Novel potent substituted 4-amino-2-thiopyrimidines as dual VEGFR-2 and BRAF kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.06.063

In the present study, we report the discovery of a novel class of substituted 4-amino-2-thiopyrimidines as antiangiogenic and antiproliferative agents. Structural hybridization between 4-substituted aminopyrimidines (VEGFR-2 inhibitors) and 2-thioxopyrimidines (BRAF inhibitors) was carried out to afford substituted 4-amino-2-thiopyrimidines as type II dual VEGFR-2/BRAF inhibitors. Our design strategy was tailored such that the 4-amino-2-thiopyrimidine scaffold is to be accommodated in the central gate area of the inactive DFG-out conformation of both enzymes. On one side, the hydrophobic substituent on the 4-amino group would occupy the hydrophobic back pocket and on the other side the substituent on the sulfide moiety should extend to fit in the hinge region (front pocket). Molecular docking simulations confirmed the ability of the designed compounds to accomplish the key interactions in VEGFR-2 and BRAF active sites. Most of the synthesized substituted 4-amino-2-thiopyrimidines demonstrated potent VEGFR-2 inhibitory activity at submicromolar concentrations. Compounds 8a, 8d, 9c and 9e showed IC₅₀ = 0.17, 0.12, 0.17 and 0.19 μM, respectively against VEGFR-2 in comparison to sorafenib (I) IC₅₀ = 0.10 μM and regorafenib (II) IC₅₀ = 0.005 μM. While compounds 9c, 9d and 10a showed IC₅₀ = 0.15, 0.22 and 0.11 μM, respectively against BRAF-WT. At 10 μM concentration 9c revealed promising in vitro broad-spectrum antiproliferative activity against cancer cell lines with growth inhibition percent ranging from 10 to 90%. Moreover, compounds 7b, 8d, 9a, 9b, 9c and 9d showed potent activity against MCF7 cell line (IC₅₀ = 17.18, 17.20, 19.98, 19.61, 13.02 and 16.54 μM, respectively). On the other hand, compounds 9c, 9d and 10d were found to be the most potent compounds against T-47D cell line (IC₅₀ = 2.18, 8.09 and 4.36 μM, respectively). Studying the effect of the most potent compounds on VEGFR-2 level in MCF7 cell line revealed that 9c and 9d showed inhibition percent of 84 and 80%, respectively, in comparison to sorafenib (I) (% inhibition = 90%).

