Design, synthesis and biological evaluation of novel pyrazoline derivatives as dual EGFR/VEGFR-2 inhibitors for targeted cancer therapy.

Three series of pyrazoline compounds (3a-f, 4a-f and 5a-f) were designed and synthesized as antiproliferative agents and potential dual EGFR/VEGFR-2 inhibitors for targeted cancer therapy. All the synthesized compounds were screened by National Cancer Institute (NCI), Bethesda, USA for anticancer activity against 60 human cancer cell lines. Compounds 2f, 3b, 3c, 3e, 3f, 4b and 4e were selected for further antiproliferative activity investigation via MTT assay. Compounds 2f, 3e and 3f demonstrated better IC₅₀ results than doxorubicin in breast T47D and MCF7 cancer cells. Compound 3e was the most potent against EGFR and VEGFR2 with IC₅₀ values of 0.142 and 0.071 μM compared to erlotinib and axitinib with IC₅₀ values of 0.064 and 0.050 μM, respectively. Moreover, it reduced VEGFR2 phosphorylation and total VEGFR2 concentration in T47D cells by approximately 3.8 and 2.9-fold, respectively. Compound 3e also caused cell cycle arrest at G0/G1 phase and demonstrated significant increase in early and late apoptosis. Additionally, it upregulated pro-apoptotic genes (caspase-3, p53 and Bax) and downregulated anti-apoptotic gene (BCL-2). Boyden chamber and wound healing assays demonstrated that compound 3e could impair cell migration and invasion in T47D cells. The selectivity indices of compound 3e towards T47D or MCF7 breast cancer cells over the normal breast cell line MCF10A were approximately 46 and 25, respectively. In-silico studies revealed that compound 3e has a bioavailability score of 0.55 and did not violate Lipiniski's rule. Also, the molecular docking studies and molecular dynamics simulations confirmed that compound 3e could interact with EGFR (PDB: 1M17) and VEGFR2 (PDB: 4AG8) analogously to erlotinib and axitinib, respectively.