1-Aryl-3-[4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl]ureas as VEGFR-2 Tyrosine Kinase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modelling Studies

The vascular endothelial growth factor receptor-2 (VEGFR-2) is a tyrosine kinase receptor involved in the growth and differentiation of endothelial cells that are implicated in tumor-associated angiogenesis. In this study, novel 1-aryl-3-[4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl]ureas were synthesized and evaluated for the VEGFR-2 tyrosine kinase inhibition. Three of these compounds showed good VEGFR-2 inhibition presenting low IC50 values (150–199 nM) in enzymatic assays, showing also a significant proliferation inhibition of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) at low concentrations (0.5–1 µM), using the Bromodeoxyuridine (BrdU) assay, not affecting cell viability. The determination of the total and phosphorylated (active) VEGFR-2 was performed by western blot, and it was possible to conclude that the compounds significantly inhibit the phosphorylation of the receptor at 1 µM pointing to their antiproliferative mechanism of action in HUVECs. The molecular rationale for inhibiting the tyrosine kinase domain of VEGFR-2 was also performed and discussed using molecular docking studies.


Introduction
Angiogenesis is the process of new blood vessel formation from preexisting vascular networks by capillary sprouting [1] and plays an important role in the pathogenesis of several disorders including cancer, vasculoproliferative ocular disorders, and rheumatoid arthritis [2]. A key regulatory pathway of angiogenesis is mediated by the vascular endothelial growth factor (VEGF), involved in the vascular permeability and an inducer of endothelial cell proliferation, migration, and survival [3], and its cell membrane tyrosine kinase receptor VEGFR-2 (also known as KDR) [4]. Upon ligand binding, VEGFR-2 undergoes autophosphorylation, triggering signaling pathways leading to endothelial cell proliferation and subsequent angiogenesis. Small molecule inhibitors act by competing with ATP for its binding site of the VEGFR-2 intracellular tyrosine kinase domain, thereby preventing the signaling pathways that lead to angiogenesis [5]. Several small molecule VEGFR-2 inhibitors have emerged as promising antiangiogenic agents for possible treatment against a wide variety of cancers ( Figure 1). Sunitinib was approved for the treatment of renal cell carcinoma and gastrointestinal stromal tumor, and Sorafenib was approved for the treatment of primary kidney cancer and hepatocellular carcinoma [6]. Recently, two new VEGFR-2 inhibitors have been approved for the treatment of advanced renal cell carcinoma: axitinib [7] and pazopanib [8]. A number of thienopyridines and thienopyrimidines ureas have also shown potent VEGFR-2 inhibition activity [9,10] including arylether derivatives [11][12][13]. In this report, we describe an ongoing effort to develop novel small molecules as VEGFR-2 inhibitors, based on the aryletherthieno [3,2-d]pyrimidine arylurea scaffold. The synthesis of the compounds and the VEGFR-2 tyrosine kinase phosphorylation inhibition evaluation using either enzymatic or cellular assays including the determination of the total and of the phosphorylated VEGFR-2 by western blot were performed. The probable binding mode of the 1-aryl-3-[4-(thieno [3,2-d]pyrimidin-4-yloxy)phenyl]ureas with the receptor using docking studies is also presented and discussed.

Synthesis.
Melting points ( ∘ C) were determined in a Stuart SMP3 and are uncorrected. 1 H and 13 C NMR spectra were recorded on a Varian Unity Plus at 300 and 75.4 MHz, respectively, or on a Bruker Avance III at 400 and 100.6 MHz, respectively. Two-dimensional 1 H-13 C correlations were performed to attribute some signals. Mass spectra (MS) EI-TOF or ESI-TOF and HRMS on the M + , [M + H] + or on [M + Na] + , were performed by the mass spectrometry service of the University of Vigo, C.A.C.T.I., Spain.

