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Synthesis, molecular Docking and biological evaluation of new 1-Aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenylureas as Potent Type II VEGFR-2 Tyrosine Kinase inhibitors

Publication . Machado, Vera A.; Peixoto, Daniela; Costa, Raquel; Calhelha, Ricardo C.; Abreu, Rui M.V.; Ferreira, Isabel C.F.R.; Soares, Raquel; Queiroz, Maria João R.P.

The vascular endothelial growth factor receptor 2 (VEGFR-2) is a tyrosine kinase receptor, expressed primarily in endothelial cells, and is activated by the specific binding of VEGF to the VEGFR-2 extracellular regulatory domain. Once activated, VEGFR-2 undergoes autophosphorylation, triggering signaling pathways leading to endothelial cell proliferation and subsequent angiogenesisY1 Small molecules may act as inhibitors by competing for the ATP-binding s'1te of the VEGFR-2 intracellular tyrosine kinase domain, thereby preventing the intracellular signa ling that leads to angiogenesis. [ZJ Here, we present the synthesis of new 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas la-c, as potent type 11 VEGFR-2 inhibitors based on molecular docking (Figure A) and biological evaluation including enzymatic assays using the VEGFR-2 tyrosine kinase domain (ICso=l0-28 nM) and studies in human umbilical vein endothelial cells (HUVECs). The latter included cell viability (MTS), proliferation (BrdU) and Western blot for total and phosphorylated VEGFR-2 (Figure B). The predicted docked poses were analyzed in detail and a plausible explanation for compounds 1 potency was obtained base9 on the simultaneous presence of a S-linker and the arylurea moiety in the meta position as a new substitution pattern for the type 11 VEGFR-2 inhibitors. These chemical features place the thieno[3,2-b]pyridine and the terminal aryl ring in close superimposition to a pyrrolo[3,2-d]pyrimidine derivative. The presence of hydrofobic substituents (F and Me) in the terminal aryl ring is also important. For these compounds a significant inhibition in HUVECs proliferation upon VEGF stimulation was observed at low concentrations (0.5-1.0 IJ.M) without affecting cell viability. Westernblot analysis demonstrated that compounds 1 significantly the inhibited VEGFR-2 phosphorylation at 1.0 jlM, thus confirming their anti-angiogenic potential.

2014Conference object

Open access

Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase

Publication . Guimarães, Rafaela; Calhelha, Ricardo C.; Froufe, Hugo J.C.; Abreu, Rui M.V.; Carvalho, Ana Maria; Queiroz, Maria João R.P.; Ferreira, Isabel C.F.R.

Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

2016Journal article

Open access

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors

Publication . Machado, Vera A.; Peixoto, Daniela; Costa, Raquel; Froufe, Hugo J.C.; Calhelha, Ricardo C.; Abreu, Rui M.V.; Ferreira, Isabel C.F.R.; Soares, Raquel; Queiroz, Maria João R.P.

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl] ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]- pyridin-7-ylthio)phenyl]ureas 4a–4h, with the arylurea in the meta position to the thioether, showed the lowest IC50 values in enzymatic assays (10–206 nM), the most potent compounds 4d–4h (IC50 10– 28 nM) bearing hydrophobic groups (Me, F, CF3 and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 lM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

2015Journal article

Open access