhibited
-1
-1
stretching vibration at 1671-1619 cm and 1650-1687 cm , respectively, also the
-1
1
characteristic band of (CN) at 2342-2200 cm . H NMR spectra for oximes 4a-e
showed the appearance of downfield singlets in the range δ 10.45-10.86 ppm related
to the hydroxyl group. The resonances of NH, S-CH , and CH protons were observed
2
3
in the expected regions at δ 11.08-11.2 ppm, δ 3.92-4.21 ppm and δ 2.12-2.14 ppm,
respectively. Moreover, all the aromatic protons appear in their expected chemical
1
3
shift. One of the characteristic features of C NMR spectra of compounds 4a-e is the
disappearance of ketonic carbonyl due to its conversation to ketoxime group (C=N-
OH) which appear at δ 152.8-153.65 ppm. Also, the methyl group attached to
ketoxime appears at δ 11.64-12.07 ppm.

2
2
.2. Biology
.2.1. In vitro one dose full NCI 60 cell panel assay
Compounds 3a, 3b, 3e and 4b have been selected for in vitro anticancer screening by
the National Cancer Institute. The results (Table 1) recorded that compound 3e
exhibited a remarkable anticancer activity against most of the tested cell lines. A
complete cell death was observed against leukemia CCRF-CEM, HL-60(TB) cell
lines, non-small cell lung cancer NCI-H522 cell line, colon cancer COLO205 cell
line, CNS cancer SF-539 cell line, melanoma MDA-MB-435 cell line and breast
cancer MDA-MB-468 cell line with growth percentages of -11.91%, -54.34%, -
1
4.53%, -16.22%, -22.86%, -46.05% and -12.81%, respectively. A significant
anticancer activity has been observed against the tested cell lines of leukemia, non-
small cell lung cancer, colon cancer, melanoma cancer, ovarian cancer, renal cancer,
prostate cancer and breast cancer cell lines with growth inhibition percentages of
5
8.41% up to 99.55% (Table 1). A moderate cell growth inhibition was observed
against the rest of cell lines with growth inhibition percentages of 24.30% up to
8.11%. Finally, compounds 3a, 3b and 4b showed a moderate cell growth inhibition
against most of the tested cell lines with growth inhibition percentages of 20.06% to
6.20%, 20.54% to 32.06% and 20.51% to 41.90%, respectively (Table 1).
4
5

Table 1: Single concentration mean graph growth inhibition of nine different cancer
cell types for compounds 3a, 3b, 3e and 4b
Subpanel cancer cell Lines
% Growth Inhibition (GI %)a
3
a
3b
3e
4b
Leukemia
CCRF-CEM
HL-60(TB)
K-562
15.89
51.46
28.47
28.72
13.93
11.31
-9.5
11.8
1.10
-3.30
-14.4
2.49
111.9
154.3
99.46
96.16
92.53
92.38
-13.0
-6.55
21.08
-5.33
-12.6
9.38
MOLT-4
RPMI-8226
SR
Non-small cell lung cancer
A549/ATCC
EKVX
14.01
-0.77
19.26
56.20
5.64
14.67
-2.14
-0.94
1.68
80.22
66.24
79.28
89.66
41.54
61.61
54.71
94.21
114.5
22.97
-2.49
14.89
9.50
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
9.04
13.18
8.86
-2.77
-15.6
-10.8
21.22
2.83
-0.95
-11.4
32.06
-7.94
-8.72
32.95
6.12
-14.5
37.64
2.31
-14.8
-5.35
23.71
-3.11
-1.38
-1.48
-11.9
116.2
78.98
96.08
59.78
93.18
83.25
74.17
-14.7
-0.39
20.51
-9.07
11.69
0.23
HT29
-4.15
-4.62
3.50
KM12
SW-620
-6.13
CNS cancer
SF-268
-6.20
11.02
5.12
-0.59
4.93
73.15
96.6
7.31
-1.33
9.14
SF-295
SF-539
2.36
122.9
41.78
61.87
83.98
SNB-19
7.65
3.48
8.92
SNB-75
-12.1
10.88
11.73
10.63
9.52
U251
15.65
Melanoma
LOX IMVI
MALME-3M
2.96
2.84
3.84
81.51
30.49
17.24
-1.95
-2.75