General Procedure for the Synthesis of Compounds 1a
and 1b. In a flask with 5 mL of DMF, thienopyrimidine (1 equiv.), 4-aminophenol (1 equiv.), and K 2 CO 3 (4 equiv.) were heated at 140 ∘ C for 2 h. After cooling, water (5 mL) and ethyl acetate (5 mL) were added. The phases were separated, and the aqueous phase was extracted with more ethyl acetate (2 × 5 mL). The organic phase was dried (MgSO 4 ) and filtered. The solvent was evaporated under reduced pressure giving a solid which was submitted to column chromatography. [3,2-         recommended by the manufacturer Invitrogen, Cat. PV3190) [14]. Briefly, assays were performed in a total of 20 L in 384-well plates using fluorescence resonance energy transfer technology. A Tyr1 substrate (coumarin-fluorescein doublelabeled peptide) at 1.0 M was incubated for 1 h with 4 g/mL VEGFR-2, 10 M ATP, and inhibitors at room temperature in 50 mM Hepes/Na (pH 7.5), 10 mM MgCl 2 , 2.0 mM MnCl 2 , 2.5 mM DTT, 0.10 mM orthovanadate, and 0.01% bovine serum albumin. The wells were incubated at 25 ∘ C for 1 hour and 5.0 L of the development reagent was added to each well. After a second incubation of 1 hour, a stop reagent was added to each well. Using a Biotek FLX800 microplate, the fluorescence was read at 445 nm and 520 nm (excitation 400 nm), and Gen5 Software was used for data analysis. The validation assay was performed using Staurosporine that presented a IC 50 value 6 nM that compares to the one reported in the literature (IC 50 7 nM) [14].

MTS Toxicity Assay.
HUVECs (2 × 10 5 cells/mL) were allowed to grow for 24 h and then incubated with the test compounds at a range concentration between 0.1 and 10 M or control (0.1% DMSO) for 24 h. After the incubation period, cells were washed and their viability was assessed using Cell Titer 96 Aqueous ONE Solution Reagent MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] colorimetric assay (Promega, Madison, USA), according to the instructions provided by the manufacturer and as previously described [15]. Optical density was measured at 492 nm. Three independent experiments were performed, and the results were expressed as mean ± SEM.

BrdU Incorporation
Assay. HUVECs (6 × 10 4 cells/mL) were grown during 24 h and then were incubated with the compounds at 0.1-10 M or control (0.1% DMSO) for 24 h. Cells were also incubated with 5 -bromodeoxyuridine (BrdU), a thymidine analogue which incorporates into DNA of dividing cells. After incubation with BrdU solution at a final concentration of 0.01 mM during the treatment period, the optical density of proliferating cells (positive for BrdU) after ELISA assay using anti-BrdU-specific antibodies (Roche Diagnostics, Mannheim, Germany) was evaluated at the microplate reader according to the manufacturer's instructions and as previously reported [16]. The results are given as percentage versus control group (100%). kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's instruction and as previously reported [16]. Immunofluorescence was visualized under a fluorescence microscope (Olympus, BH-2, UK). The percentage of stained cells was evaluated by counting the cells stained with TUNEL (apoptotic cells) divided by the total number of nuclei counterstained with DAPI (Invitrogen, CA, USA) at a 200x magnification field. One thousand nuclei were evaluated. Results were expressed as percentage of control (100%).

Western Blotting Analyses.
Proteins were isolated from HUVEC lysates using RIPA (Chemicon International, CA, USA). Proteins were quantified using a spectrophotometer (Jenway, 6405 UV/vis, Essex, UK), and 20 g of protein were subjected to 8% SDS-PAGE with a 5% stacking gel. After electrophoresis, proteins were blotted into a Hybond nitrocellulose membrane (Amersham, Arlington, VA, USA). Immunodetection for total VEGFR-2 (1 : 1000; Cell Signaling, MA, USA), phosphorylated (activated) VEGFR-2 (1 : 750; Santa Cruz Biotechnology, CA, USA), and -actin (1 : 3000; Abcam, Cambridge, UK) was accomplished with enhanced chemiluminescence (ECL kit, Amersham, Arlington, USA). The phosphorylated antibody recognizes Tyr951 kinase insert domain, a major site of VEGFR-2 phosphorylation particularly involved in angiogenic processes. The relative intensity of each protein blotting analysis was measured using a computerized software program (Bio-Rad, CA, USA), and the expression of activated and total VEGFR-2 were normalized with total VEGFR-2 and -actin bands, respectively, to compare the expression of proteins in different treatment groups.

Statistical Analyses.
All experiments were performed at least in three independent experiments. Quantifications are expressed as mean ± SEM. Statistical significance of difference between the distinct groups was evaluated by analysis of variance (ANOVA) followed by the Bonferroni test. A difference between experimental groups was considered significant with a confidence interval of 95%, whenever < 0.05.

