Fine-tuning the combination of novel spirocyclic capping moieties and heterocyclic zinc binding groups for selective HDAC6 inhibition: design, synthesis, kinetic and biological studies

Histone deacetylase 6 (HDAC6) has emerged as an attractive target for its unique cytoplasmic localization and structural features among HDAC enzymes. HDAC6 inhibitors are generally composed by three main moieties, namely a cap group, a zinc binding group (ZBG) and a linker moiety connecting them. The most frequently embedded ZBGs for HDAC inhibition are represented by hydroxamic acids which, despite their strong ability in coordinating Zn 2+ ions, are often related to toxicity concerns and suboptimal drug-like properties. Compelling new evidence supported the use of difluoromethyl-1,3,4-oxadiazoles (DFMOs) and trifluoromethyl-1,2,4-oxadiazoles (TFMOs) as novel ZBGs able to selectively target HDAC6 isoform. Building on our background and interest in the identification of novel spirocyclic cap groups for selective HDAC6 inhibition, we have herein investigated the effect of fine-tuning the combination of newly conceived spirofused capping moieties with traditional (hydroxamic) or heterocyclic (DFMOs and TFMOs) ZBGs. Western Blot analysis and NanoBRET assays confirmed robust HDAC6 target engagement in cell. Furthermore, mechanistic investigation was performed on DFMO- and TFMO-based compounds, confirming a time dependent tight-binding interaction within HDAC6 active site. Finally, we have engaged the best performing compounds in the evaluation of their effect in the human urothelial carcinoma cell line T24. Our findings suggest that compound 21e inhibits T24 cell proliferation by sequentially activating autophagy and apoptosis, two interconnected cell death pathways that may cooperate in mediating its anticancer effects. These results foster further optimization of our compounds and pave the way to more selective and safer HDAC6 inhibitors.