M14
16.40
-0.55
-3.79
1.20
3.36
5.25
4.32
12.41
-11.2
-3.68
-0.54
-0.45
15.34
-
94.83
146.0
64.11
42.23
84.75
46.34
64.04
20.6
-5.70
6.38
-3.87
2.13
20.27
-
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
-8.53
-6.48
-3.52
-2.74
6.35
-5.76
-8.04
-7.54
-2.73
8.97
61.55
99.62
38.52
48.11
76.81
24.3
-10.0
-4.14
2.46
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
-8.26
14.95
2.61
-6.05
15.88
-8.37
20.54
65.44
39.51
Renal cancer
7
86-0
11.75
18.04
-0.61
-0.45
-8.64
-0.11
-
5.27
1.90
64.53
81.52
58.41
38.45
75.44
70.53
-
-0.11
-7.72
4.19
A498
ACHN
-0.51
3.76
CAKI-1
12.89
-3.30
7.12
RXF 393
SN12C
-7.57
5.50
TK-10
1.64
12.08
9.64
UO-31
7.90
-5.03
50.65
Prostate cancer
PC-3
39.38
-8.73
11.97
-6.43
81.96
85.87
0.88
DU-145
-1.04
Breast cancer
MCF7
20.93
-6.88
0.42
2.55
4.80
81.79
80.27
89.44
63.15
64.13
112.8
8.36
19.80
3.63
MDA-MB-231/ATCC
HS 578T
BT-549
-14.8
17.77
10.55
-5.25
20.06
22.99
0.83
14.05
41.9
T-47D
MDA-MB-468
9.92

2
.2.2 In vitro five dose full NCI 60 cell panel assay
In this context, compound 3e recorded high activity against most of the cell lines
tested with GI ranging from 19 to 100 μM (Table 2, Fig. 4) and selectivity ratios
5
0
ranging between 0.75 and 1.71 at the GI50 level. Compound 3e was found to be a
broad-spectrum antitumor agent against different tumor subpanels tested without
selectivity to the cell lines tested.
Table 2: In vitro five dose full NCI 60 cell panel assay of 3e
GI50
Subpanel
MIDb
TGI
Panel
Cell line
Conc.
per
Selectivity
ratio
Conc.
per
LC50
a
b
cell line
(MID /MID ) cell line
CCRF-CEM
HL-60(TB)
K-562
31.60
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
35.00
37.70
36.40
29.90
19.00
>100
>100
79.3
>100
>100
Leukemia
3
1.60
1.71
MOLT-4
RPMI-8226
SR
>100
>100
>100
>100
>100
>100
>100
A549/ATCC
EKVX
HOP-62
Non-small
cell lung
cancer
HOP-92
22.60
89.80
>100
30.30
39.10
>100
7
0.14
0.77
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
COLO 205
>100
>100
>100
>100
>100

HCC-2998
HCT-116
HCT-15
HT29
>100
42.10
>100
55.70
63.60
46.70
68.70
43.00
29.60
64.00
28.50
55.60
41.70
48.10
37.40
24.30
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
87.60
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Colon
cancer
0
.75
72.58
KM12
SW-620
SF-268
SF-295
CNS cancer
SF-539
1
.12
SNB-19
SNB-75
U251
48.23
LOX IMVI
MALME-3M
M14
MDA-MB-
4
35
Melanoma
44.54
1.22
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
47.50
31.90
97.40
28.00
53.40
34.00
82.90
85.10
73.70
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-
Ovarian
cancer
6
6.81
0.81

RES
SK-OV-3
38.60
41.70
21.30
85.00
40.10
70.20
96.90
36.60
36.40
62.60
40.00
38.70
>100
>100
85.00
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
7
86-0
A498
ACHN
RXF-393
SN12C
TK-10
Renal
cancer
55.97
0.968
UO-31
PC-3
Prostate
cancer
4
9.5
1.09
1.60
DU-145
MCF7
MDA-MB-
2
31/ATCC
Breast
cancer
BT-549
32.00
34.00
25.40
99.70
>100
>100
>100
>100
>100
3
3.94
T-47D
MDA-MB-
4
68
MIDa
54.16

Fig. 4. Dose-antiproliferative response of compound 3e against nine different cancer
cell lines
2
.2.3. VEGFR-2 Assay
The overexpression of several kinases in endothelial cells indicated its crucial role in
angiogenesis and vasculogenesis of cancers [24]. Of these protein kinases, VEGFR-2
plays an important role in the survival/proliferation of endothelial cells and the
development of cancers [25, 26]. VEGFR-2-kinase was assayed with kinase-glo-
luminescent kinase assays against 3a, 3b, 3e, 4b and Sorafenib as a reference (Table
3
).

