Identification of 2‐Substituted‐3‐Hydroxy‐2,3‐Dihydroquinazolin‐4(1 H ) Ones as Cell‐Active Inhibitors of KDM4A Lysine Demethylase

Lysine demethylases of the KDM4 subfamily are epigenetic regulators frequently dysregulated in cancer, yet the identification of cell-active inhibitors remains challenging due to assay interference and limited chemotype diversity. Here, we report the discovery of 2-substituted-3-hydroxy-2,3-dihydroquinazolin-4(1H)-ones as a new class of KDM4A inhibitors. A focused library was identified through a screening cascade integrating biochemical, biophysical, and mass spectrometry–based assays to minimize false positives. Orthogonal validation using surface plasmon resonance, nanodifferential scanning fluorimetry, and MALDI-TOF-MS enabled reliable compound prioritization, leading to the identification of compound 6i as a validated KDM4A binder and inhibitor. Cellular target engagement was demonstrated by quantitative label-free proteomics, revealing a dose-dependent accumulation of H3K9me2 and H3K9me3 in HEK293T cells, consistent with on-target inhibition of KDM4-family demethylases. Compound 6i further exhibited favorable solubility, permeability, and metabolic stability, supporting its qualification as a promising cell-active KDM4 inhibitor scaffold.