EEFA2-IN-1 is a potent covalent inhibitor of EEF1A2, exhibiting selective antiproliferative activity against osteosarcoma (OS) cells. It induces apoptosis by inhibiting EEF1A2 and downregulating the AKT signaling pathway. In xenograft mouse models, EEFA2-IN-1 suppresses tumor growth, making it a useful compound for osteosarcoma (OS) research.

EEF1A2-IN-1 (compound 8e) exhibits potent antiproliferative activity across various cell lines with IC₅₀ values of 0.73 μM (143B), 2.27 μM (SJSA-1), 4.34 μM (MG63), 2.47 μM (MNNG/HOS), 7.25 μM (U2OS), 1.93 μM (HOS), 4.84 μM (A549), and 9.59 μM (BMSC). Over 96 hours to 10 days, EEF1A2-IN-1 suppresses colony formation in 143B cells in an EEF1A2-dependent manner, and it inhibits the proliferation of 143B, si-EEF1A2-2, and OE-EEF1A2 143B cells with IC₅₀ values of 0.73, 2.78, and 0.19 μM respectively, indicating EEF1A2’s critical role in OS 143B cell proliferation and migration. EEF1A2-IN-1, at concentrations of 0-3 μM over 0-24 hours, effectively inhibits the migration of 143B cells. Additionally, EEF1A2-IN-1 (0-3 μM) effectively suppresses the invasion of 143B cells. By targeting and inhibiting EEF1A2, EEF1A2-IN-1 induces apoptosis in 143B cells, resulting in downregulation of the Nogo-B protein and the AKT signaling pathway.

In a 143B xenograft mouse model, EEFA2-IN-1 (20 mg/kg, intraperitoneal injection, once daily for 15 days) significantly inhibits tumor growth without evident toxicity.