Targeting Class I Histone Deacetylases Triggers Antitumor Responses in Colorectal Cancer In Vitro and In Vivo.

Class I histone deacetylases (HDACs) are frequently overexpressed in colorectal cancer (CRC). Combining computational, synthetic, and biological efforts, we developed novel o-aminobenzamide-based HDAC inhibitors (HDACis) optimized for class I enzyme-specific targeting. Compounds 5d and 5i emerged as lead candidates, showing strong antiproliferative effects in CRC cells with low toxicity in healthy colon epithelium. Both compounds disrupted the G2/M checkpoint through distinct mechanisms. 5i, although less potent (HDAC1 IC50 = 1508 nM), retained selectivity, upregulated p21, and triggered pronounced apoptosis. 5d (Colrestat), one of the most selective class I HDACis to date (HDAC1 IC50 = 41.2 nM, HDAC2 IC50 = 52.5 nM, and HDAC3 IC50 = 74.3 nM), induced H3K9 acetylation, p21 upregulation, and G2/M arrest. The short-term in vitro effects of 5d were modulated by a compensatory upregulation of autophagy. However, in long-term, this protective mechanism becomes insufficient to sustain tumor survival, resulting in strong antitumor efficacy in vivo in the CAM assay for both compounds even outperforming entinostat.