Synthesis, biological evaluation, and mechanism investigation of multisubstituted quinazoline analogues as prospective inhibitors of KRAS G12D.

KRAS G12D, the most common oncogenic mutation of KRAS, represents a promising therapeutic target for solid tumors. Herein, we report the discovery of 16k, a novel KRAS G12D inhibitor based on a 6-methoxyquinazoline scaffold, identified through the scaffold hopping and structural optimization strategies. The compound 16k exhibited significant antiproliferative effects against KRAS G12D mutant tumor cells, specifically the AGS and GP2D cell lines (IC50 = 0.18 and 0.21 μM, respectively), with minimal cytotoxicity toward normal cells and effectively inhibited colony formation. Preliminary mechanistic investigations revealed that 16k markedly elevated the levels of ROS, disrupted the balance of mitochondria-associated apoptotic proteins, and induced MMP collapse by inhibiting the downstream ERK/AKT signaling pathways of KRAS in AGS and GP2D cells. Molecular dynamics simulations confirmed that 16k binds stably to KRAS G12D, with hydrogen bond interactions observed with residues His 95, Arg 68, and Asp 12, further supporting the consistency of the docking results. 16k also demonstrated favorable drug-like properties, including good hepatic microsomal metabolic stability and a safer toxicity profile in vivo. In conclusion, this work expands the structural diversity of KRAS G12D inhibitors and highlights 16k as a promising lead compound for the development of targeted therapies against G12D-mutant cancers.