Synthesis, characterization and biological evaluation of formononetin derivatives as anticancer agents

Formononetin (FMN), a natural isoflavone with broad biological activity, has emerged as a potential lead molecule as an anticancer agent. In this work, different FMN ester derivatives were synthesized via an acylation reaction (3a-d). The structural characterization and purity of the compounds were confirmed through NMR, HRMS, and HPLC analysis. Among the synthesized derivatives, the 4-Morpholinecarbonyl chloride conjugated FMN (FMN-4Morpho; 3b) demonstrated significantly (p < 0.05) enhanced cytotoxicity against multiple cancer cell lines, including A549, B16F10, and 4T1, while showing no notable toxicity up to 200 μM in non-cancerous L929 fibroblasts and RAW 264.7 macrophages, indicating good biocompatibility. The mechanistic evaluations in A549 and 4T1 cells revealed elevated ROS production, disruption of mitochondrial membrane potential, and increased apoptosis, as revealed by JC-1 and Annexin V-FITC/PI staining. Additionally, FMN-4Morpho suppressed colony formation and cell migration, downregulated cyclin A, cyclin D1, MMP-2, and MMP-9, and upregulated p53 and Bax expression, thereby lowering the Bcl-2/Bax ratio. The findings highlight FMN-4Morpho as a promising therapeutic candidate with improved anticancer efficacy.