Full text of DOI:10.1016/j.ejmech.2019.06.063

Accepted Manuscript
Novel potent substituted 4-amino-2-thiopyrimidines as dual VEGFR-2 and BRAF
kinase inhibitors
Heba T. Abdel-Mohsen, Mohamed A. Omar, Ahmed M. El Kerdawy, Abeer E.E.
Mahmoud, Mamdouh M. Ali, Hoda I. El Diwani
PII:
S0223-5234(19)30590-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.063
EJMECH 11466
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 April 2019
Revised Date: 12 June 2019
Accepted Date: 21 June 2019
Please cite this article as: H.T. Abdel-Mohsen, M.A. Omar, A.M. El Kerdawy, A.E.E. Mahmoud, M.M.
Ali, H.I. El Diwani, Novel potent substituted 4-amino-2-thiopyrimidines as dual VEGFR-2 and BRAF
kinase inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.063.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel Potent Substituted 4-Amino-2-thiopyrimidines as Dual VEGFR-2
and BRAF Kinase Inhibitors
Heba T. Abdel-Mohsen*a, Mohamed A. Omara, Ahmed M. El Kerdawyb,c,d, Abeer E.E.
Mahmoude, Mamdouh M. Alie, Hoda I. El Diwania
aDepartment of Chemistry of Natural and Microbial Products, Division of Pharmaceutical
and Drug Industries, National Research Centre, Dokki, Cairo, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-
Aini Street, Cairo, P.O. Box 11562, Egypt.
cMolecular Modeling Unit, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street,
Cairo, P.O. Box 11562, Egypt.
dDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, New
Giza, km 22 Cairo–Alexandria Desert Road, Cairo, Egypt
eDepartment of Biochemistry, Division of Genetic Engineering and Biotechnology, National
Research Centre, Cairo, Egypt
*Corresponding author: Email address: hebabdelmohsen@gmail.com
Abstract
In the present study, we report the discovery of a novel class of substituted 4-amino-2-
thiopyrimidines as antiangiogenic and antiproliferative agents. Structural hybridization
between 4-substituted aminopyrimidines (VEGFR-2 inhibitors) and 2-thioxopyrimidines
(BRAF inhibitors) was carried out to afford substituted 4-amino-2-thiopyrimidines as type II
dual VEGFR-2/BRAF inhibitors. Our design strategy was tailored such that the 4-amino-2-
thiopyrimidine scaffold is to be accommodated in the central gate area of the inactive DFG-
out conformation of both enzymes. On one side, the hydrophobic substituent on the 4-amino
group would occupy the hydrophobic back pocket and on the other side the substituent on the
sulfide moiety should extend to fit in the hinge region (front pocket). Molecular docking
simulations confirmed the ability of the designed compounds to accomplish the key
interactions in VEGFR-2 and BRAF active sites. Most of the synthesized substituted 4-
amino-2-thiopyrimidines demonstrated potent VEGFR-2 inhibitory activity at submicromolar
concentrations. Compounds 8a, 8d, 9c and 9e showed IC50 = 0.17, 0.12, 0.17 and 0.19 µM,
respectively against VEGFR-2 in comparison to sorafenib (I) IC50 = 0.10 µM and regorafenib
(II) IC50 = 0.005 µM. While compounds 9c, 9d and 10a showed IC50 = 0.15, 0.22 and 0.11
µM, respectively against BRAF-WT. At 10 µM concentration 9c revealed promising in vitro
broad‐spectrum antiproliferative activity against cancer cell lines with growth inhibition
percent ranging from 10 to 90%. Moreover, compounds 7b, 8d, 9a, 9b, 9c and 9d showed
potent activity against MCF7 cell line (IC50 = 17.18, 17.20, 19.98, 19.61, 13.02 and 16.54
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µM, respectively). On the other hand, compounds 9c, 9d and 10d were found to be the most
potent compounds against T-47D cell line (IC50 = 2.18, 8.09 and 4.36 µM, respectively).
Studying the effect of the most potent compounds on VEGFR-2 level in MCF7 cell line
revealed that 9c and 9d showed inhibition percent of 84 and 80%, respectively, in comparison
to sorafenib (I) (% inhibition = 90%).
Keywords: VEGFR-2; BRAF; antiproliferative activity, thiopyrimidines; synthesis; molecular
docking.
1. Introduction
In fundamental cellular functions, intracellular signal transduction occurs through the
transference of signals from cell surface to the nucleus resulting in cell division,
differentiation, migration and apoptosis. Receptor tyrosine kinases (RTKs), serine threonine
kinases (STKs), and G proteins are the major proteins that are involved in controlling the
process of signal transduction [1,2].
Cancer is characterized by an upregulation in many complex signaling pathways to accelerate
tumor growth, proliferation and mobility. Uncontrolled protein kinase (PK) stimulation in
response to mutations or overexpression is the main mechanism for cancer growth and
propagation [3]. Hence, PKs are considered crucial targets for the development of targeted
cancer chemotherapy with minimum normal cell toxicity [4-6].
Vascular endothelial growth factor receptor 2 (VEGFR-2) is a RTK that exists on the surface
of blood vessels and it is the principle receptor that regulates the formation of new blood
vessels (angiogenesis) [7]. VEGFR-2 plays a curial role in tumor pathophysiology as its
stimulation by VEGF activates a downstream signaling pathway resulting in tumor
angiogenesis and so enhancing the tumor nutrition and oxygen supply and, further, tumor
proliferation and metastasis [8,9]. VEGFR-2 is highly expressed in some cancer types such as
breast cancer, renal cancer and hepatocellular carcinoma [10-12]. For these reasons, there is a
long-held concept that inhibition of VEGFR-2 pathway would cause efficient anti-angiogenic
as well as antitumor response [13].
Rapidly accelerated fibrosarcoma (RAF) is a serine threonine kinase that mediates RAS-RAF-
MEK-ERK pathway [14]. RAF is mainly activated by the G protein, RAS, in response to
mutations in cancer cells or by overexpression of different RTKs such as epidermal (EGFR),
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platelet derived (PDGFR) and vascular endothelial (VEGFR) growth factors [15,16]. Once
RAF is activated, it phosphorylates mitogen-activated protein kinase (MAPK/MEK) which in
turn results in a subsequent phosphorylation of the extracellular signal-regulated kinase
(ERK). Phosphorylated ERK activates several transcription factors leading to cell growth,
survival and proliferation [17]. Many human tumors depend on activation of the RAS signal
transduction pathway to achieve cellular proliferation and survival. So, targeting this pathway
is regarded as a beneficial strategy in anticancer drug discovery [18].
There are three known RAF isoforms (ARAF, BRAF, and CRAF). BRAF is the most
susceptible to activation among RAF isoforms and so, it is the most susceptible to mutational
activation in tumor cells [14, 16, 17]. Amino acid substitution at position 600 in BRAF, from
a valine (V) to a glutamic acid (E) (V600E) is the most common mutation in BRAF [19]. The
BRAF V600E mutation leads to 500-700 fold increase in the kinase activity in comparison to
wild type BRAF. Mutations in BRAF are most common in the melanoma cell lines, some
ovarian, thyroid and colorectal cancers [19-22].
Recent strategies in anticancer drug discovery encourage the development of small molecules
that act against a specific target with high potency and efficiency [23,24]. Despite the initial
progress that was observed, targeting single oncoprotein fails to produce the desired long-term
efficiency. This is attributed to the rapid resistance development either by target protein
mutation or by the adoption of new pathways by the cancer cells that compensate for the
affected pathways. To overcome this problem, two competitive approaches have been
developed [25]. The first approach involves the use of a combined therapy that act by creating
a synergistic effect of different drugs acting on different targets. According to this approach
various drug combinations have been approved by the FDA in cancer treatment regimen. For
example, FDA approved the combination of dabrafenib, a BRAF inhibitor, with trametinib, a
MEK inhibitor, in the treatment of metastatic melanoma [26,27]. The second and more
promising approach involves the design of single-small-molecules that effectively and
simultaneously target multiple oncogenic pathways producing an overall efficient result with
less toxicity and no drug-drug interactions [28-31].
Recently, it was postulated that BRAF and VEGFR-2 have a synergistic effect on the
development, growth and progression of cancer, so, dual inhibition of BRAF and VEGFR-2 is
considered to be a promising strategy for cancer treatment [32-35]. Sorafenib, BAY 43-9006,
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(I) (Fig. 1) is a dual inhibitor of RAF and VEGFR. It inhibits VEGFR-1/2/3, BRAF, CRAF as
well as PDGFR. Several studies showed that sorafenib (I) inhibits MAPK signaling through
RAF inhibition in different cancer cell lines e.g. colon, breast, ovarian, pancreatic, melanoma
and thyroid cancer cell lines [36]. In addition, sorafenib (I) produces a significant reduction in
the level of microvessels area and density that supply the tumor resulting in tumor growth
inhibition. Currently, sorafenib (I) is approved by the FDA for advanced renal cell carcinoma
and hepatocellular carcinoma treatment [36-38]. Regorafenib, BAY 73-450 (II) (Fig. 1) is
another example of multi-targeted protein kinase inhibitor that significantly inhibited a large
set of angiogenic e.g. VEGFR-1/2/3, TIE2, FGFR1 and PDGFR‐β as well as intracellular
signaling kinases e.g. CRAF and BRAF with IC50 up to 1.5 nM [39]. In addition, RAF265
(III) (Fig. 1) was discovered as a potent dual RAF/VEGFR-2 inhibitor that effectively inhibits
the proliferation and survival of several cancer cell lines through blocking tumor growth as
well as hindering the blood supply to the cancer cells [40,41]. Despite the successful
application of sorafenib (I) and regorafenib (II) in cancer therapy, they still suffer from
serious resistance that prevents their long-term use [42]. Hence, there is a continuous need to
design and discover new scaffolds that can act on multiple targets simultaneously for cancer
treatment.
Fig.1. Structures of multi-targeted protein kinases I-III
Like all protein kinases (PKs), both VEGFR-2 and BRAF have the characteristic bi-lobed
kinase domain. The small N-terminal lobe has a predominantly conserved antiparallel β-sheet
structure which anchors and orients ATP; it is responsible for ATP recognition and binding as
it contains the glycine-rich ATP-phosphate-binding loop (P loop). On the other hand, the
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larger less conserved C-terminal lobe is mainly α-helical, and it contains the protein substrate
binding site and the activation loop (A-loop) which is characterized by the conserved triad
Asp-Phe-Gly (DFG) motif at its beginning in most PKs. The catalytic site lies in the cleft
between the two lobes and consists of residues contributed by both lobes. The kinase
inhibitors binding site can be divided into three main regions; the hinge region (front cleft), a
gate area, and a hydrophobic back cleft [43-48]. Sorafenib (I) (Fig. 1 and Fig. 2) is a type II
inhibitor of both VEGFR-2 and BRAF that interacts with the PKs in their inactive “DFG-out”
conformation occupying the front cleft, the gate area, and extend beyond the gatekeeper into the
hydrophobic back cleft. The extension into the less conserved hydrophobic back pocket enhances
its affinity and selectivity. Sorafenib (I) interacts through hydrogen bonding with Cys919
(VEGFR-2) or Cys531 (BRAF) of the hinge region, Glu885 (VEGFR-2) / Glu500 (BRAF) of the
αC helix, and Asp1046 (VEGFR-2) / Asp593 (BRAF) of the conserved DFG motif and extend
beyond the gatekeeper to interact through hydrophobic interaction with the hydrophobic back
pocket lined with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899,
Leu1019 and Ile1044 of VEGFR-2 or Val503, Leu504, Leu513, Leu566, Ile571 and Ile591 of
BRAF [49,50] (Fig. 2).
Fig. 2. Interactions of sorafenib (I) with the hot spots of VEGFR-2 and BRAF
Molecular hybridization strategy is one of the most recent approaches that are currently
applied for the discovery of novel scaffolds of dual or multitargeted small molecule inhibitors
[51]. 4-Aminopyrimidines were reported to exhibit PK inhibitory activity [52-54]. For
instance, pazopanib (IV) was reported to possess anti-angiogenic activity through the
inhibition of the angiokinases VEGFR-2, PDGFR and FGFR [53]. Moreover, MKP series e.g.
MKP115 (V) was proven to inhibit VEGFR-2 and EGFR [54]. On the other hand, 2-
thioxopyrimidines e.g. VI and VII were reported to have potent BRAF inhibitory activity
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[55,56]. According to this background, the aim of this study was to carry out molecular
hybridization of 4-aminopyrimidines (VEGFR-2 inhibitors) with 2-thiopyrimidines (BRAF
inhibitor) to yield novel substituted 4-amino-2-thiopyrimidines VIII scaffold (Fig 3.) which
should, by design, fulfill the binding requirements to act as dual VEGFR-2/BRAF inhibitors.
Based on the X-ray structures of sorafenib (I) co-crystalized with VEGFR-2 [57] and BRAF
[58] (fig. 2), our design strategy was tailored such that the core 4-amino-2-thiopyrimidine
scaffold (pink colored) is to be accommodated in the central gate area of the inactive DFG-out
conformation of both enzymes. On one side, the hydrophobic substituent on the 4-amino
group (blue colored) would occupy the hydrophobic back pocket. On the other side the
substituent (green and red colored) on the sulfide moiety should extend to fit in the hinge
region (front pocket) (Fig. 3). Organic synthesis as well as biological evaluation was
subsequently performed.
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Fig. 3. Design strategy of substituted 4-amino-2-thiopyrimidines VIII as dual VEGFR-
2/BRAF
2. Results and discussion
2.1. Chemistry
The synthesis of the target 4-amino-2-thiopyrimidines 6-10 was achieved by initial reaction of
2-thiouracil (1) with 2-bromoacetophenones 2a-h under basic conditions to give the
corresponding 2-((2-oxo-2-(4-substituted-phenyl)ethyl)thio)pyrimidin-4(3H)-ones 3 [59,60].
Subsequently, compounds 3a-h were converted to 2-((4-chloropyrimidin-2-yl)thio)-1-(4-
substituted-phenylethan-1-ones 4a-h by heating 3 in phosphorus oxychloride at 80 °C.
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Compounds 4a-h were then reacted with different primary amines 5a-e in acetic acid under
reflux to yield compounds 6-10 as single products (Scheme 1).
O
O
H
H
Br
O
N
S
O
N
S
(i)
(ii)
N
NH
R1
R1
1
2a-h
3a-h
R2
O
O
NH2
H
N
N
S
Cl
N
S
5a-e
R2
N
N
(iii)
R1
R1
4a-h
6-10
2-4a R1 = F
5a, 6 R2 = 3-COCH3
5b, 7 R2 = 4-COCH3
5c, 8 R2 = 4-COOH
5d, 9 R2 = 4-COOEt
2-4b R1 = Cl
2-4c R1 = Br
2-4d R1 = CF3
2-4e R1 = CN
2-4f R1 = NO2
5e, 10 R2 = 4-morpholine
2-4g R1 = OCH3
2-4h R1 = Ph
6a R1 = Cl
7a R1 = Cl
8a R1 = Cl
8b R1 = Br
8c R1 = CF3
8d R1 = CN
8e R1= NO2
8f R1 = OCH3
8g R1 = Ph
9a R1 = Cl
10a R1 = F
10b R1 = Cl
10c R1 = CN
6b R1 = CF3
6c R1= OCH3
6d R1 = Ph
7b R1= Br
9b R1 = CF3
9c R1 = CN
9d R1 = NO2
9e R1 = OCH3
9f R1 = Ph
7c R1 = OCH3
7d R1 = Ph
10d R1 = OCH3
Reagents and conditions: (i) anhydrous K2CO3, EtOH, reflux, 4h; (ii) POCl3, 80 °C, 30 min;
(iii) AcOH, reflux, 4h.
Scheme 1. Synthesis of substituted 4-amino-2-thiopyrimidines 6-10
2.2. Biological Evaluation
The newly synthesized substituted 4-amino-2-thiopyrimidines 6-10 were evaluated
biochemically for their VEGFR-2 inhibitory activity. Promising compounds were further
tested for their inhibitory activity against BRAF-WT. Concurrently; some compounds were
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selected to be screened for their in vitro antiproliferative activity by the Developmental
Therapeutics Program (DTP) of the National Cancer Institute (NCI) in the division of cancer
treatment and diagnosis, NIH, Bethesda, Maryland, USA at 10 µM [61]. In addition,
cytotoxicity IC50 of all the target compounds were evaluated against MCF7 cell line.
Furthermore, cytotoxicity IC50 of some selected compounds were tested against T-47D breast
cancer cell line. Candidates showed promising enzymatic as well as antiproliferative activities
were further evaluated for their effect on the level of VEGFR-2 in MCF7 cell line.
2.2.1. Biochemical kinase inhibitory activity
All the target compounds 6-10 were initially screened for their inhibitory activity against
VEGFR-2 using VEGFR-2 (KDR) Kinase Assay Kit (Bioscience). The IC50 (µM) of the
screened compounds as well as sorafenib (I) and regorafenib (II) are presented in Table 1. It
was found that most of the tested compounds exhibited potent VEGFR-2 inhibitory activity in
the single-digit, if not, sub-micromolar, range (IC50 = 0.12 – 1.96 µM) in comparison to
sorafenib (I) (IC50 = 0.10 µM) and regorafenib (II) (IC50 = 0.005 µM). Compounds 6b, 8b,
8c, 8e and 9a showed no VEGFR-2 inhibitory activity (IC50 > 10.0 µM). Compounds 8a, 8d,
9c and 9e are the most potent derivatives with IC50 of 0.17, 0.12, 0.17 and 0.19 µM,
respectively. In series 6 which possess 3-acetylphenyl moiety attached to 4-amino group, the
4-methoxyphenyl 6c and, 4-chlorophenyl 6a are the most active derivatives (IC50 of 0.42 and
0.48 µM, respectively). Introduction of a biphenyl moiety at 2-position 6d resulted in a slight
decrease in activity (IC50 = 0.74 µM) compared to 6c. On the other hand, introduction of
trifluoromethyl substituted phenyl group 6b resulted in a complete loss of activity at 10 μM.
Shifting the acetyl group from 3-position (series 6) to 4-position (series 7) resulted in a slight
increase in activity in most cases. The 4-chlorophenyl derivative 7a is the most potent
compound in this series (IC50 = 0.22 µM) followed by the biphenyl derivative 7d (IC50 = 0.28
µM) and the 4-methoxy derivative 7c (IC50 = 0.49 µM). About 5-fold decrease in activity was
observed upon replacement of the chlorine moiety in 7a with a bromine group in 7b (IC50 =
0.22 vs 0.95 µM, respectively). Further replacement of the 4-acetyl group in series 7 with a
carboxylic group in series 8 resulted in a decrease in activity in most cases. Only the p-
chlorophenyl derivative 8a showed a higher activity than its 4-acetyl congener 7a (IC50 of
0.17 µM vs 0.22 µM, respectively). Replacement of chlorine group of 8a with a 4-cyano
group in 8d resulted in a slight increase in activity (IC50 = 0.17 vs 0.12 µM, respectively). The
4-OCH3 derivative 8f and 4-ph derivative 8g showed decreased activity (IC50 = 1.43 and 0.57
µM, respectively). Complete loss of activity was observed in case of 4-Br 8b, 4-CF3 8c and 4-
NO2 8e phenyl derivatives at 10 µM. Replacement of COOH group in series 8 with an ethyl
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ester in series 9 resulted in a complete loss of activity for 9a (IC50 > 10 µM) in comparison to
8a (IC50 = 0.17 µM) and a slight decrease in activity of the p-cyanophenyl derivative 9c in
comparison to its acid congener 8d (IC50 = 0.17 µM vs 0.12 µM, respectively). While an
increase in activity was observed for 9b (IC50 = 1.96 µM), 9d (IC50 = 0.41 µM), 9e (IC50 =
0.19 µM) and 9f (IC50 = 0.34 µM) when compared to 8c (IC50 > 10 µM), 8e (IC50 > 10 µM),
8f (IC50 = 1.43 µM), and 8g (IC50 = 0.57 µM) due to their higher hydrophobic nature than
their acid congeners (series 8) what enables them of better interaction with the hydrophobic
back pocket vide infra. In series 10 where a 4-morpholino group is introduced on the amino
phenyl moiety, the 4-fluoro 10a and 4-methoxy 10d derivatives are the most potent
derivatives in this series (IC50 = 0.26 and 0.29 µM, respectively). On the other hand, more
than 3-fold decrease in potency was observed by the introduction of 4-chloro 10b or 4-cyano
10c groups (IC50 = 1.90 and 1.10 µM, respectively).
The most active compounds 7a, 7d, 8a, 8d, 9c, 9d, 9e, 10a and 10d were subsequently
screened for their inhibitory activity against BRAF-WT and the results were depicted in table
1. The tested compounds showed potent inhibitory activity with compounds 7a, 8a, 9c, 9d,
9e, 10a, 10d showing sub-micromolar IC50 of 0.64, 0.37, 0.15, 0.22, 0.41, 0.11 and 0.44 µM,
respectively, whereas, compounds 7d and 8d showed single-digit IC50 of 1.05 and 1.70 µM,
respectively. These results indicate that the designed and synthesized compounds are potent
multikinase inhibitors of VEGFR-2 and BRAF.
10