Table 3: The enzymatic inhibitory activities against VEGFR-2 for compounds 3a, 3b,
e and 4b.
3
Compound
IC (nµ)
50
3
a
386.4
> 1000
198.7
> 1000
0.17
3
b
3
e
4
b
Sorafenib
Of these, 3a and 3e were the most potent analogues with IC values of 386.4 nM and
5
0
1
98.7 nM against VEGFR-2, respectively, in comparison to Sorafenib (IC = 0.17
50
nM). The results show that introduction of 3,4,5-trimethoxy substituent on the phenyl
ring was favorable the activities while introduction of 4-methoxy substituent or oxime
moiety decrease the activity.
2
2
.2.4. Cell cycle analysis and apoptosis detection
.2.4.1. Cell cycle analysis
Studies were carried out on the effect of the 3e compound on leukemia cell cycle
growth and apoptosis. Leukemia SR cells have been incubated at an IC level of 3e
5
0
for 24 h. The study results (Fig. 5) show that the compound 3e apoptosis levels of
Leukemia SR pre-G1 was 17.34%. In Leukemia SR treated with 3e, a high percent of
cell accumulation was observed in the G2 / M phase (34.36%) indicating cell cyclic
arrest at G2 / M transformation.

Fig. 5. Cell cycle analysis in Leukemia SR cell line treated with compound 3e
2
.2.4.2. Apoptosis assay
Leukemia SR cell cycle research has shown that the signalling of preG1 apoptosis
occurs after 3e action. cells were incubated with annex V/PI for 24 h to check 3e for
apoptosis. Early and late apoptosis studies show that 3e is likely to cause significant
apoptosis with 2.37% necrosis, (Table. 4).
Table 4. Apoptosis detection of 3e in Leukemia SR cancer cell line
Compound
Apoptosis
Early
6.11
Necrosis
Total
17.34
1.79
Late
8.86
0.47
3
e / Leukemia SR
2.37
0.3
Cont. Leukemia SR
1.02

2
.3. Docking study into VEGFR-2
In the present study, compounds 3a and 3e exhibited inhibitory activity against
VEGFR-2 protien kinase with IC50 values in the nanomolar range, Table 3.
Accordingly, a comparative molecular docking study of the new compounds into the
ATP active site of VEGFR-2 (PDB code: 4ASD) was performed. The crystal struture of
the VEGFR-2 tyrosine kinase [22] co-crystallized with sorafenib was obtained from the
protein data bank (https://www.rcsb.org/structure/4asd). The docking study was
performed using AutoDock 4.2. [32]. Ligands and protein files were prepared using
AutoDock tools according to the previous reports [33,34]. Grid and docking parameter
files were prepared according to the previous reports [35,36]. The 2/3D binding modes
of the new compounds sorafenib were generated using discovery studio visualizer [25]
+
and LigPlot (v.2.1) [26].
Validation of the docking procedures was performed by re-coking sorafenib into the
active site of VEGFR-2. The results revealed the superposition of the sorafenib over
the co-crystallized ligand with RMDS of 1.01 Å. In addition, the re-docked sorafenib
showed the same binding interactions with VEGFR-2 like the co-crystallized ligand,
Fig. 6. The re-docked sorafenib exhibited three conventional hydrogen bonds with
Glu885, Cys919, and Asp1046 amino acids. In addition, several hydrophobic
interactions with Val848, Ala866, Leu889, Val916, Leu1035, Cys1045, and Phe1047
amino acids were also observed.

