New 2,4-disubstituted-2-thiopyrimidines as VEGFR-2 inhibitors: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1002/ardp.201900089

A new series of 2,4-disubstituted-2-thiopyrimidines 6a–t, 9a, and 9b was efficiently designed and synthesized as antiangiogenic and cytotoxic agents. Compounds 6j, 6l, and 6d showed IC₅₀ values of 1.23, 3.78, and 3.84 μM, respectively, against the vascular endothelial growth factor receptor-2 (VEGFR-2). Most of the synthesized 2-thiouracils showed antiproliferative activity against the HepG2 cell line (hepatocellular carcinoma) in the micromolar range, for instance, 9b, 6l, 6m, 6n, and 6j displayed IC₅₀ = 7.92, 8.35, 8.51, 9.59, and 13.06 μM, respectively, relative to sorafenib (III; IC₅₀ = 10.99 μM). Also, compounds 6j, 9a, 6m, and 6s (IC₅₀ = 15.21, 16.96, 17.68, and 18.15 μM, respectively) are the most potent compounds against the UO-31 cell line. Further evaluation of the effect of the synthesized candidates on VEGFR-2 in the HepG2 cell line demonstrated that compounds 6j and 6l exhibit VEGFR-2 inhibitory activity of 87% and 84%, respectively, relative to sorafenib (III; 92%). In silico docking of the synthesized hits into the binding site of VEGFR-2 showed their ability to perform the main binding interactions with the key amino acids in the binding site. Studying the in silico predicted ADME (absorption, distribution, metabolism, and excretion) parameters for the synthesized thiouracils demonstrated that they have favorable pharmacokinetic and drug-likeness properties. These results demonstrate that the 2,4-disubstituted thiouracils 6 and 9 have not only favorable antiangiogenic and antiproliferative activity but also satisfy the criteria required for the development of orally bioavailable drugs. Consequently, they represent a biologically active scaffold that should be further optimized for future discovery of potential hits.

Full text of DOI:10.1002/ardp.201900089

Received: 21 March 2019
Revised: 10 July 2019
Accepted: 17 July 2019
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DOI: 10.1002/ardp.201900089
F U L L P A P E R
New 2,4‐disubstituted‐2‐thiopyrimidines as VEGFR‐2
inhibitors: Design, synthesis, and biological evaluation
Heba T. Abdel‐Mohsen1
Mamdouh M. Ali3
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Adel S. Girgis2
Hoda I. El Diwani1
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Abeer E. E. Mahmoud3
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1Department of Chemistry of Natural and
Microbial Products, Division of
Abstract
Pharmaceutical and Drug Industries Research,
National Research Centre, Cairo, Egypt
A new series of 2,4‐disubstituted‐2‐thiopyrimidines 6a–t, 9a, and 9b was efficiently
designed and synthesized as antiangiogenic and cytotoxic agents. Compounds 6j, 6l,
and 6d showed IC50 values of 1.23, 3.78, and 3.84 μM, respectively, against the
vascular endothelial growth factor receptor‐2 (VEGFR‐2). Most of the synthesized 2‐
thiouracils showed antiproliferative activity against the HepG2 cell line (hepatocel-
lular carcinoma) in the micromolar range, for instance, 9b, 6l, 6m, 6n, and 6j displayed
IC50 = 7.92, 8.35, 8.51, 9.59, and 13.06 μM, respectively, relative to sorafenib (III;
IC50 = 10.99 μM). Also, compounds 6j, 9a, 6m, and 6s (IC50 = 15.21, 16.96, 17.68, and
18.15 μM, respectively) are the most potent compounds against the UO‐31 cell line.
Further evaluation of the effect of the synthesized candidates on VEGFR‐2 in the
HepG2 cell line demonstrated that compounds 6j and 6l exhibit VEGFR‐2 inhibitory
activity of 87% and 84%, respectively, relative to sorafenib (III; 92%). In silico docking
of the synthesized hits into the binding site of VEGFR‐2 showed their ability to
perform the main binding interactions with the key amino acids in the binding site.
Studying the in silico predicted ADME (absorption, distribution, metabolism, and
excretion) parameters for the synthesized thiouracils demonstrated that they have
favorable pharmacokinetic and drug‐likeness properties. These results demonstrate
that the 2,4‐disubstituted thiouracils 6 and 9 have not only favorable antiangiogenic
and antiproliferative activity but also satisfy the criteria required for the
development of orally bioavailable drugs. Consequently, they represent a biologically
active scaffold that should be further optimized for future discovery of potential hits.
2Department of Pesticide Chemistry, National
Research Centre, Cairo, Egypt
3Department of Biochemistry, Division of
Genetic Engineering and Biotechnology,
National Research Centre, Cairo, Egypt
Correspondence
Heba T. Abdel‐Mohsen, Department of
Chemistry of Natural and Microbial Products,
Division of Pharmaceutical and Drug
Industries Research, National Research
Centre, El‐Buhouth St., Dokki, P.O. Box 12622,
Cairo, Egypt.
Email: ht.abdel-mohsen@nrc.sci.eg and
hebabdelmohsen@gmail.com
Funding information
Science and Technology Development Fund,
Grant/Award Number: 15063
K E Y W O R D S
2‐thiopyrimidines, ADME, angiogenesis, hepatocellular carcinoma, VEGFR‐2 inhibitors
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1
INTRODUCTION
the required nutrition and oxygen that support its proliferation,
growth, and metastasis to other sites.[2] The angiogenic process is
Angiogenesis, the induction of new blood vessels from preexisting
ones, is an important physiological process for organ development
during embryogenesis or wound healing in adults. In some diseases,
such as cancer and rheumatoid arthritis, the angiogenic process
escalates beyond the beneficial level giving rise to pathological
angiogenesis.[1] In cancer, new blood vessels supply the tumor with
highly complex and depends on stimulation of a number of growth
factors and receptors.[3,4] Vascular endothelial growth factor (VEGF)
is the key mediator in this process. VEGF acts on one of three
structurally related VEGF‐receptor tyrosine kinase isoforms (VEGFR‐
TK), that is, VEGFR‐1 (FLT1), VEGFR‐2 (KDR/FLK1), and VEGFR‐3
(FLT4). However, there is much evidence that VEGFR‐2 represents
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Arch Pharm Chem Life Sci. 2019;e1900089.
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ABDEL‐MOHSEN ET AL.
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new microvessels, which support tumor proliferation and metasta-
sis.[17] For this reason, down‐regulation of angiogenesis through
inhibition of VEGFR‐2 is considered as a successful approach in
hindering HCC.[18,19] A wide range of antiangiogenic drugs have been
evaluated in clinical studies either as a single or an adjuvant drug in
the treatment of HCC.[19] For instance, sorafenib (III) and pazopanib
(V) were evaluated against HCC (Figures 1 and 2). Only sorafenib (III)
was approved by Food and Drug Administration (FDA) for the
treatment of patients diagnosed with HCC (Figure 1). Sorafenib (III)
was successful in inhibiting angiogenesis, proliferation, and migration
of tumor cells as well as in prolonging the survival of advanced HCC
patients. However, this survival was improved for <1 year and is
highly dependent on the sensitivity of the individuals. Moreover, it
was recognized that some resistance emerged for sorafenib (III).[20]
Hence, there is a challenging need to develop novel antiangiogenic
agents for the successful cure of liver cancer.
Sunitinib
Nintedanib
Lenvatinib
Sorafenib
FIGURE 1 Food and Drug Administration (FDA)‐approved
vascular endothelial growth factor receptor‐2 inhibitors
the main receptor in angiogenesis and vascular permeability.[5–7] For
this reason, there is a long held presumption that inhibition of
VEGFR‐2 results in prevention of pathological vasculogenesis.[8,9]
Over the years, different small molecule VEGFR‐2 inhibitors, for
example, sunitinib (I), nintedanib (II), sorafenib (III), and lenvatinib
(IV) have been approved by FDA for cancer treatments (Figure 1).[10]
Also, many studies have reported the design and synthesis of VEGFR‐
2 inhibitors with promising antiproliferative activity.[11,12] On the
basis of the X‐ray crystal structure of VEGFR‐2/ligand complexes,
VEGFR‐2 blockers are categorized into three main groups. Type I
inhibitors inhibit the target enzyme by binding within the adenosine
triphosphate (ATP)‐binding pocket of active “DFG‐in” conformation
via hydrogen bonding. Type II inhibitors stabilize the inactive “DFG‐
out” conformation by accommodating the ATP‐binding site and
expanding over the gate area into the adjacent allosteric hydrophobic
back pocket. Type III inhibitors act also on the inactive “DFG‐out”
conformation by binding noncompetitively to the hydrophobic back
pocket.[10,13–15]
Pyrimidines are an important class of the heterocyclic system
that exists in different pharmaceuticals.[21] Over the years, different
2‐thiopyrimidines were synthesized and proved to have interesting
antiproliferative activity.[22–27] For instance, we have synthesized a
series of benzimidazole–thiouracil conjugates[25] and quinoxali-
ne–thiouracil conjugates[26] of potent antitumor activity. Also, Taher
and Abou‐Seri[27] showed the promising antitumor properties of 2,4‐
disubstituted‐1,6‐dihydropyrimidine‐5‐carbonitriles. Moreover, many
studies showed the promising antiangiogenic activity of 2,4‐ or 4,6‐
disubstituted pyrimidines.[11,22,28–30] For instance, pazopanib (V) is a
multitargeted antiangiogenic agent that was approved by FDA for
the treatment of renal cancer and soft‐tissue cancer.[11,28] Also,
MPK116 (VI) and MPK121 (VII) (Figure 2) were proved to have
potent antiangiogenic activity through inhibition of EGFR and
VEGFR‐2.[29] Moreover, Hughes et al.[30] reported the promising
VEGFR‐2 inhibitory activity and in vivo antitumor activity of JNJ‐
17029259 (VIII; Figure 2).
Hepatocellular carcinoma (HCC), which is a primary cancer of the
liver, is regarded as one of the most common types of cancer
worldwide and a predominant leading cause of cancer related
mortality.[16] HCC is characterized by exaggerated formation of
Motivated by the previous studies and based on the crystal
structure of the binding site of VEGFR‐2, the main aim of the present
investigation was the design of a new series of 2,4‐disubstituted
pyrimidines as VEGFR‐2 inhibitors to target HCC (Figure 3). Our
Pazobanib (V)
MKP116 (VI)
MKP121
JNJ-17029259 (VIII)
(VII)
FIGURE 2 Known substituted pyrimidines as VEGFR‐2 inhibitors

