Design, synthesis, and biological evaluation of quinazolin-4-one-based selective HDAC6 inhibitors targeting serine 531, histidine 614 residues, and the L1 and L2 loop

Histone deacetylase 6 (HDAC6) is a crucial therapeutic target for a variety of diseases, including inflammation, autoimmune disorders, neurodegenerative diseases, cancer, viral infections, and drug addiction. Therefore, the discovery of selective HDAC6 inhibitors is essential for clinical application. In this study, we designed and synthesized a novel series of quinazolin-4-one-based selective HDAC6 inhibitors. Among them, the most potent compound, 5b (IC₅₀, 17.15 nM, HDAC6) exhibited 19-fold selectivity over HDAC1 and demonstrated significant anti-proliferative activity, with a GI₅₀ value of 2.4 μM against the MCF-7/ADR cell line. Additionally, compound 5b effectively induced the acetylation of α-tubulin, without affecting histone H3 acetylation in MCF-7/ADR cells, confirming its selectivity toward HDAC6. Compound 5b effectively suppressed cell proliferation by inducing apoptosis, as evidenced by colony formation assays and FACS analysis. Molecular docking study revealed that compound 5b effectively occupied the active site of HDAC6, supporting its strong binding affinity and selectivity. In vitro liver microsomal stability studies revealed that compound 5b was stable in both human and mouse liver microsomes. Furthermore, in an HCT116 xenograft mouse model, compound 5b significantly inhibited tumor growth without affecting body weight. The combination of in vitro and in vivo studies provides robust evidence supporting the potential of compound 5b as a highly potent and selective HDAC6 inhibitor, possessing promising anti-proliferative and apoptosis-inducing properties for further preclinical development.