Design and synthesis of novel hexahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivatives as potential anticancer agents with antiangiogenic activity via VEGFR-2 inhibition, and down-regulation of PI3K/AKT/mTOR signaling pathway.

New thieno[2,3-d]pyrimidine derivatives were designed and synthesized. The National Cancer Institute (NCI) evaluated the synthesized novel compounds against a panel of 60 tumor cell lines for their antiproliferative activity. Compounds 6b, 6f, and 6g showed potent anticancer activity at 10 µM dose, with mean GI of 20.86%, 76.41%, and 31.49%, respectively. Compound 6f was selected for five-dose concentrations evaluation. Compound 6f scored a submicromolar range of GI values against 10 cancer cell lines, indicating broad-spectrum and potent antiproliferative activity. Compound 6f TGI values were recorded in the cytostatic range of 4.02-95.1 µM. In comparison to sorafenib, the tested compounds 6b, 6f, and 6g inhibited VEGFR-2 with IC values of 0.290 ± 0.032, 0.066 ± 0.004, and 0.16 ± 0.006 µM, correspondingly. Compound 6f significantly reduced the total VEGFR-2 expression and its phosphorylation. Additionally, 6f reduced the phosphorylation of PI3K, Akt, and mTOR pathway proteins. Moreover, the migratory potential of HUVECs was significantly reduced, after 72 h of treatment with compound 6f, resulting in disrupted wound healing patterns which verified the angiogenesis suppression properties of compound 6f. Compound 6f increased the total apoptosis percentage by 21.27-fold compared to sorafenib, which caused a 24.11-fold increase in the total apoptosis percentage. This apoptotic activity was accompanied by a 7.81-fold increase in the level of apoptotic caspase-3. Furthermore, the cell cycle analysis revealed that the target derivative 6f reduced cellular proliferation and induced an arrest in HCT-15 colon cancer cell cycle at the S phase. Molecular modeling was used to determine the binding profile and affinity of derivative 6f toward the VEGFR-2 active site.