ABDEL‐MOHSEN ET AL.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
For the synthesis of the target compounds 6a–t, a series of 2‐
thioxopyrimidinones 4a–c were synthesized by one pot reaction of
thiourea (1) and ethyl cyanoacetate (2) with 4‐methylbenzaldehyde
(3a) or 4‐methoxybenzaldehyde (3b) or 2,5‐dimethoxybenzaldehyde
(3c) in the presence of anhydrous K2CO3 to yield 4‐oxo‐2‐thioxo‐6‐(p‐
tolyl)‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4a), 6‐(4‐methoxy-
phenyl)‐4‐oxo‐2‐thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile
(4b), and 6‐(2,5‐dimethoxyphenyl)‐4‐oxo‐2‐thioxo‐1,2,3,4‐tetrahydro-
pyrimidine‐5‐carbonitrile (4c), respectively.[31] Subsequently, 4a–c
were further reacted with different 2‐bromoacetophenones 5a–i
under basic conditions to give the corresponding products 6a–t in
good yields (Scheme 1).
FIGURE 3 Design of 2,4‐disubstituted thiouracils as VEGFR‐2
inhibitors
design strategy was adjusted, so that the thiouracil moiety would
accommodate the gate area of the VEGFR‐2 binding site, whereas
the substituent at the 2‐position extends to the adenine pocket and
the hydrophobic substituent at position 4 would occupy the allosteric
hydrophobic back pocket (Figure 3). Some of the newly synthesized
compounds were evaluated biochemically for their VEGFR‐2 in-
hibitory activity. In addition, newly synthesized compounds were
screened for their antiproliferative activity on the HepG2 cancer cell
line as well as for their effect on the inhibition of VEGFR‐2 in the
HepG2 cell line, and the results were compared with sorafenib (III) as
a standard. Molecular docking studies were also performed to predict
the binding interaction of the synthesized series with the active site
of VEGFR‐2. Additionally, an in silico absorption, distribution,
metabolism, and excretion (ADME) prediction study was performed
to predict the pharmacokinetic properties and the drug‐likeness
nature of the synthesized compounds.
The structures of the synthesized target compounds 6 were
confirmed by different spectroscopic measurements as well as by
elemental analyses. For instance, the infrared (IR) spectrum of 6d was
characterized by the presence of an NH stretching band at
ν = 3,435 cm−1, C≡N band at ν = 2,223 cm−1 in addition to a C═O
band at ν = 1,662 cm−1. The 1H NMR spectrum of 6d showed three
singlets at δH = 2.30, 3.86, and 4.87 ppm assignable to CH3, OCH3,
and CH2 protons, respectively. Four doublets appeared at δH = 7.05
(3J = 8.8 Hz), 7.10 (3J = 8.0 Hz), 7.58 (3J = 8.2 Hz), and 8.01 ppm
(3J = 8.8 Hz) equivalent to aromatic protons. The 13C NMR spectrum
of 6d revealed the appearance of characteristic signals at δC = 21.11
6 hr
4 hr
SCHEME 1 Synthesis of the target 2‐thiouracils 6a–t

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ABDEL‐MOHSEN ET AL.
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SCHEME 2 Synthesis of the target compounds 9a and 9b
(CH3), 38.15 (CH2), 55.76 (OCH3), 92.44 (C–CN), and 191.10 ppm
(CH2C═O), in addition to other carbon signals at δC 114.16, 116.03,
128.67, 128.70, 128.96, 130.90, 132.06, 142.16, 161.14, 163.69,
165.35, and 166.97 ppm corresponding to the molecule.
were further evaluated for their cytotoxic activity on UO‐31 cells from
renal cancer and MCF7 cells from breast cancer to determine their
IC50. Subsequently, the effect of the synthesized compounds on VEGFR‐2
level in HepG2 cell line was determined.
The target compounds 9a and 9b were synthesized by a two‐step
method (Scheme 2). Thiourea (1) was initially reacted with ethyl
benzoylacetate (7) in the presence of anhydrous KOH in ethanol to
yield 6‐phenyl‐2‐thioxo‐2,3‐dihydropyrimidin‐4(1H)‐one (8).[32] This is
followed by the reaction of 8 with 2‐bromo‐4′‐chloroacetophenone (5c)
or 2‐bromoacetylnaphthalene (5i) to yield 9a and 9b, respectively. The
structures of 9a and 9b were also confirmed by various spectroscopic
measurements and by elemental analyses. The IR spectrum of 9b showed
the presence of an NH stretching band at ν = 3,400 cm−1, in addition to a
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2.2.1
VEGFR‐2 kinase assay
Compounds 6c, 6d, 6f, 6h–n, 6p–r, 9a, and 9b were evaluated for
their VEGFR‐2 inhibitory activity using the VEGFR‐2 (KDR)
Kinase Assay Kit (Bioscience). IC50 (μM) of the tested compounds
as well as sorafenib (III) are depicted in Table 1. It was found that
most of the tested compounds showed promising VEGFR‐2
inhibitory activity at the micromolar range. Compounds 6j, 6l,
6d, and 6m are the most potent derivatives (IC50 = 1.23, 3.78,
3.84, and 4.07 μM, respectively), while compounds 6p, 6h, and 9b
revealed slightly less potent VEGFR‐2 inhibitory activity
(IC50 = 4.69, 5.05 and 7.05 μM, respectively). On the other hand,
C═O band at ν = 1,661 cm−1 1H NMR spectrum of 9b showed the
.
appearance of two singlets at δH = 5.01 and 6.64 ppm equivalent to CH2
and CH (pyrimidine), respectively, a triplet at δH = 6.92 ppm equivalent to
two aromatic protons and a triplet at 7.21 ppm equivalent to one
aromatic proton, respectively. The spectrum also displayed two multiplets
between δH = 7.62–7.73 and 8.02–8.05 ppm, integrated to four and three
aromatic protons, respectively, a doublet at δH = 8.13 ppm (3J = 7.9 Hz)
equivalent to two protons, and a singlet at δH = 8.92 ppm corresponding
to H‐1 of the naphthyl moiety.
TABLE 1 Results of in vitro biochemical VEGFR‐2 inhibitory
activity
IC50 (μM)a
on
IC50 (μM)a
on
Entry Compound VEGFR‐2
Entry Compound
VEGFR‐2
1
2
3
4
5
6
7
8
6c
6d
6f
6h
6i
>10
3.84
>10
5.05
>10
1.23
9
6m
4.07
4.23
4.69
>10
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2.2
Biological evaluation
10
11
12
13
14
15
16
6n
The synthesized compounds were evaluated for their in vitro VEGFR‐2
inhibitory activity. Meanwhile, some of the synthesized compounds were
selected by the Developmental Therapeutics Program (DTP) of the
National Cancer Institute (NCI) in the division of cancer treatment and
diagnosis, NIH, Bethesda, MD to be screened for their in vitro
antiproliferative activity at 10 μM. The synthesized compounds were
also evaluated for their antiproliferative activity against the human HCC
(HepG2 cell line). Compounds that show moderate to potent activity
6p
6q
6r
>10
6j
9a
>10
6k
6l
4.30
3.78
9b
7.05
0.10
Sorafenib (III)
aIC50 are presented as mean of two independent experiments.