Table 1. Inhibitory activity of the synthesized compounds 6-10 against VEGFR-2 and BRAF-WT
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Entry Compound
R1
R2
IC50 (µM)a
VEGFR-2
0.48 ± 0.02
> 10
IC50 (µM)a
BRAF-WT
ndb
1
Cl
3-COCH3
3-COCH3
3-COCH3
3-COCH3
4-COCH3
4-COCH3
4-COCH3
4-COCH3
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOEt
4-COOEt
4-COOEt
4-COOEt
4-COOEt
4-COOEt
4-morpholino
4-morpholino
4-morpholino
4-morpholino
-
6a
2
CF3
OCH3
Ph
nd
6b
3
0.42 ± 0.02
0.74 ± 0.10
0.22 ± 0.08
0.95 ± 0.04
0.49 ± 0.02
0.28 ± 0.01
0.17 ± 0.06
> 10
nd
6c
4
nd
6d
5
Cl
0.64 ± 0.04
7a
6
Br
nd
7b
7
OCH3
Ph
nd
7c
8
1.05 ± 0.08
0.37 ± 0.03
nd
7d
9
Cl
8a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Br
8b
CF3
CN
NO2
OCH3
Ph
> 10
nd
8c
0.12 ± 0.04
> 10
1.70 ± 0.78
nd
8d
8e
1.43 ± 0.05
0.57 ± 0.03
> 10
nd
8f
nd
8g
Cl
nd
9a
CF3
CN
NO2
OCH3
Ph
1.96 ± 0.50
0.17 ± 0.02
0.41 ± 0.03
0.19 ± 0.07
0.34 ± 0.01
0.26 ± 0.03
1.90 ± 0.08
1.10 ± 0.05
0.29 ± 0.07
0.10 ± 0.02
0.005 ± 0.002
nd
9b
0.15 ± 0.01
0.22 ± 0.02
0.41 ± 0.03
nd
9c
9d
9e
9f
F
0.11 ± 0.01
nd
10a
Cl
10b
CN
OCH3
-
nd
10c
0.44 ± 0.03
0.03 ± 0.001
0.04 ± 0.005
10d
Sorafenib
-
-
Regorafenib
aIC50 are presented as Mean ± Standard error (S.E.) of two independent experiments; b Not determined
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Based on the results of the biochemical assay against VEGFR-2 and BRAF, the most potent
compounds 9c and 10a, as representatives to this series, were further tested for their inhibitory
activity against a panel of kinases, namely PDGFR-β, FGFR1, TEI2, cKit, RAF1 to get an
overview of the kinase inhibitory profile of the designed compounds (Table 2). From the
obtained results it is obvious that both 9c and 10a are showing sub-micromolar inhibitory
activity against PDGFR-β (IC50 = 0.39 and 0.15 µM, respectively), FGFR1 (IC50 = 0.28 and
0.13 µM, respectively) and RAF1 (IC50 = 0.41 and 0.13 µM, respectively). Compound 9c
shows 1.5 to 30 fold higher selectivity toward VEGFR-2 and BRAF over the other tested
kinases; on the other hand, compound 10a shows comparable potency on all the tested kinases
(IC50 = 0.11-0.48 µM). Based on the obtained results we can conclude that some of the
designed compounds, as 9c, can act as selective dual VEGFR-2 / BRAF inhibitors while
others, as 10a, can act as multikinase inhibitors.
Table 2. Kinase inhibition profile of 9c and 10a IC50 (µM)
Entry Compound
VEGFR-2 BRAF PDGFR-β
FGFR1
0.28
TEI2 cKit
RAF1
0.41
1
2
3
0.17
0.26
0.10
0.15
0.11
0.03
0.39
0.15
0.06
1.39
0.48
nda
5.16
0.25
0.08
9c
0.13
0.13
10a
0.67
0.007
Sorafenib (I)
aNot determined.
2.2.2. In vitro antiproliferative activity
2.2.2.1. In vitro antiproliferative activity against NCI 60-cell line panel
Some of the synthesized substituted 4-amino-2-thiopyrimidine were selected to be screened
for their in vitro antiproliferative activity by the Developmental Therapeutics Program (DTP)
of the National Cancer Institute (NCI) in the division of cancer treatment and diagnosis, NIH,
Bethesda, Maryland, USA. This involves screening of the compounds at single dose of 10 µM
against a full NCI 60 cell panel including leukemia, lung, colon, brain, melanoma, ovary,
kidney, prostate and breast cancers [61]. Some selected results are presented in table 3.
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Table 3. In vitro growth inhibition % (GI %) of some selected compounds against a panel of
tumor cell lines at 10 µM
Subpanel
Compound ID
6b
6c
6d
7a
7b
7d
8c
9c
9d
10a 10b 10d
Leukemia
CCRF-CEM
HL-60(TB)
K-562
10
46
-
28
-
29
-
30
-
22
-
-
-
-
-
-
-
82
24
49
84
80
46
49
-
19
-
13
-
21
-
a
-
29
24
31
-
37
53
42
35
16
29
34
-
19
33
46
-
30
39
35
25
10
-
37
58
54
35
28
25
21
18
10
-
21
34
45
-
MOLT-4
PRMI-8226
SR
26
41
10
-
Non-small cell lung Cancer
A549/ATTC
EKVX
12
-
14
15
19
17
22
18
-
20
10
14
-
10
-
20
-
10
10
-
39
26
37
-
21
27
-
-
-
-
28
17
-
29
-
23
69
26
45
21
-
10
37
-
-
HOP-62
-
13
21
11
10
-
-
HOP-92
24
35
19
14
37
-
16
13
-
-
-
NCI-H226
NCI-H23
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCT-116
HCT-15
24
21
-
21
13
-
25
16
-
38
20
-
16
13
11
37
13
19
-
13
15
-
-
27
38
29
29
22
15
73
49
22
34
-
-
-
-
-
11
42
13
16
-
-
-
-
29
28
24
24
-
-
-
-
-
-
-
28
-
23
22
11
14
15
-
-
10
-
26
13
19
32
-
18
-
14
-
HT29
-
-
-
-
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KM12
-
-
15
-
11
-
24
-
45
10
21
-
-
-
-
-
-
-
SW-620
-
-
-
-
26
-
CNS Cancer
SF-268
-
-
-
-
-
-
-
22
24
17
10
-
-
35
-
15
-
-
-
-
-
-
-
SF-295
15
-
-
-
14
-
-
-
-
28
11
-
SF-539
12
13
31
-
11
-
-
13
-
-
-
15
19
36
-
SNB-19
10
32
12
-
-
-
37
65
16
SNB-75
-
-
13
-
-
28
-
-
U251
-
-
-
15
-
Melanoma
LOX IMVI
MALME-3M
M14
13
-
23
-
-
-
14
-
-
-
20
19
49
32
29
19
62
22
61
10
-
15
29
-
-
11
-
17
-
-
14
-
30
-
-
-
15
15
-
-
-
16
15
-
-
-
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
-
-
-
-
-
-
16
10
19
37
19
28
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11
41
13
29
-
-
14
-
22
-
-
65
-
17
-
-
-
-
53
-
-
-
-
-
29
35
24
34
23
20
12
10
30
44
-
18
12
19
-
20
-
-
12
-
14
-
13
-
21
21
35
-
19
-
-
17
-
12
-
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
-
18
57
-
-
12
12
-
18
-
-
-
-
20
-
-
15
-
-
-
-
-
-
-
32
40
29
46
18
31
-
-
77
58
31
27
62
26
17
-
24
24
11
14
-
-
14

