Examining the 2-aryl-5-nitrobenzofuran-based hydrazones for anti-breast (MCF-7) cancer activity, potential to induce cell cycle arrest and inhibit receptor tyrosine kinases (VEGFR-2 & EGFR)

The development of small molecules capable of inhibiting multiple oncogenic pathways, such as those driven by the vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinases, represents a promising strategy in Anticancer drug discovery. A strategically iodinated benzaldehyde precursor 1 was synthesized and condensed with phenylhydrazine derivatives to yield the iodophenol-hydrazones 2a and 2b. Subsequent Sonogashira coupling and cycloisomerization afforded novel benzofuran-hydrazone hybrids 3a-k with diverse aryl substituents. Compounds 2 and 3 were screened for in vitro cytotoxicity against the MCF-7 breast Cancer cell line and non-cancerous Vero cells. The mechanism of action was investigated through cell cycle analysis, annexin staining, enzymatic inhibition assays against VEGFR-2 and EGFR, including comprehensive in silico studies. Compound 3k featuring a 4-(trifluoromethylphenyl)hydrazone arm and a 2-(3,5-dimethoxyphenyl) substituent emerged as the most potent cytotoxic agent against MCF-7 cells (IC₅₀ = 4.21 μM) with a favorable selectivity index (SI = 2.1). This compound induced cell cycle arrest at the G0/G1 phase. Apoptosis was assessed on compounds 3c and 3k using the Muse® Annexin V & Dead Cell Kit on the MCF-7 cells. Treatment with 3c showed 70.5 % and 28.1 % rise in early and late Apoptosis, respectively. Derivative 3k, on the Other hand, resulted in a 76.2 % increase in early Apoptosis. In enzymatic assays, compound 2b showed strong VEGFR-2 inhibition (IC₅₀ = 2.86 μM), while several benzofuran hybrids, notably 3j (IC₅₀ = 3.35 μM) and 3k (IC₅₀ = 3.37 μM), also displayed potent activity. Compound 3f was the most effective EGFR Inhibitor (IC₅₀ = 5.29 μM). Several derivatives, including 3k, demonstrated promising dual VEGFR-2 and EGFR inhibitory profiles. Molecular modelling rationalized these findings, highlighting key structure-activity relationships (SARs). The ADMET predictions indicated excellent drug-like properties, particularly for the non-Pgp substrate series. The finding successfully identified compound 3k as a lead dual inhibitor of VEGFR-2 and EGFR with significant Anticancer activity and selectivity.

2-Aryl-5-nitrobenzofuran-based hydrazones; Apoptosis; Cell cycle arrest; Computational studies; Cytotoxicity; Tyrosine kinases (VEGFR-2 & EGFR).