ABDEL‐MOHSEN ET AL.
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compounds 6c, 6f, 6i, 6q, 6r, and 9a were found to possess weak
activity (IC50 > 10 μM). From the obtained results, it is obvious
that replacement of the 4‐methylphenyl group at position 4 of 6d
TABLE 2 Results of in vitro GI% of selected NCI cell lines at 10 µM
of the synthesized compounds
GI%
(IC50 = 3.84 μM) with the 4‐methoxyphenyl group in 6j (IC50
=
Subpanel
Leukemia
CCRF‐CEM
HL‐60(TB)
K‐562
6c 6f 6g 6h 6i 6j 6k 6l 6s 9a
1.23 μM) resulted in an apparent increase in the activity. No
activity was observed for derivatives exhibiting the p‐cyanophe-
nyl group at the 2‐position, compounds 6c, 6i, and 6r showed
IC50 > 10 μM. Complete loss of activity was observed in the case
of replacement of the 4‐methoxyphenyl group of 6h (IC50 = 5.05
μM) by the 2,5‐dimethoxyphenyl group in 6q (IC50 > 10 μM).
–
–
–
–
–
–
–
12 16
–
–
–
–
–
–
–
–
–
–
–
–
–
16 17 10 18
11 10
16 13
–
–
–
MOLT‐4
–
–
–
12
–
–
–
–
–
SR
L
20 19 13 19 14
11 nd
Non‐small‐cell lung cancer
A549/ATTC
–
–
–
–
–
12
–
15 16 14 11
–
–
–
–
–
–
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2.2.2
In vitro antiproliferative activity
HOP‐62
HOP‐92
–
19 19 20 15 18
In vitro one dose (10–5 M) cytotoxic activity on NCI‐60 cell panel
Some of the synthesized 2‐thiouracils were selected by NCI,
Bethesda, MD (www.dtp.nci.nih.gov) to be evaluated for their
cytotoxic activity at single dose (10−5 M) against full NCI‐60 cancer
cell lines derived from nine different cancer types. Some results of
the growth inhibition percent (GI%) of the treated cells are depicted
in Table 2. The obtained results revealed that the tested 2‐thiouracils
have a different selectivity pattern against the NCI cancer cell lines.
It is obvious that NCI‐H522 from non‐small‐cell lung cancer, SNB‐75
from CNS, UACC‐62 from melanoma, SK‐OV‐3 from ovarian and
UO‐31 from renal cancer are the most sensitive cell lines to the
synthesized compounds. Compound 6c showed a lethal effect on one
of the leukemia cell lines (SR). Compounds 6j and 6k showed a wide
range of activity against most of the tested cell lines.
16 16 18 13 14 16 21 14 20
17 19 20 18 20 12 18 13
18
NCI‐H226
NCI‐H23
–
–
–
–
–
–
–
–
NCI‐H522
Colon cancer
HCT‐116
18 16 13 26 14 13 28 68 20 18
11 12
–
11 17
–
12
–
–
10 13
33 18
CNS cancer
SNB‐75
12 20 22 23 12 31 27
Melanoma
LOX IMVI
UACC‐257
UACC‐62
Ovarian cancer
IGROV1
–
–
–
11 10
–
–
12
–
35
–
–
–
18 10 13 11 10 19
–
10
–
15 17 12 15 27 17 30 17
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
21
–
–
–
–
–
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2.2.3
In vitro cytotoxic activity against HepG2,
OVCAR‐4
OVCAR‐5
15
UO‐31, and MCF7 cell lines
17 17 11 15 14 17
18
30
SK‐OV‐3
Renal cancer
A498
18 12 19 27 15 27 24
The synthesized 2,4‐disubstituted‐2‐thiouracils 6b–t, 9a, and 9b
were evaluated for their in vitro cytotoxic activity against the human
HCC (HepG2 cell line), using the sulforhodamine‐B (SRB) assay and
applying sorafenib (III) as a reference standard.[33,34] The IC50 values
are presented in Table 3. Some compounds were further selected to
be tested for their cytotoxic activity against the renal cancer (UO‐31
cell line) and breast cancer (MCF7 cell line) to determine their IC50
and the results are presented in Table 3.
–
–
–
–
19 16
16 11
–
–
–
–
–
21 12
–
27
–
ACHN
12 10
–
–
–
–
CAK1‐1
SN12C
14 24 14 14 18 14
15 15 10 17 17
27
–
–
–
10
UO‐31
Prostate cancer
23 17 22 21 14 31 23 24 32 26
PC‐3
–
–
–
–
10 17
–
–
10 –
Structure–activity relationship
Breast cancer
MCF7
Most of the synthesized 2‐thiouracils showed antiproliferative activity
against the HepG2 cell line at the micromolar range. Compounds 9b, 6l,
6m, 6n, and 6j displayed IC50 = 7.92, 8.35, 8.51, 9.59, and 13.06 μM,
respectively, relative to sorafenib (III; IC50 = 10.99 μM). On the basis of
the observed IC50 results, it was found that compounds having the 4‐
methoxyphenyl group at C‐4 of the pyrimidine moiety, 6f–n, showed
cytotoxic potency up to IC50 = 8.35 μM against the HepG2 cancer cell line
in comparison to compounds having the 4‐methylphenyl group at C‐4,
6b–e (IC50 = 41.46–105.66 μM) or the 2,5‐dimethoxyphenyl group 6o–t
(IC50 = 22.82–84.12 μM). In series 6f–n, replacement of 4‐H of the
benzoyl moiety of 6f (IC50 = 20.88 μM) by 4‐F in 6g (IC50 = 54.40 μM), 4‐
CN in 6i (IC50 = 65.13 μM), or 4‐phenyl 6k (IC50 = 41.39 μM) decreased
–
–
–
–
–
–
–
10
–
–
11 11 20 12 22
MDA‐MB‐231/ATTC
HS 578T
11
–
–
11
–
–
–
17
–
16 14 20 10 11 17
18 15 23 16 18
15 12
T‐47D
–
–
–
Abbreviations: CNS, central nervous system; GI%, growth inhibition
percent; L, lethal effect; NCI, National Cancer Institute; nd, not
determined; –, growth inhibition is below 10%.
the cell inhibitory activity whereas introduction of 4‐Cl in 6h (IC50
=
15.22 µM) or 4‐methoxy in 6j (IC50 = 13.06 µM) increased the antipro-
liferative activity. The presence of the 2‐methoxybenzoyl moiety at
position 2 of thiouracil in 6l (IC50 = 8.35 µM) or 3,4‐dichlorobenzoyl in 6m

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TABLE 3 Results of in vitro cytotoxic activity of the target
2‐thiouracils against HepG2, UO‐31, and MCF7 cell lines
potent cytotoxicity on the UO‐31 cell line. Compound 6s
(IC50 = 17.36 μM) was found to be the most potent compound against
the MCF7 cell line, while compounds 6l and 6k (IC50 = 20.22 and
24.52 μM) showed less potent cytotoxic activity on MCF7. In contrast,
the thiouracils, 6d, 6e, 6m, and 6n displayed no cytotoxic activity on
MCF7 at 50 μM.
IC50 (μM)a on IC50 (μM)a
IC50 (μM)a
on MCF7
Entry Compound
HepG2
on UO‐31
1
6b
6c
6d
6e
6f
41.46 0.56
52.89 3.80
31.16 2.09 42.08 2.34
28.05 2.44 nd
2
3
105.66 5.11 27.46 1.93 >50
4
81.23 8.90
20.88 0.92
54.40 0.39
15.22 0.85
65.13 3.76
13.06 0.94
41.39 2.11
8.35 0.50
8.51 0.46
9.59 0.36
76.44 1.50
22.82 1.75
57.46 0.67
60.77 0.52
84.12 5.34
52.94 3.90
31.39 1.66
7.92 0.62
26.12 1.80 >50
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2.2.4
In vitro cell based VEGFR‐2 inhibitory
5
30.56 2.55 nd
activity
6
6g
6h
6i
25.76 1.36 nd
The inhibitory activities of the synthesized compounds on VEGFR‐2
in the HepG2 cell line at their previously determined cytotoxicity
IC50 were evaluated using the enzyme‐linked immunosorbent assay
(ELISA) kit (Table 4). It was interesting to find that more than half of
the tested compounds exert moderate to strong inhibition of VEGFR‐
2 ranging from 32% to 87% in comparison to sorafenib (III), which
showed 92% inhibition. Compounds 6j and 6l exerted the most
promising VEGFR‐2 inhibitory activity of 87% and 84%, respectively.
7
23.24 0.78 nd
8
33.57 2.72 nd
9
6j
15.21 0.59 32.39 1.76
26.49 0.27 24.52 2.18
20.34 1.94 20.22 1.05
17.68 1.40 >50
10
11
12
13
14
15
16
17
18
19
20
21
22
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
21.27 2.13 >50
nd
nd
29.53 2.37 nd
Structure–activity relationship
31.07 1.91 29.00 1.43
22.96 1.54 nd
In a similar manner, most of the compounds possessing a 4‐
methoxyphenyl substituent at position 4, 6f–n, showed favorable
18.15 0.89 17.36 1.87
TABLE 4 Effect of the synthesized compounds on the level of
VEGFR‐2 in the HepG2 cell line
Entry Compound Amount of VEGFR‐2 (ng/ml)a % Inhibitionb
nd
nd
9a
9b
16.96 1.23 27.81 1.65
>50
>50
1
6b
126.38 14.00
177.00 21.03
80.37 9.76
167.16 18.33
132.70 15.85
60.75 9.34
173.00 18.20
24.36 2.33
82.11 7.50
29.46 4.27
31.75 4.11
40.36 5.11
72.00 9.11
111.36 9.60
141.50 16.21
182.20 16.49
178.12 19.00
65.21 8.00
46.65 6.82
14.60 1.02
185.20 21.85
32
4
Sorafenib (III) 10.99 0.48
2.51 0.21
4.33 0.35
2
6c
Abbreviation: nd, not detemined.
aIC50 are presented as mean standard error (SE) of three independent
experiments.
3
6d
57
10
28
67
7
4
6f
5
6g
(IC50 = 8.51 µM) resulted in much more promising activity, compared with
unsubstituted phenyl 6f or 4‐substituted phenyl 6g–k derivatives. Also,
the presence of the naphthyl moiety in 6n instead of unsubstituted
phenyl 6f or 4‐substituted phenyl derivatives 6g–k showed better
cytotoxic activity, for example IC50 = 9.59 μM of 6n compared with
IC50 = 13.06–65.13 μM of 6f–k. The same effect was observed in 9a and
9b derivatives, where 9b with a naphthyl moiety showed promising
activity (IC50 = 7.92 μM) in comparison with the 4‐chlorobenzoyl deriva-
tive 9a (IC50 = 31.39 μM). In 6p–t, the presence of the 4‐F group in the
benzoyl moiety of 6p (IC50 = 22.82 μM) is much more favorable over the
4‐Cl, 4‐CN, 4‐OMe, 4‐Ph substituents in 6q–t (IC50 = 52.94–84.12 μM).
On the basis of NCI results and the cytotoxicity results on the HepG2
cell line, some compounds were selected to be further evaluated for their
cytotoxic activity on the UO‐31 cell line from renal cancer and the MCF7
cell line from breast cancer to determine their IC50 and the results are
depicted in Table 3. The obtained results revealed that most of the
tested compounds showed moderate to potent cytotoxic activity on
6
6h
7
6i
8
6j
87
56
84
83
78
61
40
24
2
9
6k
10
11
12
13
14
15
16
17
18
19
20
21
6l
6m
6n
6p
6q
6r
6s
6t
4
9a
65
75
92
–
9b
Sorafenib
DMSO
UO‐31 (IC50 = 15.21–33.57 μM). Compounds 6j, 9a, 6m, and 6s (IC50
=
Abbreviations: DMSO, dimethyl sulfoxide; VEGFR‐2, vascular endothelial
growth factor receptor‐2.
15.21, 16.96, 17.68, and 18.15 μM, respectively) are the most potent
compounds in the tested series. Compounds 6l, 6n, 6r, and 6h
(IC50 = 20.34, 21.27, 22.96, and 23.24 μM, respectively) showed less
aData were expressed as mean standard error (SE) of two experiments.
bPercentage of inhibition as compared with control cancer cells.