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SK-OV-3
Renal Cancer
786-0
21
-
22
-
-
11
-
-
-
-
-
-
-
-
-
-
-
-
-
22
50
20
43
-
-
13
36
24
41
43
20
-
-
-
A498
-
22
-
40
12
23
-
19
11
21
-
19
10
24
-
55
26
16
-
39
-
61
15
16
-
13
10
23
-
-
ACHN
19
42
11
17
-
11
16
10
-
CAKI-1
35
14
22
-
19
-
RXF 393
SN 12C
13
-
-
14
-
11
-
-
26
10
52
17
-
-
TK-10
-
-
-
-
UO-31
57
55
40
33
38
27
32
41
27
26
35
Prostate Cancer
PC-3
30
-
37
-
25
-
30
-
25
-
19
16
12
-
60
21
33
16
27
16
12
-
15
-
DU-145
Breast Cancer
MCF7
26
22
11
-
11
27
19
27
45
34
12
41
14
-
19
-
18
12
-
-
-
33
19
-
22
22
-
31
22
39
31
34
38
-
17
11
-
MDA-MB-231/ATTC
HS 578T
BT-549
22
-
24
10
-
10
-
-
-
19
44
21
-
50
90
52
28
60
51
-
-
T-47D
37
16
49
14
71
27
29
13
-
-
67
13
MDA-MB-468
-
19
a Growth inhibition % produced by the compound is below 10%
From the obtained results in table 3, it is obvious that each of the screened substituted 4-
amino-2-thiopyrimidines has different degree of selectively against 60 cell lines. CCRF-CEM,
K-562, MOLT-4 and PRMI-8226 from leukemia; NCI-H522 from non-small cell lung cancer;
SK-MEL-5 and UACC-62 from melanoma; A498 and UO-31 from renal cancer; PC-3 from
prostate cancer and T-47D and MDA-MB-468 from breast cancer are the most sensitive cell
15

