Design, synthesis, and biological evaluation of secondary amine-containing flavonoids with anti-osteosarcoma activity associated with modulation of AKT signaling and EMT-related gene expression.

The development of more effective therapeutics for osteosarcoma is frequently hindered by chemoresistance, metastatic progression, and chemotherapy-associated toxicity. Targeting key oncogenic signaling pathways, such as the PI3K/AKT axis - a key driver of osteosarcoma progression, migration, EMT-like processes, and chemoresistance - represents a rational therapeutic strategy for improving treatment outcomes. In this study, five flavonoid-based derivatives bearing a trimethoxy moiety on the B-ring and distinct secondary amines at C-7 of the A-ring were designed, synthesized, and screened against the 143B osteosarcoma cell line with high propensity to metastasize. Derivatives 6a and 6b demonstrated potent anticancer activity, exhibiting high selectivity toward the osteosarcoma cell line and low toxicity toward non-malignant MRC-5 lung fibroblasts. Interestingly, both compounds synergistically interacted with doxorubicin, substantially reducing the required dose of the chemotherapeutic agent. Mechanistically, both compounds, as single agents and in combination with doxorubicin, were associated with reduced AKT phosphorylation and downregulation of downstream mediators associated with tumor progression and epithelial-mesenchymal transition (EMT), including ZEB1, MMP2, and MMP9. Collectively, these findings show that 6a and 6b exert anti-osteosarcoma effects that are consistent with modulation of the AKT signaling pathway and EMT-related gene expression. Here, we demonstrate that secondary amine functionalities in the flavonoid backbone provide a rational framework for the development of targeted therapeutic strategies for osteosarcoma.