New 2-benzylideneamino-4,5-diphenylfuran-3‑carbonitrile derivatives and their benzylamino analogues: Synthesis, in vitro cytotoxicity, protein kinase inhibitory activity and in silico insights.

4,5-Diphenyl-2-substituted furan-3‑carbonitrile Schiff bases 3a-c and 6a-d and their benzylamine derivatives 4a-c and 7a-d were designed, synthesized, and assessed for their cytotoxicity in the NCI-60 Human Tumor Cell Lines Screen. The benzylamines 4a-c and 7a-d displayed potent GI% relative to their Schiff base precursors. Compound 7c that showed potent GI% against twenty-one cell lines ranging from 50.17 to 174.62 % was further subjected to five dose assay, where it displayed significant GI50versus leukemia (Hela-60), colon (HCT-116) and melanoma (LOX-IMVI). The safety of 7c towards normal cells was determined by screening its cytotoxicity against HCT-116, LOX-IVMI and two normal cell lines WI-38 and Vero cells. Likewise, 7c displayed a superior inhibitory activity towards wild type EGFR and CDK2. Furthermore, evaluation of 7c in HCC827 (exon 19 deletion) mutation, which is a cell model highly sensitive to tyrosine kinase inhibitors, showed that the tested compound exhibited lower inhibition than erlotinib. Western blot analysis of 7c revealed its efficacy to decrease EGFR, pEGFR, CDK2 and pCDK2 protein expression levels. Interestingly, compound 7c caused a cell cycle arrest in HCT-116 and LOX-IMVI cells at G1 and S phases, respectively, along with apoptosis induction. Moreover, 7c increased Bax and decreased Bcl-2 levels by 2.53 and 0.49 folds, respectively in HCT-116 cells. Furthermore, 7c caused an increase in the levels of caspase 3 (4.68 folds) and caspase 9 (4.54 folds) in HCT-116 cells. Additionally, in silico studies of 7c showed a plausible binding mode that correlates with its potent inhibitory activity against the two enzymes, whereas ADME prediction revealed a favorable oral absorption.