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lines to the tested compounds. Compound 9c showed broad spectrum of potent anti-cancer
activity against several NCI cell panels with mean growth inhibition of 33%. At 10 µM
concentration 9c showed 82, 84 and 80% inhibition against the leukemia cell lines CCRF-
CEM, MOLT-4 and RPMI-8226, respectively. Against non-small cell lung cancer cell line
NCI-H522, it exerted 73% inhibition. Against melanoma cell lines SK-MEL-5 and UACC-62,
it showed 62 and 61% inhibition, respectively. Against ovarian cancer cell lines OVCAR-8
and NCI/ADR-RES, it showed 77 and 58% inhibition, respectively. Against renal cancer cell
lines A498 and UO-31, it showed 50 and 52% inhibition, respectively. Against prostate
cancer cell line PC-3, it exerted 60% inhibition. Finally, against breast cancer cell lines
MCF7, BT-549, T-47D, MDA-MB-468, it showed 33, 50, 90, 52% inhibition.
2.2.2.2. In vitro cytotoxic activity against breast cancer cell lines
The newly synthesized substituted 4-amino-2-thiopyrimidines 6-10 as well as sorafenib (I)
were evaluated for their in vitro cytotoxic activity on MCF7 cell line as a representative cell
line which expresses both VEGFR-2 [10,11] and BRAF WT [62,63], using Sulfo-Rhodamine-
B (SRB) assay [64]. Table 4 shows the IC50 results of the tested compounds indicating that
compounds 7b, 8d, 9a, 9b, 9c and 9d (IC50 of 17.18, 17.20, 19.98, 19.61, 13.02 and 16.54
µM, respectively) showed the most potent activity against MCF7 cell line. On the other hand,
compounds 6b, 6d, 10a and 10d (IC50 of 28.21, 24.37, 26.88 and 23.01 µM, respectively)
showed slightly less potent activity. The reference standard, sorafenib (I) showed an IC50 of
4.33 µM in this assay.
Based on the results of the NCI 60 cell panel, selected compounds were further screened for
their activity against T-47D, which expresses BRAF WT [63], to determine their IC50.
Compounds 9c, 9d and 10d (IC50 = 2.18, 8.09 and 4.36 µM) were found to be the most potent
compounds in comparison to sorafenib (I) (IC50 = 1.60 µM). While 6b, 6c, 6d, 7a and 10a
(IC50 = 17.08, 13.23, 12.05, 14.45 and 14.35 µM) were found to be of less potency against T-
47D cell line.
Table 4. Cytotoxic activity of the synthesized substituted 4-amino-2-thiopyrimidines on
MCF7 and T-47D cell lines.
Entry
Compound IC50 (µM)a MCF7
IC50 (µM)a T-47D
32.95
1
2
53.63
28.21
6a
6b
17.08
16

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3
67.07
24.37
55.19
17.18
80.31
66.14
37.16
81.25
67.55
17.20
53.24
37.80
49.04
19.98
19.61
13.02
16.54
32.11
68.51
26.88
73.27
69.05
23.01
13.23
12.05
14.45
ndb
nd
6c
4
6d
5
7a
6
7b
7
7c
8
nd
7d
9
nd
8a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
nd
8b
nd
8c
nd
8d
nd
8e
nd
8f
nd
8g
nd
9a
32.06
2.18
8.09
43.39
nd
9b
9c
9d
9e
9f
14.35
nd
10a
10b
10c
10d
Sorafenib
nd
4.36
4.33
1.60
aIC50 are presented as mean of three independent experiments; bnot determined
2.2.3. In vitro cell based VEGFR-2 inhibitory activity
Substituted 4-amino-2-thiopyrimidines which demonstrated promising enzymatic as well as
anti-proliferative activity on MCF7 cell line were further assayed for their effect on the
inhibition of VEGFR-2 in MCF7 cell line at 10 µM employing ELISA assay kit. The
observed results are depicted in table 5. It was obvious that most of the tested compounds
showed moderate to potent inhibition of VEGFR-2 e.g. 33-84% in comparison to sorafenib (I)
which showed 90% inhibition. The only exceptions are compounds 6c and 9e. Compounds 9c
17

ACCEPTED MANUSCRIPT
and 9d have the most promising VEGFR-2 inhibitory activity of 84 and 80%, respectively.
The derivatives 7b, 8d, 9b showed slightly less potent activity of 76, 77, 76%, respectively,
while compounds 6d, 8a, 8f, 10a, 10d showed moderate inhibitory activity of 36, 35, 40, 33,
52%.
Table 5. Results of the effect of substituted 4-amino-2-thiopyrimidines on VEGFR-2 level in
MCF7 cell line
Entry Compound
Amount of VEGFR-2 (ng/mL)a
237.66 ± 28.13
180.78 ± 20.14
68.50 ± 7.15
% Inhibitionb
1
2
16
36
76
35
77
40
76
84
80
12
33
52
90
0
6c
6d
3
7b
4
184.90 ± 21.00
66.00 ± 7.66
8a
5
8d
6
169.17 ± 18.32
68.00 ± 7.13
8f
7
9b
8
46.00 ± 4.11
9c
9
57.10 ± 6.81
9d
10
11
12
13
14
250.40 ± 25.00
191.11 ± 21.70
135.70 ± 16.00
28.26 ± 3.09
9e
10a
10d
Sorafenib
DMSO
283.80 ± 29.67
aData were expressed as mean ± standard error (S.E.) of two experiments. bPercentage of
inhibition as compared with control cancer cells
These biological results indicate that 9c is a promising antiproliferative agent with a dual
VEGFR-2/BRAF inhibitory activity.
2.4. Molecular Docking Studies and Structure Activity Relationship
In order to study the binding characteristics of the newly synthesized compounds in the
binding site of both VEGFR-2 and BRAF, molecular docking studies were carried out using
Molecular Operating Environment (MOE, 2010.10) software. The two X-ray crystallographic
structures of VEGFR-2 (PDB ID: 4ASD) [57] and BRAF (PDB ID: 1UWH) [58] in their
18