ABDEL‐MOHSEN ET AL.
7 of 15
|
inhibitory activity over the ones having the 4‐methylphenyl
substituent, 6b–d or 2,5‐dimethoxyphenyl substituted thiouracils,
6p–t. Compounds 6g and 6i were exceptions, as 6b, 6p, and 6r
TABLE 5 Docking energy scores (S) in kcal/mol of the 2,4‐
disubstituted‐2‐thiouracils 6, 9, and sorafenib (III) in theVEGFR‐2
active site
showed
a higher percentage of VEGFR‐2 inhibition. Also, the
Energy
score (S;
Energy
score (S;
presence of the 4‐ or 2‐methoxybenzoyl group in 6j and 6l, 3,4‐
dichlorobenzoyl in 6m or naphthyl in 6n, showed improved inhibition
of VEGFR‐2 (% inhibition = 87, 84, 83, and 78, respectively). Also, 9b
with the naphthyl moiety showed 75% inhibition compared with the
p‐chlorobenzoyl substituted derivative 9a, which showed 65%
inhibition. In the 6p–t series, compounds 6p and 6q with p‐fluoro
or p‐chlorobenzoyl substituents showed 61% and 40% inhibition,
respectively, while 6r–t showed poor % inhibition of VEGFR‐2.
Entry Compound kcal/mol)
Entry Compound kcal/mol)
1
6a
6b
6c
6d
6e
6f
−13.49
−13.18
−15.20
−13.44
−15.31
−14.03
−13.62
−13.69
−15.41
−13.74
−15.20
−13.97
13
14
15
16
17
18
19
20
21
22
23
6m
6n
−15.28
−14.65
−13.48
−13.97
−13.87
−15.63
−14.19
−15.34
−12.91
−13.83
−15.19
2
3
6o
4
6p
5
6q
6
6r
7
6g
6h
6i
6s
8
6t
|
2.3
Molecular docking studies
9
9a
The interaction of the synthesized 2,4‐disubstituted thiouracils 6a–t,
9a and 9b with human VEGFR‐2 kinase was predicated using in silico
molecular docking studies. The Molecular Operating Environment
(MOE, 2010.10) was utilized for this study and the X‐ray crystal
structure of VEGFR‐2 tyrosine kinase in its inactive conformation
(DFG‐out) co‐crystallized with sorafenib (III) was obtained from the
Protein Data Bank at the Research Collaboration for Structural
Bioinformatics (RCSB; www.rcsb.org; PDB ID: 4ASD).[35] In the
present study, redocking of the co‐crystallized ligand, sorafenib (III)
in the VEGFR‐2 active site was carried out to validate the docking
protocol. This step successfully reproduced the experimental inter-
action of sorafenib (III) with the active‐site hotspots (Glu885,
Cys919, and Asp1046; Figure 4) and showed a root‐mean‐square
deviation of 0.470 Å between the docked pose obtained in the
validation step and the co‐crystallized ligand. This result indicated
the validity of the docking protocol for further docking experiments.
Energy scores (S) ranging from −12.91 to −15.63 kcal/mol in
comparison to sorafenib (III; −15.19 kcal/mol; Table 5) were observed
upon docking of the synthesized 2,4‐disubstituted thiouracils 6 and 9
(for further information, see Supporting Information). Analysis of the
binding modes of the synthesized compounds showed that 2,4‐
10
11
12
6j
9b
6k
6l
Sorafenib
disubstituted‐2‐thiouracils 6a–t, 9a, and 9b have a general binding
pattern to VEGFR‐2 activity. This involves the accommodation of the 4‐
oxopyrimidine moiety in the gate area between the ATP‐binding site
and the allosteric hydrophobic back pocket through hydrogen‐bonding
interactions by its NH and/or C═N functional groups with the side
chain of Glu885, and/or the backbone of Asp1046, respectively. The
benzoyl moiety at position 2 of 2‐thiouracil occupies the adenine
pocket of the ATP‐binding site and is involved in hydrophobic
interactions with Leu840, Val848, Lys868, and Phe1047 amino acids.
The presence of the CN group at the 4‐position of benzoyl moiety has a
great influence on the binding affinity to the VEGFR‐2 binding site as it
is involved in hydrogen bond interaction with the Cys919 in the hinge
region (Figure 5). The phenyl moiety at position 4 extends to the
allosteric hydrophobic back pocket and is involved in hydrophobic
interactions with Leu889, Ile892, Val898, Val899, and Lue1019 that
FIGURE 4 Two‐dimensional diagram of sorafenib (III) in VEGFR‐2
FIGURE 5 Two‐dimensional diagram of 6i in VEGFR‐2 active site
active site (distances in Å)
(distances in Å)

8 of 15
ABDEL‐MOHSEN ET AL.
|
The presence of the diphenyl substituent at position 2 of 6e,
6k, and 6t increases the binding affinity to the binding site of
VEGFR‐2 (energy scores = −15.31, −15.20, and −15.34 kcal/mol,
respectively). This can be attributed to the additional hydro-
phobic interactions with amino acids in the hinge region. Only 6k
showed moderate VEGFR‐2 activity, IC50 = 4.30 μM and 56%
VEGFR‐2 inhibition in HepG2 cell line, whereas 6r showed weak
inhibitory activity.
|
2.3.1
In silico prediction of physicochemical,
pharmacokinetic, and ADME properties
To study the physicochemical and pharmacokinetic properties of the
synthesized 2,4‐disubstituted thiouracils 6a–t, 9a, and 9b, in silico
calculations of the physicochemical and ADME parameters of 6 and 9
were performed employing the SwissADME server.[36,37] Some
selected results are presented in Table 6 (for more details see
Supporting Information). Analysis of the obtained results showed
that the 2,4‐disubstituted thiouracils 6a–t, 9a, and 9b have molecular
weights between 356.83 and 483.54 g/mol, ilogP (octanol–water
partition coefficient)[38] in the range of 2.22–3.15, topological polar
surface area (TPSA) between 88.12 and 154.16 Å and the number of
hydrogen bond acceptors are <10 whereas the number of hydrogen
bond donors are <5. They show moderate water solubility and most
of them show high gastrointestinal tract (GIT) absorption. All the
compounds are not predicted to penetrate the blood–brain barrier,
therefore, no adverse effects are expected in the central nervous
system. Also, they are non‐substrates for P‐glycoprotein (P‐gp),
which is one of the most important efflux transporters that is located
in different organs and participates in pumping of xenobiotic outside
the cells to enhance their clearance.[39]
FIGURE 6 Two‐dimensional diagram of 6j in VEGFR‐2 active site
(distances in Å)
increase the compounds’ binding affinity (Figures 5–7; for more details,
see Supporting Information).
Compounds 6j, 6k, 6l, 6m, and 6n with a p‐methoxy‐substituted
phenyl moiety at position 4 showed binding affinities of −13.74,
−15.20, −13.97, −15.28, and −14.65 kcal/mol, respectively, with a
highly promising VEGFR‐2 inhibitory activity (IC50 = 1.23, 4.30, 3.78,
4.07, and 4.23 μM, respectively) as well as promising VEGFR‐2
inhibitory activity in HepG2 cell line (87%, 56%, 84%, 83%, and 78%,
respectively). Figures 6 and 7 show the 2D binding modes of 6j and 6l
in the VEGFR‐2 binding site.
Although compounds 6c, 6i, and 6r with 4‐cyano‐substituted
phenyl group at position 2 showed high binding affinities, with energy
scores = −15.20, −15.41, and −15.63 kcal/mol, respectively, as well as
good binding modes, they showed unexpected weak practical
VEGFR‐2 inhibitory activity (IC50 > 10 μM).
Most of the tested compounds exhibit promising predicted
physicochemical properties to be orally bioavailable. This can be
easily represented by the bioavailability radar.[37] Taking the
bioavailability radar plot of the most potent compounds 6j and 6l
(Figure 8) as examples, the pink‐colored zone is the optimum space
for six physicochemical properties namely size, lipophilicity, polarity,
solubility, flexibility, and saturation that are optimal for oral
bioavailability.[37] All of the 2,4‐thiouracils are nearly fully located
in the pink area (for more details, see the Supporting Information),
only the degree of saturation is slightly deviated out from the pink
area because the fraction of sp3 hybridized carbons is less than 0.25.
Interestingly, 6a–t, 9a, and 9b fulfill the drug‐likeness rules including
Lipinski’s rule,[40] Veber rule,[41] Ghose‐filter,[42] Egan,[43] and
[44]
Muegge’s
filter and they do not exhibit Pan Assay Interference
substructures.[45]
Inspired by the results of the in silico ADME prediction study,
we can conclude that the synthesized 2,4‐disubstituted thiour-
acils 6a–t, 9a, and 9b not only have a promising cytotoxic and
antiangiogenic activity but also have favorable physicochemical,
pharmacokinetic, and drug‐like properties for future optimization
for drug discovery.
FIGURE 7 Two‐dimensional diagram of 6l in the VEGFR‐2 active
site (distances in Å)

