Novel Pyrano[2,3-c]pyrazole derivatives: Synthesis, spectroscopic characterization, and In Silico evaluation of interactions with VEGFR-1 and aromatase

Abstract

Eighteen new pyrano[2,3-c]pyrazole derivatives (1–18) were synthesized via an eco-friendly multicomponent reaction and fully characterized by IR, NMR, and MS spectroscopy. All compounds were evaluated for cytotoxicity against MCF-7 (breast), HepG-2 (liver), and HCT-116 (colon) cancer cell lines using the standard MTT assay, with normal BJ-1 fibroblasts included as a non-cancerous control. Several derivatives (especially 6, 8, 9, 12, 17, and 18) exhibited significant antiproliferative activity, with compound 18 being the most potent. Molecular docking and molecular dynamics simulations, combined with MM/GBSA free energy calculations, revealed favorable interactions between the active compounds and both vascular endothelial growth factor receptor-1 (VEGFR-1) and aromatase, suggesting a dual inhibitory mechanism. Consistently, enzyme-linked immunosorbent assay (ELISA) confirmed that the active compounds inhibit VEGFR-1 and aromatase. For example, compound 8 significantly reduced VEGFR-1 levels in HepG-2 and HCT-116 cells, compound 9 was most effective in MCF-7 cells, and compounds 17 and 18 exhibited aromatase inhibition comparable to letrozole (Femara). Mechanistic studies indicated that the active pyrano[2,3-c]pyrazoles induce apoptosis in cancer cells, as evidenced by increased caspase-3, -8, -9, p53, and Bax levels and decreased Bcl-2. Western blot analysis further validated these pro-apoptotic effects for key compounds (8, 17, 18). Notably, compound 18, and specifically its R-enantiomer, emerged as a lead candidate, exhibiting the strongest cytotoxic activity, dual inhibition of VEGFR-1 and aromatase, and the ability to induce cell-cycle arrest. This work highlights the integration of structural chemistry and computational modeling in developing potent dual-action anticancer agents and identifies compound 18 as a promising anticancer agent for further development.