Docking Simulations Using AutoDock4.
A VEGFR-2 crystal structure (PDB: 2XIR) was extracted from the Protein Data Bank (PDB) (http://www.rcsb.org/). The cocrystallized ligand was extracted from the PDB file, and AutoDockTools was used to assign polar hydrogens and Gasteiger charges to the compounds [17]. AutoGrid4 was used to create affinity grid maps for all the atom types. The affinity grids enclosed an area of 100 × 100 × 100 with 0.375Å spacing, centered on the coordinates = 86.3, = 51.2, and = 48.3. AutoDock4 (version 4.1) with the Lamarckian genetic algorithm was used with the following docking parameters: 100 docking runs, population size of 200, random starting position and

Results and Discussion
3.1. Synthesis. We were able to promote the regioselective attack of the hydroxyl group of the 4-aminophenol in the chlorine nucleophilic displacement of two commercial 4chlorinated thieno[3,2-d]pyrimidines, using stoichiometric amounts of the reagents, in the synthesis of the aminated compounds 1a and 1b in excellent yields (Scheme 1). This regioselectivity constitutes an important achievement that avoids the reaction of 4-nitrophenols followed by reduction of the nitro compounds to the corresponding amino compounds, as often described in the literature [12,13]. With 4-aminophenol as a reagent, it is possible to use DMF as solvent and heat only at 140 ∘ C instead of diphenylether at 180 ∘ C which is needed for the nucleophilic displacement of the chlorine using 4-nitrophenol. The amino compounds 1a and 1b, obtained in one step, were then reacted with different arylisocyanates to give the corresponding new  Then, the ability of compounds 2a-c to inhibit VEGFstimulated proliferation of HUVECs was evaluated using the BrdU incorporation assay (Figure 2(b)). Inhibition of the VEGFR-2 activity was strongly reflected at the cellular level, with all the three compounds showing a statistical significant inhibition activity against VEGF-stimulated HUVEC proliferation at 0.5 M (15.2 ± 2.7% for 2a; 12.8 ± 2.4% for 2b, and 14.7 ± 1.7% for 2c) when comparing to control, which is consider to be 100.00 ± 2.8%. The number of proliferating HUVECs decreased in a dose-dependent manner (Figure 2(b)).
Interestingly, incubation of HUVECs with compounds 2a, 2b, and 2c for 24 h with concentrations 0.1, 0.5, and 1 M (due to cytotoxic effect for higher concentrations, Figure 2(a)) using the TUNEL assay resulted in an increase in apoptosis, reaching statistical significance only for 2a at the highest concentration tested (32.8 ± 1.23% increase) comparing with untreated cells (Figure 2(c)).
To investigate the cellular inhibition of VEGFR-2 by the compounds in HUVEC cultures, immunoblotting assays for total and phosphorylated (active) VEGFR-2 were performed (Figure 3(a)). As illustrated in Figure 3(b), incubation with compounds 2a and 2b significantly decrease the formation of the active form of the receptor at 1 M concentration, when comparing with the expression of VEGFR-2 ( * < 0.05). Compound 2c did not significantly inhibit the activation of the VEGFR-2, although a dose-dependent decrease was observed.
VEGF signaling pathway through the VEGFR-2 activation displays a crucial role in endothelial cells, namely, survival, proliferation, invasion, and apoptosis. Our data suggest that compounds 2a-c exert direct actions in HUVECs by inhibiting their growth mainly at 0.5 and 1 M, slightly increasing the percentage of apoptotic cells without cytotoxic effects at the same range of concentrations. The referred cellular-based results are triggered by the inhibition of the phosphorylation (activation) of the VEGFR-2, which is overexpressed in several pathological conditions, such as cancer.

Molecular Modelling Studies.
To better understand the molecular basis of compounds 2 observed VEGFR-2 inhibition activity, docking simulations using AutoDock4 [17] were carried out against the VEGFR-2 kinase domain (PDB code 2XIR). The terminal aryl group is positioned in a hydrophobic region formed by several hydrophobic residues (Ile888, Leu889, Ile892, Val898, Val 899, and Leu1019). From Figure 4(a), it is clearly observed that the 4-methoxyphenyl and 4-cyanophenyl substituents occupy part of a large hydrophobic pocket that is able to accommodate them, providing an explanation for compounds 2b and 2c being almost equipotent to 2a that bears no substituents in the phenyl ring. Substitutions of hydrophobic groups in ortho or meta positions of the phenyl ring will probably be worth exploring for synthesis of even more potent compounds in this series.
The lack of inhibition by compounds 2d-f for VEGFR-2 was analysed by superimposing the docked pose of compound 2c (the most potent in the enzymatic assays, 150 nM) and the corresponding 7-methylated compound 2f (Figure 4(b)). The methyl group seems to displace the thieno[3,2-d]pyrimidine ring away from Cys919. This displacement probably interferes with the formation of the H-bond between the N atom in position 4 of the