ABDEL‐MOHSEN ET AL.
9 of 15
|
TABLE 6 Results of in silico physicochemical and pharmacokinetic properties of 6a–t, 9a, and 9b
No. of
No. of H‐
bond
No. of H‐
bond
Fraction
Csp3
rotatable
bonds
BBB
P‐gp
Product MW
acceptors
donors
MR
TPSA
iLogP GI absorption permeability
substrate
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
6a
6b
6c
6d
6e
6f
361.42 0.1
379.41 0.1
5
5
5
6
6
6
6
6
6
7
7
7
6
6
7
7
7
7
8
8
5
6
4
5
5
5
4
5
6
5
5
6
5
6
5
5
6
7
6
7
7
6
3
5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
101.41 111.91 2.22 High
101.37 111.91 2.61 High
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
386.43 0.1
391.44 0.14
437.51 0.08
377.42 0.1
395.41 0.1
411.86 0.1
411.86 0.1
407.44 0.14
453.51 0.08
407.44 0.14
446.31 0.1
427.48 0.08
407.44 0.14
425.43 0.14
441.89 0.14
432.45 0.14
437.47 0.18
483.54 0.11
356.83 0.06
427.48 0.08
106.13 135.7
2.5
Low
107.9 121.14 2.49 High
126.85 111.91 3.15 Low
102.94 121.14 2.26 High
6g
6h
6i
102.9
121.14 2.52 High
107.95 121.14 2.43 High
107.95 121.14 2.43 High
109.43 130.37 2.44 High
128.37 121.14 3.15 Low
109.43 130.37 2.47 High
112.96 121.14 3.05 Low
120.44 121.14 2.65 High
109.43 130.37 2.39 High
109.39 130.37 2.74 Low
114.44 130.37 3.04 Low
114.14 154.16 2.73 Low
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
115.92 139.6
134.87 130.37 3.13 Low
96.74 88.12 2.55 High
120.44 121.14 2.65 High
2.67 Low
9a
9b
Abbreviations: BBB, blood–brain barrier; MR, magnetic resonance; P‐gp, P‐glycoprotein; TPSA, topological polar surface area.
FIGURE 8 Bioavailability radar plot from SwissADME online web tool for 6j and 6l. ADME, absorption, distribution, metabolism, and
excretion
|
3
CONCLUSION
inhibitory activity as well as interesting antiproliferative activity on
HepG2 and UO‐31 cell lines. Compounds 6j and 6l showed VEGFR‐2
inhibitory activity of IC50 = 1.23 and 3.78 μM, respectively, and
IC50 = 13.06 and 8.35 μM, respectively, against HepG2 cells. The
same derivatives 6j and 6l were found also to have potent VEGFR‐2‐
In this manuscript, new mimics of 2‐thiouracils were designed and
synthesized as antiangiogenic and cytotoxic agents. The synthesized
compounds were proved to have moderate to potent VEGFR‐2

10 of 15
ABDEL‐MOHSEN ET AL.
|
inhibitory activity of 87% and 84%, respectively, relative to sorafenib
(III; % inhibition = 92%) on VEGFR‐2 in the HepG2 cell line. Molecular
docking of the synthesized series on the active site of VEGFR‐2
rationalized the promising VEGFR‐2 activity of this series to the
hydrogen‐bonding interactions with Glu885 and Asp1046 in the gate
area of the binding site. This is in addition to hydrophobic
interactions by their substitutions at position 2 with the hydrophobic
side chains of Leu840, Val848, Lys868, and Phe1047 amino acids in
the hinge region. The binding affinity was further increased by
accommodation of the phenyl group at position 4 of the thiouracil
moiety in the allosteric hydrophobic back pocket of the active site
and their involvement in hydrophobic interactions with the hydro-
phobic side chains of amino acids Ile888, Leu889, Ile892, Val898,
Val899, and Leu1019. In silico prediction of physicochemical,
pharmacokinetic properties of the 2,4‐disbstituted thiouracils
showed that they are promising candidates for drug discovery.
methanol to give the target compounds 6a–t, 9a, and 9b in good
yields.
6‐Oxo‐2‐((2‐oxo‐2‐phenylethyl)thio)‐4‐(p‐tolyl)‐1,6‐dihydropyrimi-
dine‐5‐carbonitrile (6a)
According to the general procedure, 4‐oxo‐2‐thioxo‐6‐(p‐tolyl)‐
1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4a; 243.28 mg, 1 mmol),
2‐bromoacetophenone (5a; 199.04 mg, 1 mmol), and anhydrous
K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol (20 ml) to
produce 6a (CAS number 791828‐69‐8) as a pale yellow powder
(189 mg, 52%), mp 220–222°C; Rf = 0.25 (pet ether/EtOAc = 1:5).
2‐((2‐(4‐Fluorophenyl)‐2‐oxoethyl)thio)‐6‐oxo‐4‐(p‐tolyl)‐1,6‐dihy-
dropyrimidine‐5‐carbonitrile (6b
)
According to the general procedure, 4‐oxo‐2‐thioxo‐6‐(p‐tolyl)‐
1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4a; 243.28 mg, 1 mmol),
2‐bromo‐4′‐fluoroacetophenone (5b; 217.0 mg, 1 mmol) and anhy-
drous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol (20 ml) to
produce 6b as a pale yellow powder (212 mg, 56%); mp 245–247°C;
Rf = 0.24 (pet ether/EtOAc = 1:4); δH (500 MHz; DMSO‐d6) 2.30 (3H,
s, CH3), 4.91 (2H, s, CH2), 7.10 (2H, d, 3J = 8.1 Hz), 7.37 (2H, t‐like,
3J = 8.9 Hz), 7.55 (2H, d, 3J = 8.2 Hz), and 8.11 ppm (2H, dd,
3J = 8.9 Hz, 3J = 5.5 Hz); δC (125 MHz; DMSO‐d6) 21.00, 38.24,
92.44, 115.88 (d, J = 21.3 Hz), 115.90, 128.54, 128.80, 131.43 (d,
J = 10.0 Hz), 131.98, 132.50 (d, J = 1.3 Hz), 142.01, 161.03, 164.31,
165.98 (d, J = 218.8 Hz), 166.32, and 191.38 ppm; Anal. Calcd for
C20H14FN3O2S: C, 63.31; H, 3.72; N, 11.08. Found: C, 63.64; H, 3.95;
N, 11.36.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General remarks
All chemicals were purchased from commercial suppliers. Analytical
thin‐layer chromatography was performed on precoated silica gel 60
F245 aluminium plates (Merck) with visualization under ultraviolet
light. Melting points (mps) were determined on a Stuart SMP30
melting‐point apparatus with open capillary tubes and are uncor-
rected. Elemental and spectral analyses of the compounds were
performed in the Microanalytical Laboratory, National Research
Centre, and the Microanalytical Unit, Faculty of Pharmacy, Cairo
University, Egypt. IR spectra (4,000–400 cm−1) were recorded using
KBr pellets in a Jasco FT/IR 300E Fourier transform infrared
spectrophotometer. 1H NMR and 13C NMR spectra were recorded
at 300/400/500 (75/100/125) MHz on a Bruker instrument using
DMSO‐d6 as a solvent. Chemical shifts are given in parts per million
(ppm) relative to TMS as internal standards. Coupling constants are
reported in Hertz.
2‐((2‐(4‐Cyanophenyl)‐2‐oxoethyl)thio)‐6‐oxo‐4‐(p‐tolyl)‐1,6‐dihy-
dropyrimidine‐5‐carbonitrile (6c)
According to the general procedure, 4‐oxo‐2‐thioxo‐6‐(p‐tolyl)‐
1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4a; 243.28 mg, 1 mmol),
2‐bromo‐4′‐cyanoacetophenone (5d; 224.1 mg, 1 mmol), and anhy-
drous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol (20 ml) to
produce 6c as a white powder (251 mg, 65%); mp 242–244°C;
Rf = 0.20 (pet ether/EtOAc = 1:4); δH (500 MHz; DMSO‐d6) 2.31 (3H,
s, CH3), 4.92 (2H, s, CH2), 7.07 (2H, d, 3J = 8.1 Hz), 7.49 (2H, d,
3J = 8.1 Hz), 7.98 (2H, d, 3J = 8.3 Hz), and 8.12 ppm (2H, d, 3J = 8.4 Hz);
δC (125 MHz; DMSO‐d6) 21.12 (CH3), 38.20 (CH2), 92.44, 115.44,
115.77, 118.10, 128.40, 128.76, 128.86, 129.27, 132.77, 139.06,
141.88, 164.88, 166.98, 169.59, and 192.52 ppm; Anal. Calcd for
C21H14N4O2S: C, 65.27; H, 3.65; N, 14.50. Found: C, 65.38; H, 3.86;
N, 14.39.
The InChI codes, together with some biological activity data, are
provided as Supporting Information.
|
4.1.2
General procedure for the synthesis of 6a–t,
9a, and 9b
A suspension of 4‐oxo‐2‐thioxo‐6‐substituted‐1,2,3,4‐tetrahydropyr-
imidine‐5‐carbonitrile 4 (1 mmol), or 6‐phenyl‐2‐thioxo‐2,3‐dihydro-
pyrimidin‐4(1H)‐one (8; 1 mmol), 2‐bromoacetophenones 5 (1 mmol)
and anhydrous K2CO3 (138.21 mg, 1 mmol) in ethanol (20 ml) was
refluxed for 4 hr. The reaction mixture was cooled to room
temperature (rt) and poured on water and neutralized with 2 N
HCl. The precipitated product was filtered and dried to give the
crude product, which was further purified by crystallization from
2‐((2‐(4‐Methoxyphenyl)‐2‐oxoethyl)thio)‐6‐oxo‐4‐(p‐tolyl)‐1,6‐dihy-
dropyrimidine‐5‐carbonitrile (6d)
According to the general procedure, 4‐oxo‐2‐thioxo‐6‐(p‐tolyl)‐
1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4a; 243.28 mg, 1 mmol),
2‐bromo‐4′‐methoxyacetophenone (5e; 229.1 mg, 1 mmol), and an-
hydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol (20 ml)
to produce 6d as a white powder (250 mg, 64%); mp 243–245°C;
Rf = 0.26 (pet ether/EtOAc = 1:4); ύ max (atr)/cm−1 3,435 (NH), 3,077,