Fig. 6: Binding mode/interactions of redocked/co-crystallized sorafenib into the active of
VEFGR-2 (PDB code: 4ASD): A) 3D binding modes showing superimposition of the re-
docked ligand (Sorafenib, shown as sticks, colored in yellow) over the native
cocrystallized ligand (sorafenib, shown as sticks, colored by element) into the active site
of VEGFR-2, RMSD = 1.01 Å; B) 2D binding mode of the re-docked sorafenib into
VEGFR-2 showing H-bonding and hydrophobic interactions, hydrogen atoms were
omitted for clarity.
The results of the docking study also revealed nice fitting of the new compounds (3a-
e and 4-e) into the active site of VEGFR-2, Fig. 7. The new compounds showed
binding free energies (G ) in the range of -9.80 to -11.25 kcal/mol and inhibition
b
constant (K ) in the range of 5.69-66.05 nM, compared to a binding free energy of -
i
1
2.12 kcal/mol and inhibition constant of 1.31 nM for sorafenib. Generally, the
compounds 4a-e exhibited higher affinities toward VEGFR-2 than their parent
derivatives 3a-e. This could be attributed to the presence of hydroxyimino moiety in
compound 4a-e which formed a cluster of hydrogen bonds with amino acids in
VEGFR-2. The best fit conformation of most of the new compounds exhibited
binding orientations similar to sorafenib and displayed similar binding interactions
with the key amino acids in the active site of VEGFR-2. The 4-(un)substituted-
phenyl-1,6-dihydropyrimidine moiety of the new compounds displayed multiple
hydrophobic interactions similar to the 4-chloro-3-(trifluoromethyl)phenyl moiety in
sorafenib. Moreover, the acetyl/ hydroxyimino moities of the new compounds formed

different hydrogen-bonding interactions like the N-methylcarboxamide moiety in
sorafenib, Fig. 3. However, 3b and 4b exhibited different binding orientations from
sorafenib which can account of their weak inhibitory activity against VEGFR-2,
Table 3.
Fig. 7. 3D binding modes of compounds 3a-e (shown as yellow lines) and compounds
4
a-e (shown as blue lines) overlaid with the native co-recrystallized sorafenib (shown
as sticks colored by element) into the active site of VEGFR-2, receptor shown as H-
bond surface, hydrogen atoms were hidden for clarity
The results of the docking study of compounds 3a-e revealed binding free energy in
the range of -9.80 to -10.86 kcal/mol compared to -12.12 kcal/mol for sorafenib.
Among these derivatives, compound 3e exhibited a binding free energy of -10.19
kcal/mol. These results were quite matched with the IC50 values of sorafenib and
compounds 3, Table 3. Investigation of the binding mode of compound 3e revealed
similar binding orientation/interactions to those of the co-crystallized sorafenib. Three
conventional hydrogen bonds were observed between compound 3e and Lys868,
Glu885, and Asp1046 with bond length in the range of 1.59-2.61Å. Moreover,
compound 3e formed three carbon-hydrogen bonds with Leu840, Phe918, and
CYS919 amino acids with bond length in the range of 1.97-2.30 Å. Like sorafenib,
compound 3e also displayed several hydrophobic interactions with, Val848, Ala866,

Val899, Val916, His1026, Leu1035, and Cys1045 amino acids in VEGFR-2, Fig. 8.
The high similarity between hydrogen bonding/hydrophobic interactions of compound
3
e and sorafenib could account for its high inhibitory activity against VEGFR-2,
Table 3.
Fig. 8. Binding modes/interactions of compound 3e into VEGFR (PDB code: 4ASD):
A) 3D binding mode of compound 3e (shown as stick, colored by element), receptor
surface shown as H-bond donor (pink) and acceptor (light green); B) 2D binding
mode of compound 3e (shown as stick, colored by element); hydrogen atoms were
omitted for clarity.
In addition, compound 3a exhibited three conventional hydrogen bonds with Glu885,
Cys919, and Asp1046 amino acids in VEGFR, Fig. 9. These hydrogen bonds were
identical with the three hydrogen bonds formed by sorafenib, Fig. 6. In addition,
compound 3a showed several types of hydrophobic interactions with Val848, Ala866,
Leu889, Leu1019, Leu1035, Ile1044, and Cys1045 amino acids, like those observed
with sorafenib.