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inactive conformation (DFG-out) were downloaded from the Protein Data Bank (PDB) [65].
The downloaded proteins are co-crystalized with a type II PK inhibitor, sorafenib (I).
Molecular docking protocol was initially validated by re-docking of the co-crystalized ligand,
sorafenib (I), in the binding sites of VEGFR-2 and BRAF. These two simulations successfully
reproduced the binding pattern of the co-crystalized ligand in VEGFR-2 and BRAF binding
sites with energy scores of −15.19 and −14.01 kcal/mol, respectively, and with an RMSD of
0.470 and 0.580 Å, respectively, between the docked poses and the co-crystalized ligand. In
addition, the docking poses reproduced all the key interactions achieved by the co-crystallized
ligand with the binding site hot spots in VEGFR-2 (Glu885, Cys919 and Asp1046) and BRAF
(Glu500, Cys531 and Asp593). The validation step results indicate the suitability of the used
molecular docking protocol for the molecular docking study of the newly synthesized
compounds in the binding sites of VEGFR-2 and BRAF (for further details see SI).
Generally, the newly synthesized compounds 6-10 showed comparable binding pattern in
both proteins with predicted docking energy scores in the binding site of both VEGFR-2 and
BRAF ranging from −13.00 to −15.19 kcal /mol and −11.41 to −13.96 kcal /mol, respectively
in comparison to the native ligand binding score of −15.19 and −14.01 kcal /mol, respectively
(for further details see the SI). The designed compounds showed a general promising binding
pattern as they interact with the key amino acids in the VEGFR-2 and BRAF binding sites.
The 4-amino-2-thiopyrimidine scaffold is accommodated in the gate area at the binding site
center interacting through hydrogen bonding by its 4-amino group with the side chain
carboxylate of Glu885 and Glu500 of the αC helix in VEGFR-2 and BRAF, respectively, and
by its pyrimidine N atom at position 3 with Asp1046 and Asp593 of the conserved DFG motif
in VEGFR-2 and BRAF, respectively. This orientation fits the hydrophobic substituent on the
4-amino group in the hydrophobic allosteric back pocket achieving hydrophobic interactions
with the hydrophobic side chains of Ile888, Leu889, Ile892, Val898, Val899, Leu1019 and
Ile1044 amino acids lining the back pocket of VEGFR-2 and Val503, Leu504, Leu513,
Leu566, Ile571 and Ile591 of BRAF. On the other side, the substituent on the sulfide moiety
extends to fit in the front pocket which could achieve a hydrogen bond interaction with the
hinge region Cys919 and Cys531 amino acid in VEGFR-2 and BRAF, respectively, when it
has the appropriate functional group as OCH3. Some compounds achieve additional hydrogen
bond interaction with Cys1045 (VEGFR-2) by their carbonyl group on the 2-sulfide moiety
(For further details see SI)
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Based on the molecular docking study and biological evaluations we can conclude the
following structure-activity relationships; hydrophobic substitution on the 4-aminophenyl
moiety increases the binding to the proteins through increasing the hydrophobic interaction
with the hydrophobic allosteric back pocket. This SAR rationalizes the significant potency of
series 9 due to the hydrophobic nature of its ethyl ester moiety fitted in the back pocket. On
the other hand, the hinge region (front pocket) tolerates both hydrophobic and hydrophilic
moieties. Hydrophobic substituent (Cl, Br, CF3 and Ph) is tolerated through hydrophobic
interaction with the hydrophobic side chains of amino acids Leu840, Val848, Ala866, Val899,
Phe918, Leu1035 and Phe1047 in VEGFR-2 and Ile462, Val740, Trp530, Phe582 and Phe594
in BRAF. Hydrophilic substituents (CN, OCH3) are tolerated through either their hydrogen
bond interaction with Cys919 in VEGFR-2 and Cys531 in BRAF or through bulk solvent
exposure. (Fig. 4,5) (For further details see SI).
(A)
20

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(B)
Fig. 4. 2D diagram (A) and 3D representation (B) of compound 9c showing its interaction
with the VEGFR-2 active site.
(A)
21

ACCEPTED MANUSCRIPT
(B)
Fig. 5. 2D diagram (A) and 3D representation (B) of compound 9c showing its interaction
with the BRAF active site.
3. Conclusion
A hybridization strategy was adopted between substituted 4-aminopyrimidines (VEGFR-2
inhibitors) and substituted 2-thioxopyrimidines (BRAF inhibitors) to afford substituted 4-
amino-2-thiopyrimidines as type II dual VEGFR-2/BRAF inhibitors. In vitro evaluation of
their kinase inhibitory activity in VEGFR-2 and BRAF showed that most of the synthesized
compounds possess dual kinase inhibitory activity with single-digit micromolar to sub-
micromolar IC50 range. Compound 9c displayed promising inhibitory activity on VEGFR-2
(IC50 = 0.17 µM), as well as, on BRAF (IC50 = 0.15 µM). In addition, it showed potent anti-
proliferative activity with inhibition percentage up to 90% against wide range of cancer cell
lines when tested at 10 µM. Moreover, it showed comparable reduction in the VEGFR-2
level in MCF7 cell line to that of sorafenib (I) (84 vs 90%, respectively). Molecular docking
of the designed compounds in VEGFR-2 and BRAF active sites revealed that they are able
to accomplish the key binding interactions in the binding sites of both kinases.
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4. Experimental
4.1. Chemistry
4.1.1. General remarks
All chemicals were purchased from commercial suppliers. Analytical thin layer
chromatography (TLC) was performed on precoated silica gel 60 F245aluminium plates
(Merck) with visualization under UV light. Melting points were determined on a Stuart
SMP30 melting point apparatus with open capillary tubes and are uncorrected. Elemental
analyses and spectral data of the compounds were performed in the Micro analytical labs,
National Research Centre and Micro Analytical Laboratory Centre, Faculty of Science, Cairo
University, Cairo, Egypt. IR spectra (4000–400 cm-1) were recorded using KBr pellets in a
Jasco FT/IR 300E Fourier transform infrared spectrophotometer on a Perkin Elmer FT-IR
1650(spectrophotometer). 1HNMR and 13CNMR spectra were recorded at 400 (100) MHz and
500 (125) MHz on Bruker instruments 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 (Hz).
4.1.1.1. General procedure I for the synthesis of 2-((2-oxo-2-(4-substituted-
phenyl)ethyl)thio)pyrimidin-4(3H)-ones 3a-h
A suspension of 2-thiouracil (1), 2-bromoacetophenones 2a-h and anhydrous K2CO3 were
stirred under reflux in ethanol for 4h. The reaction mixture was then cooled to rt and poured
on water and neutralized with 2N HCl. The precipitated solid was filtered and dried to give
the crude products which were further purified by crystallization form methanol to give
analytically pure products 3a-h.
2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)pyrimidin-4(3H)-one (3a)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
fluoroacetophenone (2a) (2.17g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10 mmol) were
reacted in ethanol (40 mL) to give 3a as a pale yellow powder (2.17 g, 82%); mp 191-192 °C
(lit. [59] 192-194 °C).
2-((2-(4-Chlorophenyl)-2-oxoethyl)thio)pyrimidin-4(3H)-one (3b)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
chloroacetophenone (2b) (2.34 g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10 mmol) were
23

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reacted in ethanol (40 mL) to give 3b as a pale yellow powder (2.25 g, 80%); mp 185-187 °C.
(lit. [59] 177-180 °C).
2-((2-(4-Bromophenyl)-2-oxoethyl)thio)pyrimidin-4(3H)-one (3c)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
bromoacetophenone (2c) (2.77 g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10 mmol) were
reacted in ethanol (40 mL) to give 3c as pale yellow powder (3.01 g, 93%); mp 206-208 °C
(lit. [60] 208-210 °C).
2-((2-Oxo-2-(4-(trifluoromethyl)phenyl)ethyl)thio)pyrimidin-4(3H)-one (3d)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
(trifluoromethyl)acetophenone (2d) (2.67 g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10
mmol) were reacted in ethanol (40 mL) to give 3d as a pale yellow powder (2.73 g, 87%); mp
159-161 °C.
4-(2-((6-Oxo-1,6-dihydropyrimidin-2-yl)thio)acetyl)benzonitrile (3e)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
cyanoacetophenone (2e) (2.24 g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10 mmol) were
1
reacted in ethanol (40 mL) to give 3e as a yellow powder (1.50 g, 55%); mp 246-248 °C; H
NMR (500 MHz; DMSO-d6) δH 4.82 (s, 2H, CH2), 6.04 (d, 3J = 7.0 Hz, 1H), 7.80 (d, 3J = 6.5
Hz, 2H), 8.04 (d, 3J = 8.5 Hz, 2H), 8.17 (d, 3J = 8.5 Hz, 1H), 12.93 ppm (br., 1H).
2-((2-(4-Nitrophenyl)-2-oxoethyl)thio)pyrimidin-4(3H)-one (3f)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
nitroacetophenone (2f) (2.24 g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10 mmol) were
reacted in ethanol (40 mL) to give 3f (2.12 g, 73%); mp 200-202 °C. (lit. [59] 196-198 °C).
2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)pyrimidin-4(3H)-one (3g)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
methoxyacetophenone (2g) (2.29 g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10 mmol) were
reacted in ethanol (40 mL) to give 3g (2.33 g, 84%); mp 186-188°C (lit. [59] 183-184 °C).
2-((2-([1,1'-Biphenyl]-4-yl)-2-oxoethyl)thio)pyrimidin-4(3H)-one (3h)
According to the general procedure I; 2-thiouracil (1) (1.28 g, 10 mmol), 2-bromo-4′-
phenylacetophenone (2h) (2.75 g, 10 mmol) and anhydrous K2CO3 (1.38 g, 10 mmol) were
24