ABDEL‐MOHSEN ET AL.
11 of 15
|
2,915 (CH), 2,223 (CN), 1,662 (C═O), 1,538, and 1,473; δH (400 MHz;
DMSO‐d6) 2.30 (3H, s, CH3), 3.86 (3H, s, OCH3), 4.87 (2H, s, CH2),
7.05 (2H, d, 3J = 8.8 Hz), 7.10 (2H, d, 3J = 8.0 Hz), 7.58 (2H, d,
3J = 8.2 Hz), and 8.01 ppm (2H, d, 3J = 8.8 Hz); δC (100 MHz; DMSO‐
d6) 21.11 (CH3), 38.15 (CH2), 55.76 (OCH3), 92.44, 114.16, 116.03,
128.67, 128.70, 128.96, 130.90, 132.06, 142.16, 161.14, 163.69,
165.35, 166.97, and 191.10 ppm; Anal. Calcd for C21H17N3O3S: C,
64.44; H, 4.38; N, 10.73. Found: C, 64.75; H, 4.12; N, 10.98.
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6h as a pale yellow powder (190 mg, 46%); mp
233–235°C; Rf = 0.29 (pet ether/EtOAc = 1:5); ύ max (atr)/cm−1 3,125
(CH), 2,213 (CN), 1,654 (C═O), 1,575, and 1,480; δH (300 MHz;
DMSO‐d6) 3.77 (3H, s, OCH3), 4.84 (2H, s, CH2), 6.77 (2H, d,
3J = 8.8 Hz), 7.60 (2H, d, 3J = 8.8 Hz), 7.75 (2H, d, 3J = 8.5 Hz), and
7.95 ppm (2 H, d, 3J = 8.5 Hz); Anal. Calcd for C20H14ClN3O3S: C,
58.33; H, 3.43; N, 10.20. Found: C, 58.14; H, 3.58; N, 10.59.
2‐((2‐((1,1′‐Biphenyl)‐4‐yl)‐2‐oxoethyl)thio)‐6‐oxo‐4‐(p‐tolyl)‐1,6‐di-
hydropyrimidine‐5‐carbonitrile (6e)
2‐((2‐(4‐Cyanophenyl)‐2‐oxoethyl)thio)‐4‐(4‐methoxyphenyl)‐6‐oxo‐
1,6‐dihydropyrimidine‐5‐carbonitrile (6i
)
According to the general procedure, 4‐oxo‐2‐thioxo‐6‐(p‐tolyl)‐
1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4a; 243.28 mg, 1 mmol),
2‐bromo‐4′‐phenylacetophenone (5f; 275.14 mg, 1 mmol), and anhy-
drous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol (20 ml) to
produce 6e as a pale yellow powder (166 mg, 38%); mp 210–212°C;
Rf = 0.18 (pet ether/EtOAc = 1:2); ύ max (atr)/cm−1, 3,432 (NH),
3,029, 2,974 (CH), 2,214 (CN), 1,694 and 1,641 (C═O), 1,534, and
1,468; δH (400 MHz; DMSO‐d6) 2.22 (3H, s, CH3), 4.83 (2H, s, CH2),
7.07 (2H, d, 3J = 8.0 Hz), 7.44–7.46 (1H, m), 7.50–7.56 (4H, m), 7.76
(2H, d, 3J = 7.4 Hz), 7.81 (2H, d, 3J = 8.3 Hz), and 8.10 ppm (2H, d,
3J = 8.1 Hz); Anal. Calcd for C26H19N3O2S: C, 71.38; H, 4.38; N, 9.60.
Found: C, 71.14; H, 4.66; N, 9.54.
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromo‐4′‐cyanoacetophenone (5d; 224.05 mg, 1 mmol),
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6i as a pale yellow powder (200 mg, 50%); mp
195–197°C; Rf = 0.28 (pet ether/EtOAc = 1:5); ύ max (atr)/cm−1 3,434
(NH), 3,085, and 2,926 (CH), 2,211 (CN), 1,696 and 1,651 (C═O),
1,542 and 1,474; δH (400 MHz; DMSO‐d6) 3.78 (3H, s, OCH3), 4.62
(2H, s, CH2), 6.86 (2H, d, 3J = 8.6 Hz), 7.61 (2H, d, 3J = 8.6 Hz), 7.98
(2H, d, 3J = 8.2 Hz), 8.14 (2H, d, 3J = 8.1 Hz); δC (100 MHz; DMSO‐d6)
38.87, 55.97, 85.54, 114.07, 114.39, 114.86, 118.97, 129.56, 129.59,
130.51, 131.12, 133.37, 161.53, 162.26, 163.07, 167.50, and 171.00;
Anal. Calcd for C21H14N4O3S: C, 62.68; H, 3.51; N, 13.92. Found: C,
62.65; H, 3.86; N, 13.74.
4‐(4‐Methoxyphenyl)‐6‐oxo‐2‐((2‐oxo‐2‐phenylethyl)thio)‐1,6‐dihy-
dropyrimidine‐5‐carbonitrile (6f
)
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromoacetophenone (5a; 199.02 mg, 1 mmol), and anhy-
drous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol (20 ml) to
produce 6f (CAS Number 540773‐82‐8) as a pale yellow powder
(208 mg, 55%); mp 229–231°C; Rf = 0.28 (pet ether/EtOAc = 1:5).
4‐(4‐Methoxyphenyl)‐2‐((2‐(4‐methoxyphenyl)‐2‐oxoethyl)thio)‐6‐
oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6j)
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromo‐4′‐methoxyacetophenone (5e; 229.07 mg, 1 mmol)
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted ethanol
(20 ml) to produce 6j as a pale yellow powder (200 mg, 49%); mp
243–245°C; Rf = 0.23 (pet ether/EtOAc = 1:5); ύ max (atr)/cm−1 3,443
(NH), 3,125 and 2,920 (CH), 2,221 (CN), 1,654 (C═O), 1,541, and
1,468; δH (500 MHz; DMSO‐d6) 3.79 (3H, s, OCH3), 3.88 (3H, s,
OCH3), 4.88 (2H, s, CH2), 6.83 (2H, d, 3J = 8.0 Hz), 7.09 (2H, d,
3J = 8.0 Hz), 7.71 (2H, d, 3J = 8.0 Hz), 8.04 (2H, d, 3J = 8.0 Hz); δC
(125 MHz; DMSO‐d6) 38.44 (CH2), 55.88 (OCH3), 56.13 (OCH3),
92.44, 114.11, 114.53, 116.68, 127.70, 129.10, 131.12, 131.29,
161.81, 162.49, 164.03, 165.86, 167.59, and 191.51; Anal. Calcd for
C21H17N3O4S: C, 61.91; H, 4.21; N, 10.31. Found: C, 61.78; H, 4.45;
N, 10.53.
2‐((2‐(4‐Fluorophenyl)‐2‐oxoethyl)thio)‐4‐(4‐methoxyphenyl)‐6‐oxo‐
1,6‐dihydropyrimidine‐5‐carbonitrile (6g)
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromo‐4′‐fluoroacetophenone (5b; 217.04 mg, 1 mmol),
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6g as a pale yellow powder (170 mg, 43%); mp
231–233°C; Rf = 0.25 (pet ether/EtOAc = 1:5); ύ max (atr)/cm−1 3,401
(NH), 3,145 and 2,927 (CH), 2,217 (CN), 1,655 (C═O), 1,501, and
1,459; δH (400 MHz; DMSO‐d6) 3.77 (3H, s, OCH3), 4.90 (2H, s, CH2),
6.81 (2H, d, 3J = 8.7 Hz), 7.38 (2H, t, 3J = 8.7 Hz), 7.67 (2H, d,
3J = 8.6 Hz), and 8.11–8.34 ppm (2H, m); Anal. Calcd for
C20H14FN3O3S: C, 60.75; H, 3.57; N, 10.63. Found: C, 60.97; H,
3.32; N, 10.68.
2‐((2‐((1,1′‐Biphenyl)‐4‐yl)‐2‐oxoethyl)thio)‐4‐(4‐methoxyphenyl)‐6‐
oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6k
)
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromo‐4′‐phenylacetophenone (5f; 275.14 mg, 1 mmol),
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6k as a pale yellow powder (240 mg, 53%); mp
218–220°C; Rf = 0.27 (pet ether/EtOAc = 1:5); δH (500 MHz; DMSO‐
d6) 3.66 (3H, s, OCH3), 4.96 (2H, s, CH2), 6.77 (2H, d, 3J = 8.0 Hz), 7.44
2‐((2‐(4‐Chlorophenyl)‐2‐oxoethyl)thio)‐4‐(4‐methoxyphenyl)‐6‐oxo‐
1,6‐dihydropyrimidine‐5‐carbonitrile (6h)
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromo‐4′‐chloroacetophenone (5c; 233.49 mg, 1 mmol),

