Design, synthesis, and biological evaluation of selective HDAC6 inhibitors featuring an N-benzyl-2-(aryl/heteroaryl)acetamide cap.

Histone deacetylase 6 (HDAC6) is predominantly localized in the cytoplasm with nucleocytoplasmic shuttling capability. Featuring two tandem catalytic domains and a C-terminal ubiquitin-binding domain, it primarily deacetylates non-histone substrates such as α-tubulin and HSP90. This distinct subcellular localization and substrate profile endow HDAC6 with the unique capacity to orchestrate pivotal roles in cytoskeletal regulation, the cellular stress response, protein degradation, and cell migration, thereby positioning it as a highly attractive target for anti-tumor drug development. Utilizing the previously identified hit compound Hit-063 as a starting point and guided by the classic HDAC inhibitor pharmacophore model, a series of novel compounds incorporating an N-benzyl-2-(aryl/heteroaryl)acetamide fragment were rationally designed and synthesized. Among these analogs, compound 27 (Cmpd_27) was identified as the most promising lead, exhibiting potent nanomolar inhibition against HDAC6 with an IC50 value of 4.29 ± 0.12 nM. Notably, Cmpd_27 demonstrated superior isoform selectivity, with selectivity indices exceeding 150-fold over HDAC1 and HDAC3, as further validated by Western blot analysis. At the cellular level, Cmpd_27 triggered G2/M phase arrest and induced apoptosis in HCT-116 cells, leading to a significant suppression of cell proliferation (IC50 = 0.69 ± 0.04 μM). Moreover, insights derived from quantum chemical analysis of frontier molecular orbitals and molecular dynamics (MD) simulations provide a robust theoretical foundation for the rational design and structural optimization of next-generation selective HDAC6 inhibitors.