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reacted in ethanol (40 mL) to give 3h as a pale yellow powder (3.02 g, 94 %); mp 198-200 °C
(lit. [59] 195-197 °C).
4.1.1.2. General procedure II for the synthesis of 2-((4-chloropyrimidin-2-yl)thio)-1-(4-
substituted phenyl)-ethan-1-ones 4a-h.
A mixture of 2-((2-oxo-2-(4-substituted phenyl)ethyl)thio)pyrimidin-4(3H)-ones 3a-h and
POCl3 were stirred at 80 °C for 30 min. The reaction mixture was cooled to room temperature
and poured on crushed ice with continuous stirring. The precipitated solid was filtered and
dried to give the crude products 4a-h which were directly used without further purification.
2-((4-Chloropyrimidin-2-yl)thio)-1-(4-fluorophenyl)ethan-1-one (4a)
According to the general procedure II; 2-((2-(4-fluorophenyl)-2-oxoethyl)thio)pyrimidin-
4(3H)-one (3a) (2.64 g, 10 mmol) was reacted with POCl3 (10 mL) to yield 4a as a pale
1
yellow powder (2.26 g, 80%); mp 68-70 °C; H NMR (400 MHz; DMSO-d6) δH 4.83 (s, 2H,
CH2), 7.37-7.41 (m, 3H), 8.12-8.15 (m, 2H), 8.53 ppm (d, 3J = 5.2 Hz, 1H).
1-(4-Chlorophenyl)-2-((4-chloropyrimidin-2-yl)thio)ethan-1-one (4b)
According to the general procedure II; 2-((2-(4-chlorophenyl)-2-oxoethyl)thio)pyrimidin-
4(3H)-one (3b) (2.20 g, 7.84 mmol) was reacted with POCl3 (10 mL) to yield 4b as a pale
1
yellow powder (2.30 g, 98%); mp 114-116 °C; H NMR (400 MHz; DMSO-d6) δH 4.82 (s,
2H, CH2), 7.38 (d, 3J = 5.6 Hz, 1H), 7.63 (d, 3J = 8.4 Hz, 2H), 8.06 (d, 3J = 8.8 Hz, 2H), 8.53
ppm (d, 3J = 5.3 Hz, 1H).
1-(4-Bromophenyl)-2-((4-chloropyrimidin-2-yl)thio)ethan-1-one (4c)
According to the general procedure II; 2-((2-(4-bromophenyl)-2-oxoethyl)thio)pyrimidin-
4(3H)-one (3c) (1.10 g, 3.38 mmol) was reacted with POCl3 (6 mL) to yield 4c as a pale
1
yellow powder (0.90 g, 77%); mp 116-118 °C; H NMR (500 MHz; DMSO-d6) δH 4.82 (s,
2H, CH2), 7.38 (d, 3J = 5.5 Hz, 1H), 7.78 (d, 3J = 8.5 Hz, 2H), 7.98 (d, 3J = 8.5 Hz, 2H), 8.53
ppm (d, 3J = 5.0 Hz, 1H).
2-((4-Chloropyrimidin-2-yl)thio)-1-(4-(trifluoromethyl)phenyl)ethan-1-one (4d)
According
to
the
general
procedure
II;
2-((2-oxo-2-(4-
(trifluoromethyl)phenyl)ethyl)thio)pyrimidin-4(3H)-one (3d) (3.14 g, 10 mmol) was reacted
1
with POCl3 (10 mL) to yield 4d as a pale green powder (2.66 g, 80%); mp 125-127 °C; H
25

ACCEPTED MANUSCRIPT
NMR (400 MHz; DMSO-d6) δH 4.88 (s, 2H, CH2), 7.38 (d, 3J = 5.2 Hz, 1H), 7.94 (d, 3J = 8.0
Hz, 2H), 8.23 (d, 3J = 8.0 Hz, 2H), 8.54 ppm (d, 3J = 5.2 Hz, 1H).
4-(2-((4-Chloropyrimidin-2-yl)thio)acetyl)benzonitrile (4e)
According to the general procedure II; 4-(2-((6-oxo-1,6-dihydropyrimidin-2-
yl)thio)acetyl)benzonitrile (3e) (0.50 g, 1.84 mmol) was reacted with POCl3 (6 mL) to yield
1
4e as a pale yellow powder (0.40 g, 75%); mp 109-111 °C; H NMR (500 MHz; DMSO-d6)
δH 4.86 (s, 2H, CH2), 7.38 (d, 3J = 5.5 Hz, 1H), 8.05 (d, 3J = 9.0 Hz, 2H), 8.19 (d, 3J = 8.5 Hz,
2H), 8.53 ppm (d, 3J = 5.0 Hz, 1H).
2-((4-Chloropyrimidin-2-yl)thio)-1-(4-nitrophenyl)ethan-1-one (4f)
According to the general procedure II; 2-((2-(4-nitrophenyl)-2-oxoethyl)thio)pyrimidin-
4(3H)-one (3f) (2.91 g, 10 mmol) was reacted with POCl3 (10 mL) to yield 4f as a yellow
powder (2.62 g, 85%); mp 115-117 °C; 1H NMR (500 MHz; DMSO-d6) δH 4.89 (s, 2H, CH2),
7.38 (d, 3J = 5.0 Hz, 2H), 8.27 (d, 3J = 9.0 Hz, 1H), 8.37 (d, 3J = 8.5 Hz, 2H), 8.53 ppm (d, 3J
13
= 5.0 Hz, 1H); C NMR (125 MHz; DMSO-d6) δC 38.89, 117.47, 123.93, 129.73, 140.75,
150.08, 159.55, 160.11, 170.98, 192.95 ppm.
2-((4-Chloropyrimidin-2-yl)thio)-1-(4-methoxyphenyl)ethan-1-one (4g)
According to the general procedure II; 2-((2-(4-methoxyphenyl)-2-oxoethyl)thio)pyrimidin-
4(3H)-one (2.30 g, 8.32 mmol) (3g) was reacted with POCl3 (10 mL) to yield 4g as a pale
green powder (2.20 g, 94%), mp 162-164 °C; 1H NMR (400 MHz; DMSO-d6) δH 3.84 (s, 3H,
3
3
3
OCH3), 4.78 (s, 2H, CH2), 7.06 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 5.2 Hz, 1H), 8.02 (d, J =
8.8 Hz, 2H), 8.53 ppm (d, 3J = 5.2 Hz, 1H).
1-([1,1'-Biphenyl]-4-yl)-2-((4-chloropyrimidin-2-yl)thio)ethan-1-one (4h)
According to the general procedure II; 2-((2-([1,1'-biphenyl]-4-yl)-2-oxoethyl)thio)pyrimidin-
4(3H)-one (3h) (3.22 g, 10 mmol) was reacted with POCl3 (10 mL) to yield 4h as a pale
1
yellow powder (2.70 g, 79%); mp 114-116 °C; H NMR (400 MHz; DMSO-d6) δH 4.88 (s,
2H, CH2), 7.38 (d, 3J = 5.2 Hz, 1H), 7.42-7.45 (m, 1H), 7.51 (t like, 3J = 7.6 Hz, 2H), 7.77 (d,
3
3
3
3J = 7.6 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 8.13 (d, J = 8.4 Hz, 2H), 8.55 ppm (d, J = 5.2
Hz, 1H).
4.1.1.3. General procedure III for the synthesis of 2-((4-substituted-pyrimidin-2-yl)thio)-
1-phenylethan-1-one derivatives (6-10).
26