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(1H, t, 3J = 8.0 Hz), 7.52–7.55 (2H, m), 7.67–7.69 (2H, m), 7.79–7.81
(2H, m), 7.87 (2H, d, 3J = 8.0 Hz), and 8.14 (2H, d, 3J = 8.1 Hz); δC
(125 MHz; DMSO‐d6) 38.60 (CH2), 55.75 (OCH3), 91.85, 114.05,
115.30, 127.35, 127.44, 127.50, 129.04, 129.56, 129.63, 131.09,
135.04, 139.19, 145.47, 162.44, 165.25, 166.57, 169.43, and 192.86;
Anal. Calcd for C26H19N3O3S: C, 68.86; H, 4.22; N, 9.27. Found: C,
68.65; H, 4.39; N, 9.47.
4‐(2,5‐Dimethoxyphenyl)‐6‐oxo‐2‐((2‐oxo‐2‐phenylethyl)thio)‐1,6‐di-
hydropyrimidine‐5‐carbonitrile (6o)
According to the general procedure, 6‐(2,5‐dimethoxyphenyl)‐4‐oxo‐
2‐thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4c; 289.31 mg,
1 mmol), 2‐bromoacetophenone (5a; 199.4 mg, 1 mmol), and anhy-
drous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol (20 ml) to
produce 6o as
a
canary yellow powder (150 mg, 37%); mp
max (atr)/cm−1
224–226°C; Rf = 0.25 (pet ether/EtOAc = 1:5);
ύ
4‐(4‐Methoxyphenyl)‐2‐((2‐(2‐methoxyphenyl)‐2‐oxoethyl)thio)‐6‐
3,441 (NH), 3,110 and 2,927 (CH), 2,226 (CN), 1,644 (C═O), 1,537,
and 1,468; δH (500 MHz; DMSO‐d6) 3.48 (3H, s, OCH3), 3.69 (3H, s,
OCH3), 4.89 (2H, s, CH2), 6.72 (1H, br.), 6.96–7.02 (2H, m), 7.46 (2H,
t, 3J = 7.7 Hz), 7.62 (1H, t, 3J = 7.4 Hz), and 7.95 (2H, d, 3J = 7.6 Hz); δC
(125 MHz; DMSO‐d6) 38.95 (CH2), 55.71, 56.11, 96.93, 113.52,
114.98, 115.49, 118.36, 125.39, 128.71, 129.15, 134.04, 136.15,
150.84, 153.11, 160.96, 166.13, 166.67, and 193.14 ppm; Anal. Calcd
for C21H17N3O4S: C, 61.91; H, 4.21; N, 10.31. Found: C, 61.64; H,
4.57; N, 10.12.
oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6l
)
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromo‐2′‐methoxyacetophenone (5g; 229.1 mg, 1 mmol),
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6l as a pale yellow powder (150 mg, 37%); mp
163–165°C; Rf = 0.23 (pet ether/EtOAc = 1:5); δH (300 MHz; DMSO‐
d6) 3.79 (3H, s, OCH3), 3.88 (3H, s, OCH3), 4.45 (2H, s, CH2), 6.93 (2H,
d, 3J = 8.7 Hz), 7.00 (1H, t, 3J = 7.6 Hz), 7.17 (1H, d, 3J = 8.3 Hz), 7.55
(2H, d, 3J = 7.5 Hz), and 7.72 ppm (2H, d, 3J = 8.6 Hz); δC (75 MHz;
DMSO‐d6) 38.20 (CH2), 55.24, 55.83, 88.07, 112.27, 113.31, 120.34,
120.46, 127.07, 129.66, 129.73, 129.90, 133.70, 158.05, 160.50,
165.89, 170.43, 170.85, and 196.46 ppm; Anal. Calcd for
C21H17N3O4S: C, 61.91; H, 4.21; N, 10.31. Found: C, 61.76; H,
4.45; N, 10.47.
4‐(2,5‐Dimethoxyphenyl)‐2‐((2‐(4‐fluorophenyl)‐2‐oxoethyl)thio)‐6‐
oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6p)
According to the general procedure, 6‐(2,5‐dimethoxyphenyl)‐4‐oxo‐
2‐thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4c; 289.31 mg,
1 mmol), 2‐bromo‐4′‐fluoroacetophenone (5b; 217.04 mg, 1 mmol)
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6p as a yellow powder (180 mg, 42%); mp
227–229°C; Rf = 0.37 (pet ether/EtOAc = 1:5); ύ max (atr)/cm−1 3,439
(NH), 3,076, 3,014, 2,928 (CH), 2,229 (CN), 1,693 and 1,661 (C═O),
1,540 (C═N), and 1,496; δH (400 MHz; DMSO‐d6) 3.53 (3 H, s, OCH3),
3.69 (3H, s, OCH3), 4.84 (2H, s, CH2), 6.65 (1H, s), 6.98–7.00 (2H, m),
7.26 (2H, t, 3J = 8.8 Hz), 8.01–8.04 (2H, m); Anal. Calcd for
C21H16FN3O4S: C, 59.29; H, 3.79; N, 9.88. Found: C, 59.54; H,
3.97; N, 9.65.
2‐((2‐(3,4‐Dichlorophenyl)‐2‐oxoethyl)thio)‐4‐(4‐methoxyphenyl)‐6‐
oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6m)
According to the general procedure, 4‐(4‐methoxyphenyl)‐6‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromo‐3′,4′‐dichloroacetophenone (5g; 267.93 mg,
1 mmol) and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted
in ethanol (20 ml) to produce 6m as a pale yellow powder (250 mg,
56%); mp 223–225°C; Rf = 0.30 (pet ether/EtOAc = 1:5); ύ max (atr)/
cm−1 3,379 (NH), 2,907 (CH), 2,215 (CN), 1,674 (C═O), 1,578, and
1,480; δH (300 MHz; DMSO‐d6) 3.79 (3H, s, OCH3), 4.79 (2H, s, CH2),
6.81 (2H, d, 3J = 8.0 Hz), 7.62 (2H, d, 3J = 8.0 Hz), 7.80 (1H, d,
3J = 8.1 Hz), 7.95 (1 H, d, 3J = 7.9 Hz), 8.21 (1H, s); Anal. Calcd for
C20H13Cl2N3O3S: C, 53.82; H, 2.94; N, 9.42. Found: C, 53.52; H, 2.69;
N, 9.73.
2‐((2‐(4‐Chlorophenyl)‐2‐oxoethyl)thio)‐4‐(2,5‐dimethoxyphenyl)‐6‐
oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6q
)
According to the general procedure, 6‐(2,5‐dimethoxyphenyl)‐4‐oxo‐
2‐thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4c; 289.31 mg,
1 mmol), 2‐bromo‐4′‐chloroacetophenone (5c; 233.5 mg, 1 mmol) and
anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6q as a white powder (210 mg, 48%); mp
236–238°C; Rf = 0.18 (pet ether/EtOAc = 1:5); δH (500 MHz;
DMSO‐d6) 3.54 (3H, s, OCH3), 3.69 (3H, s, OCH3), 4.83 (2H, s,
CH2), 6.64 (1H, s, CH), 6.97–7.01 (2H, m), 7.49 (2H, d, 3J = 8.5 Hz),
and 7.94 (2H, d, 3J = 8.5 Hz); δC (125 MHz; DMSO‐d6) 38.20 (CH2),
55.30, 55.65, 96.49, 112.93, 114.87, 114.96, 117.35, 124.88, 128.71,
130.06, 134.51, 138.44, 150.28, 152.59, 160.53, 165.52, 166.21, and
192.08; Anal. Calcd for C21H16ClN3O4S: C, 57.08; H, 3.65; N, 9.51.
Found: C, 57.24; H, 3.33; N, 9.72.
4‐(4‐Methoxyphenyl)‐2‐((2‐(naphthalen‐2‐yl)‐2‐oxoethyl)thio)‐6‐oxo‐
1,6‐dihydropyrimidine‐5‐carbonitrile (6n)
According to the general procedure, 6‐(4‐methoxyphenyl)‐4‐oxo‐2‐
thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4b; 259.28 mg,
1 mmol), 2‐bromoacetylnaphthalene (5i; 249.11 mg, 1 mmol), and
anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6n as a pale yellow powder (210 mg, 49%); mp
190–192°C; Rf = 0.28 (pet ether/EtOAc = 1:5); ύ max (atr)/cm−1 3,476
(NH), 2,916 (CH), 2,207 (CN), 1,672 (C═O), 1,551, and 1,467; δH
(300 MHz; DMSO‐d6) 3.67 (3H, s, OCH3), 4.77 (2H, s, CH2), 6.70 (2H,
d, 3J = 8.7 Hz), 7.62–7.68 (4H, m), 8.01 (3H, br.), 8.07 (1H, d,
3J = 7.9 Hz), and 8.86 (1H, s); Anal. Calcd for C24H17N3O3S: C,
67.43; H, 4.01; N, 9.83. Found: C, 67.31; H, 4.36; N, 9.65.
2‐((2‐(4‐Cyanophenyl)‐2‐oxoethyl)thio)‐4‐(2,5‐dimethoxyphenyl)‐6‐
oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6r)
According to the general procedure, 6‐(2,5‐dimethoxyphenyl)‐4‐oxo‐
2‐thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4c; 289.31 mg,

