Fair and Square: Design, Synthesis and Biological Evaluations of Squaric Acid Derivatives as Novel HDAC8 Inhibitors

Histone deacetylase 8 (HDAC8) is a clinically validated target in neuroblastoma, where isoform selective inhibition offers a strategy to suppress tumour growth while limiting off-target toxicity. Hydroxamic acids remain the dominant zinc-binding group (ZBG) in HDAC inhibitors but are also associated with metabolic instability, suboptimal pharmacokinetics and nonspecific metal chelation. In this study, we report the biological evaluation of a focussed library of 51 squaric acid derivatives as alternative, non-hydroxamic HDAC inhibitors. While a subset of these compounds has been described previously in a synthetic context, their HDAC inhibitory activity and selectivity have not been reported to date. The compounds organised into four structural subfamilies were screened against HDAC8 with moderate isoform selectivity. Structure activity relationships, supported by molecular docking and in silico techniques, highlight the influence of substitution pattern, electronic properties and molecular rigidity on HDAC8 inhibition. Collectively, these findings establish squaric acid derivatives as a viable non-hydroxamic acid scaffold for the development of selective HDAC inhibitors.