Design and synthesis of Entadamide A derivatives targeting breast cancer: Insights into In silico, In vitro and In vivo models.

In this study, a series of Entadamide A (methylthio acrylamide) derivatives (3a-3x) have been designed and synthesized via Steglich esterification reaction with various cinnamic acids, long chain fatty acids, and benzoic acids. All these derivatives were systematically characterized (using NMR and Mass spectroscopy) and evaluated for their anticancer properties against various cancer cells. Among the compounds synthesized, long chain fatty acid derivatives (especially compounds 3u and 3x) manifested excellent anticancer properties against the breast cancer cell lines (MCF-7) with IC values of 2.82 μM and 2.18 μM. Further mechanistic investigations demonstrated that compounds 3u and 3x significantly induced apoptosis while downregulating anti-apoptotic gene expression and also markedly reduced the expression of epithelial-to-mesenchymal transition (EMT) markers and stemness-associated genes. Functional assays confirmed that treatment with compounds 3u and 3x inhibited cell migration and sphere-forming capability in a dose-dependent manner. In 4T1-induced orthotopic mouse models, both compounds, 3u and 3x significantly suppressed tumor growth kinetics and reduced tumour volume compared to controls. The molecular docking study was performed to analyse the probable binding affinities of compounds with ABCB1, ABCG2 and Bmi1/Ring1B-UbcH5c ubiquitin ligase complex proteins. Collectively, these findings indicate that the Entadamide A derivatives 3u and 3x exert potent anti-tumour activity in breast cancer models and provide a strong foundation for further translational development.