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

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

Novel Potent Substituted 4-Amino-2-thiopyrimidines as Dual VEGFR-2

and BRAF Kinase Inhibitors

Heba T. Abdel-Mohsen*^a, Mohamed A. Omar^a, Ahmed M. El Kerdawy^b,c,d, Abeer E.E.

Mahmoud^e, Mamdouh M. Ali^e, Hoda I. El Diwani^a

^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 IC₅₀up 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

H

Br

O

N

S

O

N

S

(i)

(ii)

N

NH

R¹

1

2a-h

3a-h

R²

O

NH₂

H

N

S

Cl

N

S

5a-e

R²

N

(iii)

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 IC₅₀of all the target compounds were evaluated against MCF7 cell line.

Furthermore, cytotoxicity IC₅₀of 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 IC₅₀of 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 (IC₅₀of 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 (IC₅₀of

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-OCH₃derivative 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-CF₃8c and 4-

NO₂8e 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 IC₅₀of 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 IC₅₀of 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

ACCEPTED MANUSCRIPT

Entry Compound

R¹

R²

IC₅₀(µM)^a

VEGFR-2

0.48 ± 0.02

> 10

IC₅₀(µM)^a

BRAF-WT

nd^b

1

Cl

3-COCH₃

4-COCH₃

4-COOH

4-COOEt

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

^aIC₅₀are presented as Mean ± Standard error (S.E.) of two independent experiments; ^bNot 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

nd^a

5.16

0.25

0.08

9c

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

-

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

22

15

73

49

22

34

-

11

42

13

16

-

29

28

24

-

28

-

23

22

11

14

15

-

10

-

26

13

19

32

-

18

-

14

-

HT29

-

13

ACCEPTED MANUSCRIPT

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

-

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

35

-

19

-

17

-

12

-

OVCAR-3

OVCAR-4

OVCAR-5

OVCAR-8

NCI/ADR-RES

-

18

57

-

12

-

18

-

20

-

15

-

32

40

29

46

18

31

-

77

58

31

27

62

26

17

-

24

11

14

-

14

ACCEPTED MANUSCRIPT

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

-

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

^aGrowth 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

ACCEPTED MANUSCRIPT

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 IC₅₀results of the tested compounds indicating that

compounds 7b, 8d, 9a, 9b, 9c and 9d (IC₅₀of 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 (IC₅₀of 28.21, 24.37, 26.88 and 23.01 µM, respectively)

showed slightly less potent activity. The reference standard, sorafenib (I) showed an IC₅₀of

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)^aMCF7

IC₅₀(µM)^aT-47D

32.95

1

2

53.63

28.21

6a

6b

17.08

16

ACCEPTED MANUSCRIPT

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

nd^b

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

^aIC₅₀are 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

% Inhibition^b

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

ACCEPTED MANUSCRIPT

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)

19

ACCEPTED MANUSCRIPT

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, CF₃and 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

ACCEPTED MANUSCRIPT

(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 IC₅₀range. 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.

22

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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-d₆as 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 K₂CO₃were

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

ACCEPTED MANUSCRIPT

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) δ_H4.82 (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

ACCEPTED MANUSCRIPT

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

POCl₃were 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) δ_H4.83 (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) δ_H4.82 (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) δ_H4.82 (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) δ_H4.88 (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)

δ_H4.86 (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) δ_H4.89 (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) δ_C38.89, 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) δ_H3.84 (s, 3H,

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) δ_H4.88 (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

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; R_f= 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) δ_H2.53 (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; R_f= 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

d₆) δ_H2.51 (s, 3H, CH₃), 4.81 (s, 2H, CH₂), 6.47 (d, J = 5.5 Hz, 1H), 7.16 (t, J = 8.0 Hz,

3

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) δ_C26.61, 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; R_f= 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) δ_H2.53 (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) δ_C

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; R_f= 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) δ_H2.51 (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) δ_C26.66, 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; R_f= 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) δ_H2.39 (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

Article Doi

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

DOI: 10.1016/j.ejmech.2019.06.063

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

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