Fig. 9. Binding modes/interactions of compound 3a into VEGFR(PDB code: 4ASD):
A) 3D binding mode of compound 3a (shown as stick, colored by element), receptor
surface shown as H-bond donor (pink) and acceptor (light green); B) 2D binding
mode of compound 3a (shown as stick, colored by element); hydrogen atoms were
omitted for clarity.
On the other hand, the results of the docking study of compounds 3b and 4b revealed
binding free energies of-10.86 and -10.69 kcal/mol. However, compounds 3b and 4b
exhibited weaker inhibitory activities toward VEGFR-2 compared to compounds 3a,e,
Table 3. Molecular docking analyses of the best-fit conformations of compounds 3b
and 4b revealed completely different binding orientations from those of sorafenib and
compounds 3a,e, Fig. 10. This difference in the orientation could account for their
weak inhibitory activity against VEGFR-2.
Fig. 10. 3D Binding modes of compound 3b and 4b into VEGFR(PDB code: 4ASD):
A) 3D binding mode of compound 3b (shown as stick, colored by element), overlaid
with sorafenib (shown as orange line); B) 3D binding mode of compound 4b (shown
as stick, colored by element), overlaid with sorafenib (shown as orange line), receptor
shown as H-bond surface.
Furthermore, LigPlot views of the best fit conformation of compounds 3b and 4b were
generated showing different types of interactions with VEGFR-2, Fig. 11. Compounds
3b and 4b displayed two and five hydrogen bonds with VEGFR-2, respectively. The
fewer number of hydrogen bonds observed for compound 3b could also account for its
weak inhibitory activity against VEGFR-2, Table 3. In addition, compound 4b
displayed one hydrogen bond with Glu885, while the remaining four hydrogen bonds

were formed with Ile1025, His1026, and Arg1027 indicating different interaction
pattern from sorafenib and compounds 3a,e. The difference in the binding interactions
of compound 4b could also account for its weak inhibitory activity toward VEGFR-2.
Fig. 11: LigPlot view of compound 3b and4b:A) LigPlot view of compound 3b into the
binding site ofVEGFR-2 (PDB code: 4ASD) showing two conventional hydrogen
bonds with Glu885 and Asp1046; B) LigPlot view of compound 4binto VEGFR-2
showing five hydrogen bonds with Glu885, Ile1025, His1026 and Arg1027;
hydrophobic interactions shown as brick red dotted lines and hydrogen bonding
interactions shown as olive green dotted lines.

3
- CONCLUSION
A series of novel 1,6-dihydropyrimidine-2-thio derivatives targeting VEGFR-2 were
developed and synthesized. Compounds 3a, 3b, 3e and 4b have been selected for in
vitro anticancer screening by the National Cancer Institute. The results recorded that
compound 3e exhibited a remarkable anticancer activity against most of the tested cell
lines. where a complete cell death was observed against. In vitro five dose full NCI 60
cell panel assay results revealed that 3e showed high activity against most of the cell
lines tested with GI50 ranging from 19 to 100 μM and selectivity ratios ranging
between 0.75 and 1.71 at the GI50 level. Compound 3e was found to be a broad-
spectrum antitumor agent against different tumor subpanels tested without selectivity
to the cell lines tested. A molecular docking study was performed to evaluate binding
modes, affinities and interactions of the new compounds into the active site of
VEGFR-2. The results of the docking study revealed nice fitting of the new
compounds into the active site of VEGFR-2 with binding free energies in the range of
-9.80 to -11.25 kcal/mol compared to -12.12 kcal/mol for sorafenib. Compounds 4a-e
with the hydroxyimino group exhibited higher binding affinities toward VEGFR-2
than their parent derivatives 3a-e. Compound 3a and 3e which showed similar
binding modes and interactions to sorafenib. On the other hand, compounds 3b and
4
b exhibited completely different orientations from sorafenib which resulted in
different interaction patterns into VEGFR-2.

4
4
. EXPERIMENTAL
.1. Chemistry
General details [See appendix A]
Compounds 1a-e and 2 [28] were prepared according to previous reported procedures.
4
.1.1. General procedure for synthesis of N-(4-acetylphenyl)-2-(5-cyano-4-oxo-6-
(substituted phenyl)-1,6-dihydropyrimidin-2-ylthio) acetamide (3a-e)
A mixture of appropriate pyrimidine derivative 1a-e (1 mmol), compound 2 (1 mmol)
and anhydrous K CO (0.167 g, 1.2 mmol) in 50 mL acetone was heated at reflux for
2
3
4
-8 hours, then 20 mL of distilled water was added, the precipitate was filtered off,
washed with distilled water, dried and crystallized from absolute ethanol affording the
target products 3a-e
4
.1.1.1.
N-(4-Acetylphenyl)-2-(5-cyano-4-oxo-6-phenyl-1,6-dihydro-
pyrimidin-2-ylthio)acetamide (3a)
Yellowish white powder in (0.38 g, 95% yield); mp: 200-202 C; IR (KBr), vmax/cm-1;
°
1
3
453 (N-H), 2212 (CN), 1674 (CO-NH), 1592 (C=N); H NMR (DMSO-d ) δ (ppm):
6
2
.49 (s, 3H, CH ), 3.87 (s, 2H, CH ), 7.36-7.46 (m, 3H, Ar-H+NH), 7.67 (d, 2H, J =
3
2
8
.0 Hz, Ar-H), 7.74 (d, 2H, J = 8.00 Hz, Ar-H), 7.89 (d, 2H, J = 8.00 Hz, Ar-H),
1
1.17 (s, 1H, NH); 13C NMR (DMSO-d ) δ (ppm): 26.75, 35.64, 89.83, 118.48,
6
1
20.04, 128.45, 128.49, 129.87, 130.25, 131.92, 137.63, 143.73, 167.60, 168.66,
70.52, 171.82, 196.92. HRMS (ESI) calcd. for C H N O S [M-H]: 403.0870,
1
2
1
15
4
3
found: 403.0872.