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1 mmol), 2‐bromo‐4′‐cyanoacetophenone (5d; 224.1 mg, 1 mmol), and
anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6r as a pale yellow powder (215 mg, 50%); mp
200–202°C; Rf = 0.20 (pet ether/EtOAc = 1:5); δH (400 MHz; DMSO‐
d6) 3.56 (3H, s, OCH3), 3.68 (3H, s, OCH3), 4.82 (2H, s, CH2), 6.54 (1H,
s, CH), 6.98 (2H, br.), 7.87 (2H, d, 3J = 8.1 Hz), and 8.02 ppm (2H, d,
3J = 8.1 Hz); Anal. Calcd for C22H16N4O4S: C, 61.10; H, 3.73; N, 12.96.
Found: 61.38; H, 3.54; N, 12.65.
for C18H13ClN2O2S: C, 60.59; H, 3.67; N, 7.85. Found: C, 60.23; H,
3.25; N, 7.54.
2‐((2‐(Naphthalen‐2‐yl)‐2‐oxoethyl)thio)‐4‐phenylpyrimidin‐6(3H)‐
one (9b
)
According to the general procedure, 6‐phenyl‐2‐thioxo‐2,3‐dihydro-
pyrimidin‐4(1H)‐one (8; 204.25 mg, 1 mmol), 2‐bromoacetylnaphtha-
lene (5h; 249.11 mg, 1 mmol), and anhydrous K2CO3 (138.21 mg,
1 mmol) were reacted in ethanol (20 ml) to produce 9b as a white
powder (185 mg, 50%); mp 218–220°C; Rf = 0.30 (pet ether/EtOAc =
1:5); ύ max (atr)/cm−1 3,400 (NH), 2,916 and 2,874 (CH), 1,661
(C═O), 1,532, and 1,458; δH (300 MHz; DMSO‐d6) 5.01 (2H, s, CH2),
6.65 (1H, s), 6.92 (2H, t‐like, 3J = 7.3 Hz), 7.21 (1H, t‐like, 3J = 7.0 Hz),
7.62–7.73 (4H, m), 8.02–8.05 (3H, m), 8.13 (1H, d, 3J = 7.9 Hz), 8.92
(1H, s), and 12.84 ppm (1H, NH); Anal. Calcd for C22H16N2O2S: C,
70.95; H, 4.33; N, 7.52. Found: C, 71.21; H, 4.07; N, 7.82.
4‐(2,5‐Dimethoxyphenyl)‐2‐((2‐(4‐methoxyphenyl)‐2‐oxoethyl)thio)‐
6‐oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6s)
According to the general procedure, 6‐(2,5‐dimethoxyphenyl)‐4‐oxo‐
2‐thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4c; 289.31 mg,
1 mmol), 2‐bromo‐4′‐methoxyacetophenone (5e; 229.1 mg, 1 mmol),
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6s as a pale yellow powder (195 mg, 45%), mp
188–190°C; Rf = 0.18 (pet ether/EtOAc = 1:5); δH (400 MHz; DMSO‐
d6) 3.53 (3H, s, OCH3), 3.77 (3H, s, OCH3), 3.89 (3H, s, OCH3), 4.80
(2H, s, CH2), 6.75 (1H, s), 6.96–7.92 (2H, m), 7.10 (2H, d, 3J = 8.6 Hz),
and 8.50 (2H, d, 3J = 8.6 Hz); Anal. Calcd for C22H19N3O5S: C, 60.40;
H, 4.38; N, 9.61. Found: C, 60.73; H, 4.59; N, 9.43.
|
4.2
Biological evaluation
|
4.2.1
Biochemical VEGFR‐2 inhibition assay
This assay was carried out using VEGFR‐2 (Bioscience) and Kinase‐
Glo Plus Luminescence Kinase (Promega) kits according to the
manufacturers’ protocol. This assay measures the kinase activity by
quantitating the amount of ATP remaining in the solution following a
kinase reaction. The luminescent signal from the assay is related to
the amount of ATP present and is inversely proportional to the
amount of kinase activity. A stock solution of the synthesized
compounds and sorafenib (III) in DMSO, for example, 10% was
initially prepared. Serial dilutions were carried out and 5 µl of the
dilution was added to a 50 µl reaction. All the enzymatic reactions
were conducted at 30°C for 45 minutes. The 50 µl reaction mixture
contained 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/ml bovine
serum albumin, 1 mM dithiothreitol, 0.2 mg/ml Poly(Glu, Tyr)
substrate, 10 μM ATP and protein. After the enzymatic reaction,
50 µL of Kinase‐Glo Plus Luminescence Kinase assay solution was
added to each reaction, and the plate was incubated for 15 minutes
at room temperature. The luminescence signal was measured using a
Promega multimode microplate reader. The difference between
luminescence intensities in the absence of kinase (Lut) and in the
presence of kinase (Luc) was defined as 100% activity (Lut – Luc).
Using the luminescence signal (Lu) in the presence of the compound,
% activity was calculated as: % activity = {(Lut − Lu)/(Lut − Luc)} ×
100%, where Lu is the luminescence intensity in the presence of the
compound. The luminescence data were analyzed using GraphPad
Prism and IC50 values were calculated as the average value from two
independent experiments.
2‐((2‐((1,1′‐Biphenyl)‐4‐yl)‐2‐oxoethyl)thio)‐4‐(2,5‐dimethoxyphe-
nyl)‐6‐oxo‐1,6‐dihydropyrimidine‐5‐carbonitrile (6t)
According to the general procedure, 6‐(2,5‐dimethoxyphenyl)‐4‐oxo‐
2‐thioxo‐1,2,3,4‐tetrahydropyrimidine‐5‐carbonitrile (4c; 289.31 mg,
1 mmol), 2‐bromo‐4′‐phenylacetophenone (5f; 275.14 mg, 1 mmol),
and anhydrous K2CO3 (138.21 mg, 1 mmol) were reacted in ethanol
(20 ml) to produce 6t as a yellow powder (210 mg, 43%); mp
192–194°C; Rf = 0.24 (pet ether/EtOAc = 1:5); δH (400 MHz; DMSO‐
d6) 3.47 (3H, s, OCH3), 3.72 (3H, s, OCH3), 4.92 (2H, s, CH2), 6.74 (1H,
s, CH), 6.95 (1H, dd, 3J = 8.8 Hz, 4J = 2.0 Hz), 7.02 (1H, d, 3J = 9.0 Hz),
7.43–7.47 (1H, m), 7.53 (2H, t‐like, 3J = 7.7 Hz), 7.73–7.77 (4H, m),
and 8.05 (2H, d, 3J = 8.3 Hz); δC (100 MHz; DMSO‐d6) 38.40 (CH2),
55.34, 55.79, 96.55, 113.14, 114.74, 115.12, 117.87, 126.90, 127.07,
128.61, 129.09, 129.24, 134.62, 138.88, 145.00, 150.47, 152.72,
158.65, 160.67, 165.77, and 192.48 ppm; Anal. Calcd for
C27H21N3O4S: C, 67.07; H, 4.38; N, 8.69. Found: C, 67.35; H, 4.59;
N, 8.46.
2‐((2‐(4‐Chlorophenyl)‐2‐oxoethyl)thio)‐4‐phenylpyrimidin‐6(3H)‐
one (9a)
According to the general procedure, 6‐phenyl‐2‐thioxo‐2,3‐dihydro-
pyrimidin‐4(1H)‐one (8; 204.25 mg, 1 mmol), 2‐bromo‐4′‐chloroace-
tophenone (5c; 233.49 mg, 1 mmol) and anhydrous K2CO3
(138.21 mg, 1 mmol) were reacted in ethanol (20 ml) to produce 9a
(CAS number 284665‐83‐4) as a white powder (150 mg, 42%); mp
230–232°C; Rf = 0.27 (pet ether/EtOAc = 1:5); ύ max (atr)/cm−1 3,476
(NH), 2,916 (CH), 1,648 (C═O), 1,564, and 1,487; δH (300 MHz;
DMSO‐d6) 4.87 (2H, s, CH2), 6.63 (1H, s), 7.17 (2H, t‐like, 3J = 7.6 Hz),
7.35 (1H, t‐like, 3J = 7.3 Hz), 7.58 (2H, t‐like, 3J = 7.5 Hz), 7.69–7.72
(2H, m), 8.10 (2H, d, 3J = 7.5 Hz), and 12.50 ppm (1H, br.); Anal. Calcd
|
4.2.2
In vitro antiproliferative activity
The anticancer activity of the synthesized compounds was measured
in vitro using the sulforhodamine‐B (SRB) assay according to Skehan
et al.[33] Briefly, cells were inoculated in a 96‐well microtiter plate
(104 cells/well) for 24 hr before treatment with the tested

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ABDEL‐MOHSEN ET AL.
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compounds to allow attachment of cell to the wall of the plate. Test
compounds were dissolved in DMSO at 1 mg/ml immediately before
use and diluted to the appropriate volume just before addition to the
cell culture. Different concentrations of tested compounds and
sorafenib (III) were added to the cells. Three wells were prepared for
each individual dose. Monolayer cells were incubated with the
compounds for 48 hr at 37°C and in an atmosphere of 5% CO2. After
48 hr, the cells were fixed, washed, and stained for 30 min with 0.4%
(w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by
four washes with 1% acetic acid, and the attached stain was
recovered with Tris‐EDTA buffer. Color intensity was measured in
an ELISA reader. The relation between the surviving fraction and
drug concentration is plotted to get the survival curve after the
specified time. The concentration required for 50% inhibition of cell
viability (IC50) was calculated and the results were compared to the
effect of the reference drug, sorafenib.
was determined by the position of co‐crystallized ligand (sorafenib
III). Triangle matcher placement method and the London dG scoring
function was applied for docking. Redocking of the co‐crystallized
ligand (sorafenib III) in the binding site was initially conducted to
validate the docking simulation. An energy score (S) = −15.19 kcal/
mol and an RMSD of 0.470 Å from the co‐crystallized sorafenib were
observed. The newly synthesized compounds were subsequently
docked in VEGFR‐2 active site applying the same parameters.
|
4.3.1
In silico prediction of physicochemical,
pharmacokinetic properties and ADME parameters
SwissADME, a free web tool available from the Swiss Institute of
Bioinformatics,[36,37] was utilized for estimation of the physicochem-
ical and pharmacokinetic properties and for prediction of the ADME
parameters and drug likeness of synthesized compounds. The
structures of 2,4‐disubstituted thiouracils 6a–t, 9a, and 9b were
initially converted to SMILES notations. Accessing http://www.
swissadme.ch in a web browser, the submission page is directly
displayed. The SMILES list was directly pasted to the online server
for computation.
|
4.2.3
In vitro cell based VEGFR‐2 inhibitory
activity
The effect of the synthesized compounds on the level of VEGFR‐2 in
the HepG2 cell line was determined. The cells in culture medium
were treated with 20 µl of the IC50 values of the compounds or the
standard reference drug, sorafenib (III) dissolved in DMSO, then
incubated for 48 hr at 37°C, in a humidified 5% CO2 atmosphere. The
cells were harvested and homogenates were prepared in saline using
a tight pestle homogenizer until complete cell disruption. The kit uses
a double‐antibody sandwich ELISA to assay the level of human
VEGFR‐2 in samples. Antibody specific for VEGFR‐2 was pre‐coated
onto 96‐well plates. Standard and test samples were added to the
wells; then a horseradish peroxidase (HRP)‐conjugated antibody
specific VEGFR‐2 was added subsequently and incubated. Unbound
conjugates were washed away. The TMB substrate was used to
visualize the HRP enzymatic reaction. TMB is catalyzed by HRP to
produce a blue color product that changed into yellow after adding
stop solution. The density of yellow is proportional to the human
VEGFR‐2 amount. The optical density was determined spectro-
photometrically at a wavelength 450 nm. The expression of VEGFR‐2
in samples was calculated (ng/ml) as duplicate determinations and
the data were compared with sorafenib (III) as standard VEGFR‐2
inhibitor.
ACKNOWLEDGMENT
The authors are grateful to the Science and Technology Development
Fund (STDF) for their funding through the project ID 15063. Special
thanks to the National Cancer Institute (NCI), Bethesda, MD for
performing the anticancer screening for the selected compounds
over their panel of cell lines.
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Heba T. Abdel‐Mohsen
http://orcid.org/0000-0002-7610-8312
Adel S. Girgis http://orcid.org/0000-0003-4407-9745
Abeer E. E. Mahmoud
http://orcid.org/0000-0002-4335-4824
http://orcid.org/0000-0002-4931-4153
http://orcid.org/0000-0001-7525-9437
Mamdouh M. Ali
Hoda I. El Diwani
|
4.3
Molecular docking studies
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How to cite this article: Abdel‐Mohsen HT, Girgis AS,
Mahmoud AEE, Ali MM, El Diwani HI. New 2,4‐disubstituted‐
2‐thiopyrimidines as VEGFR‐2 inhibitors: Design, synthesis,
and biological evaluation. Arch Pharm Chem Life Sci.
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