Development of Indole-3-yl-methylene-thiobarbital Derivatives as Inhibitors of HDAC8 Enzyme Activity

Histone deacetylase 8 (HDAC8), a zinc-dependent class I enzyme, plays key roles in regulating gene expression and is implicated in various malignancies. Although hydroxamic acid-based inhibitors, like PCI-34051, have demonstrated potent HDAC8 inhibition, limitations including poor selectivity, instability, and off-target effects underscore the need for novel scaffolds. Here, we designed and synthesized a series of indole-based (thio)barbiturate derivatives to identify selective HDAC8 inhibitors. Compound 3d showed promising potency (IC₅₀ = 2.9 μM) and selectivity for HDAC8 over other isoforms. Structure–activity relationship studies revealed that thiobarbituric acid and 2-methylindole scaffolds enhanced HDAC8 inhibition. Remarkably, compound 3r emerged as the most potent inhibitor (IC₅₀ = 0.08 μM), exhibiting slow-binding kinetics, nanomolar potency, and high selectivity over HDAC1, 2, 3, and 6. Furthermore, compound 3r selectively induced SMC3 hyperacetylation in THP-1 cells, confirming its functional HDAC8 inhibition. Computational modeling and molecular dynamics simulations rationalized the experimental SAR and suggested an allosteric mechanism of inhibition.