4
.1.2.2.
dihydropyrimidin-2-ylthio)acetamide (3b)
Yellow powder in (0.37 g, 86% yield); mp: 195-197 C; IR (KBr), v /cm : 3321
N-(4-Acetylphenyl)-2-(5-cyano-6-(4-methoxyphenyl)-4-oxo-1,6-
°
-1
max
(
N-H), 2204 (CN), 1699 (CO-CH ), 1674 (CO-NH), 1653 (C=N), 1606 (C=C), 1313
3
1
(
C-O). H NMR (DMSO-d ) δ (ppm): 2.4 (s, 3H, CH ), 3.75 (s, 3H, OCH ), 3.84 (s,
6
3
3
2
2
H, CH ), 6.92 (d, 2H, J = 8.00 Hz, Ar-H), 7.68 (d, 2H, J = 8.00 Hz, Ar-H), 7.74 (d,
2
1
3
H, J = 8.0 Hz, Ar-H), 7.90 (2H, d, J = 8.0 Hz, Ar-H), 11.28 (s, 1H, NH); C NMR
(
DMSO-d ) δ (ppm): 26.49, 35.35, 55.30, 88.70, 113.48, 118.14, 120.14, 129.61,
6
1
29.85, 131.62, 143.20, 143.51, 160.67, 166.54, 168.44, 170.23, 171.30, 196.50.
HRMS (ESI) calcd for C H N O S [M-H]: 433.0976, found: 433.0979.
2
2
17
4
4
4
.1.1.3.
N-(4-Acetylphenyl)-2-(5-cyano-6-(3-methoxyphenyl)-4-oxo-1,6-
dihydropyrimidin-2-ylthio)acetamide (3c)
Yellowish white powder in (0.36 g, 84% yield); mp: 205-207 C; IR (KBr), vmax/cm-1:
°
1
3
329 (N-H), 2212 (CN), 1674 (CO-NH), 1595 (C=N), 1303 (C-O); H NMR (DMSO-
d ) δ (ppm): 2.50 (s, 3H, CH ), 3.74 (s, 3H, OCH ), 3.87 (s, 2H, CH ), 7.02 (d, 1H, J
6
3
3
2
=
8.00 Ar-H), 7.30-7.33 (m, 3H, Ar-H), 7.67 (d, 2H, J = 8.0 Hz, Ar-H), 7.89 (d, 2H, J
1
3
=
8.0 Hz, Ar-H), 11.18 (s, 1H, NH), C NMR (DMSO-d ) δ (ppm): 26.54, 35.39,
6
5
1
5.24, 89.70, 113.61, 115.66, 118.27, 119.77, 120.60, 129.39, 129.64, 131.71, 138.74,
43.49, 158.97, 167.18, 168.41, 170.20, 171.53, 196.70. HRMS (ESI) calcd for
C H N O S [M-H]: 433.0976, found: 433.0979.
2
2
17
4
4
4
.1.1.4.
dihydropyrimidin-2-ylthio)acetamide (3d)
Yellowish brown powder in (0.24 g, 51% yield); mp: 180-181 C; IR(KBr), vmax/cm-1:
321 (N-H), 2200 (CN), 1675 (C=O), 1653 (CO-NH), 1646 (C=N), 1606 (C=C),
N-(4-Acetylphenyl)-2-(5-cyano-6-(3,4-dimethoxyphenyl)-4-oxo-1,6-
°
3