Synthesis and structural proof of novel oxazolo[5,4-d]pyrimidine derivatives as potential VEGFR2 inhibitors. In vitro...

The present study aimed to design and synthesize novel 6-N-benzyloxazolo[5,4-d]pyrimidin-7(6H)-imines 3a-j as possible inhibitors of the vascular endothelial growth factor receptor 2 (VEGFR2). The structures of newly synthesized compounds were confirmed via spectral and crystallographic data. NOESY spectroscopy was very useful in distinguishing between 6-N-benzyl-7(6H)-imine 3a and isomeric 7-N-benzyl-7-amine 4a, obtained by Dimroth rearrangement. Molecular docking at the VEGFR2 active site was performed, indicating that 7(6H)-imines should have a similar binding mode as type II VEGFR2 inhibitors. All derivatives were preliminary evaluated for in vitro cytotoxic activity against four human cancer cell lines, including lung cancer (A549), colorectal cancer (HT-29), melanoma (A375), breast cancer (MCF7), using tivozanib as a reference drug, and some of them were subjected to VEGFR2 inhibition, anti-angiogenic activity, and human serum albumin (HSA) binding assays. Only 6-N-2,4-dimethoxybenzyl derivative 3h appeared to be as active as tivozanib against all tested anticancer cell lines but equally toxic to healthy normal human dermal fibroblasts (NHDF). Derivatives 3f (6-N-2-methybenzyl) and 3b (6-N-4-methylbenzyl) have revealed slightly worse activity than 3h. They were cytotoxic agents comparable to tivozanib against three anticancer lines, but only 3b showed no cytotoxicity against NHDF. Both 3b and 3h proved to be effective VEGFR2 inhibitors with IC₅₀ values comparable to that of tivozanib. Notably, 4a did not actually show an anticancer effect against the tested cancer lines, in contrast to isomeric 3a. In an angiogenesis assay, 3f and 3h significantly suppressed the tube formation ability of human dermal microvascular endothelial cells (HMEC-1), indicating their anti-angiogenic potential. The interactions between these compounds and HSA appeared to occur at two specific binding sites.