ACCEPTED MANUSCRIPT
A
mixture
of
2-((4-chloropyrimidin-2-yl)thio)-1-(4-substituted-phenyl)ethan-1-one
derivatives 4a-h (0.50 mmol) and substituted aniline 5a-e (0.50 mmol) were refluxed in acetic
acid (15 mL) for 4h. The reaction mixture was poured into ice-water, filtered, washed
thoroughly with water, dried and crystallized from methanol to obtain the target compounds
6-10.
2-((4-((3-Acetylphenyl)amino)pyrimidin-2-yl)thio)-1-(4-chlorophenyl)ethan-1-one (6a).
According to the general procedure III, 1-(4-chlorophenyl)-2-((4-chloropyrimidin-2-
yl)thio)ethan-1-one (4b) (149.59 mg, 0.50 mmol) and 3´-aminoacetophenone (5a) (67.58 mg,
0.50 mmol) were reacted in acetic acid (15 mL) to give 6a as an off white powder (155.00
mg, 78%); mp 164-166 °C; Rf = 0.46 (Pet Ether / EtOAc = 1:2); ύ max (atr)/cm-1, 3329, 3082,
3032, 2913, 1674, 1570 and 1477; 1H NMR (400 MHz; DMSO-d6) δH 2.53 (s, 3H, CH3), 4.76
(s, 2H, CH2), 6.48 (d, 3J = 6.0 Hz, 1H), 7.18-7.20 (m, 1H), 7.53 (d, 3J = 7.6 Hz, 1H), 7.58 (d,
3J = 8.4 Hz, 2H), 7.82 (d, 3J = 8.0 Hz, 1H), 7.97 (d, 3J = 8.4 Hz, 2H), 8.04 (s, 1H), 8.07 (d, 3J
= 5.6 Hz, 1H) and 9.84 ppm (br., 1H); Anal. Calcd for C20H16ClN3O2S: C, 60.38; H, 4.05; N,
10.56. Found: C, 60.65; H, 4.32; N, 10.25.
2-((4-((3-Acetylphenyl)amino)pyrimidin-2-yl)thio)-1-(4-trifluoromethylphenyl)ethan-1-one
(6b).
According to the general procedure III, 2-((4-chloropyrimidin-2-yl)thio)-1-(4-
(trifluoromethyl)phenyl)ethan-1-one (4d) (166.36
mg, 0.50 mmol) and 3´-
aminoacetophenone (5a) (67.58 mg, 0.50 mmol) were reacted in acetic acid (15 mL) to give
6b as a yellow powder (172.00 mg, 80%); mp 140-142 °C; Rf = 0.26 (Pet Ether / EtOAc =
1:2); ύ max (atr)/cm-1, 3433, 2925, 1740, 1635, 1517 and 1458; 1H NMR (500 MHz; DMSO-
3
3
d6) δH 2.51 (s, 3H, CH3), 4.81 (s, 2H, CH2), 6.47 (d, J = 5.5 Hz, 1H), 7.16 (t, J = 8.0 Hz,
3
3
3
1H), 7.48 (d, J = 8.0 Hz, 1H), 7.80 (dd, J = 8.0 Hz, 4J = 1.5 Hz, 1H), 7.87 (d, J = 8.5 Hz,
2H), 8.03-8.04 (m, 1H), 8.07 (d, 3J = 6.0 Hz, 1H), 8.12 (d, 3J = 8.0 Hz, 2H) and 9.84 ppm (s,
1H); 13C NMR (125 MHz; DMSO-d6) δC 26.61, 38.46, 103.52, 119.07, 122.56, 124.35,
125.66, 125.69, 128.88, 128.97, 137.14, 139.21, 139.46, 142.30, 155.48, 159.45, 168.79,
193.29 and 197.47 ppm; Anal. Calcd for C21H16F3N3O2S: C, 58.46; H, 3.74; N, 9.74. Found:
C, 58.28; H, 3.97; N, 9.56.
2-((4-((3-Acetylphenyl)amino)pyrimidin-2-yl)thio)-1-(4-methoxyphenyl)ethan-1-one (6c).
According to the general procedure III, 2-((4-chloropyrimidin-2-yl)thio)-1-(4-
methoxyphenyl)ethan-1-one (4g) (147.38 mg, 0.50 mmol) and 3´-aminoacetophenone (5a)
27

ACCEPTED MANUSCRIPT
(67.58 mg, 0.50 mmol) were reacted in acetic acid (15 mL) to give 6c as an off white powder
(138.00 mg, 70%); mp 139-140 °C; Rf = 0.37 (Pet Ether / EtOAc = 1:2); ύ max (atr)/cm-1,
1
3338, 2923, 1668, 1600, 1561 and 1481; H NMR (500 MHz; DMSO-d6) δH 2.53 (s, 3H,
3
CH3), 3.85 (s, 3H, OCH3), 4.73 (s, 2H, CH2), 6.49 (d, J = 5.0 Hz, 1H), 7.03 (d, 3J = 8.0 Hz,
2H), 7.17 (d, 3J = 8.0 Hz, 1H), 7.54 (d, 3J = 7.5 Hz, 1H), 7.88 (d, 3J = 7.5 Hz, 1H), 7.97 (d, 3J
13
= 8.0 Hz, 2H), 8.05-8.08 (m, 2H) and 9.85 ppm (s, 1H); C NMR (125 MHz; DMSO-d6) δC
26.69, 37.93, 55.60, 103.46, 113.95, 118.96, 122.56, 124.28, 128.69, 128.95, 130.63, 137.24,
139.62, 155.48, 159.46, 163.34, 169.19, 191.89 and 197.58 ppm; Anal. Calcd for
C21H19N3O3S: C, 64.11; H, 4.87; N, 10.87. Found: C, 64.38; H, 4.54; N, 10.55.
1-([1,1'-Biphenyl]-4-yl)-2-((4-((3-acetylphenyl)amino)pyrimidin-2-yl)thio)ethan-1-one (6d).
According to the general procedure III, 1-([1,1'-biphenyl]-4-yl)-2-((4-chloropyrimidin-2-
yl)thio)ethan-1-one (4h) (170.41 mg, 0.50 mmol) and 3´-aminoacetophenone (5a) (67.58 mg,
0.50 mmol) were reacted in acetic acid (15 mL) to give 6d as a pale yellow powder (166.00
mg, 76%); mp 182-184 °C; Rf = 0.30 (Pet Ether / EtOAc = 1:2); ύ max (atr)/cm-1, 3430, 3076,
1
2955, 2924, 1675, 1646, 1601 and 1542; H NMR (500 MHz; DMSO-d6) δH 2.51 (s, 3H,
CH3), 4.82 (s, 2H, CH2), 6.50 (d, 3J = 6.0 Hz, 1H), 7.17 (t, 3J = 8.0 Hz, 1H), 7.44 (t like, 3J =
7.5 Hz, 1H), 7.49-7.53 (m, 3H), 7.76 (d like, 3J = 7.5 Hz, 2H), 7.81 (d, 3J = 8.5 Hz, 2H), 7.87
(dd, 3J = 8.0 Hz, 4J = 1.5 Hz, 1H), 8.05-8.06 (m, 3H), 8.09 (d, 3J = 6.0 Hz, 1H) and 9.86 ppm
(s, 1H); 13C NMR (125 MHz; DMSO-d6) δC 26.66, 38.31, 103.49, 119.03, 122.55, 124.31,
126.90, 127.02, 128.46, 128.97, 129.13, 134.65, 137.20, 138.85, 139.57, 155.48, 159.47,
169.06, 193.11 and 197.53 ppm; Anal. Calcd for C26H21N3O2S: C, 71.05; H, 4.82; N, 9.56.
Found: C, 71.24; H, 4.57; N, 9.74.
2-((4-((4-Acetylphenyl)amino)pyrimidin-2-yl)thio)-1-(4-chlorophenyl)ethan-1-one (7a).
According to the general procedure III, 1-(4-chlorophenyl)-2-((4-chloropyrimidin-2-
yl)thio)ethan-1-one (4b) (149.59 mg, 0.50 mmol) and 4´-aminoacetophenone (5b) (67.58 mg,
0.50 mmol) were reacted in acetic acid (15 mL) to give 7a as an off white powder (150.00
mg, 75%); mp 210-212 °C; Rf = 0.38 (Pet Ether / EtOAc = 1:2); ύ max (atr)/cm-1, 3429, 3087,
2962, 1664, 1633, 1564 and 1457; 1H NMR (400 MHz; DMSO-d6) δH 2.39 (s, 3H, CH3), 4.80
(s, 2H, CH2), 6.54 (m, 1H), 7.51-7.60 (m, 6H), 8.05-8.12 (m, 3H) and 9.98 ppm (br., 1H);
Anal. Calcd for C20H16ClN3O2S: C, 60.38; H, 4.05; N, 10.56. Found: C, 60.12; H, 4.23; N,
10.79.
2-((4-((4-Acetylphenyl)amino)pyrimidin-2-yl)thio)-1-(4-bromophenyl)ethan-1-one (7b).
28