SYNTHESIS OF NOVEL HETEROCYCLIC COMPOUNDS BASED ON RATIONAL DESIGN AND EVALUATION OF THEIR ANTITUMOR AND ANTIANGIOGENIC POTENCIAL IN HUMAN ENDOTHELIAL AND TUMOR CELL LINES WITH TYROSINE  KINASE MEMBRANE RECEPTORS AS TARGETS

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New di(heteroaryl)thioethers 1,3-diarylureas in the thieno[3,2-b]pyridine series as VEGFR2 tyrosine kinase inhibitors: docking, synthesis, enzymatic and cellular assays

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

Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a tyrosine kinase receptor, expressed primarily in endothelial cells, and is activated by the specific binding of VEGF, produced and released by the tumor, to the VEGFR2 extracellular regulatory domain, undergoing autophosphorylation, triggering signaling pathways leading to endothelial cell proliferation towards the tumor [!].Small molecules may act as inhibitors by competing for the ATP-binding site of the VEGFR2 intracellular tyrosine kinase domain, thereby preventing the intracellular signaling that leads to angiogenesis [2]. Herein, we report the synthesis using rational design of new 1-aryl-3-[3-thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas (la-c) as VEGFR2 tyrosine kinase inhibitors. The compounds presented, with the arylurea in the meta position to the thioether and with F or a Me group, showed very low !Cso values (11-28 nM) in enzymatic assays as predicted by molecular docking. To examine the activity of compounds 1 in endothelial cells, VEGF-stimulated (60 ng/mL) Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in M199 medium in the absence (C) or presence of each compound at different concentrations. A remarkable reduction in the proliferation of HUVECs using the BrdU incorporation assay was observed for all compounds at I !JM, for compound la being observed a higher antiproliferative effect. Further studies are ongoing to examine whether these molecules affect the expression and activity of VEGFR2 and the signaling pathways, using western blotting assays. Given the established role of VEGFR2 in proliferation and migration of endothelial cells, these molecules are promising anti-angiogenic agents that can be used for therapeutic purposes in pathological conditions where angiogenesis is exacerbated, such as cancer.

2013Conference object

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

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