Our study investigates the biochemical and functional impact of selective histone deacetylase 6 (HDAC6) inhibitors, a promising class of novel therapeutics, in several cancer models. Selective HDAC6 inhibitors (Tubathian A, Tubastatin A, Tubacin and Ricolinostat) and a non‐selective HDAC inhibitor (Vorinostat) were evaluated on cancer cell lines derived from multiple tumour types in both an in vitro and in vivo setting as potential cancer therapeutics. Selective HDAC6 inhibitors resulted in α‐tubulin acetylation with no impact on histone acetylation but failed to show any anti‐cancer properties. Only the use of high concentrations of selective HDAC6 inhibitors resulted in co‐inhibition of other HDAC enzymes and consequently in reduced growth, migratory and/or invasive activity of cancer cells in vitro as well as in vivo. The specificity of HDAC6 inhibition was confirmed using a CRISPR/Cas9 knockout cell line. Our results suggest that selective HDAC6 inhibitors may fall short as potential single agent anti‐cancer drugs and prove that many previous data regarding this promising class of compounds need to be interpreted with great care due to their use in high concentrations resulting in low selectivity and potential off‐target effects.

The synthesis of novel isoform-selective HDAC inhibitors is considered to be an important, emerging field in medicinal chemistry. In this paper, the preparation and assessment of thirteen selective HDAC6 inhibitors is disclosed, elaborating on a previously developed thiaheterocyclic Tubathian series. All compounds were evaluated in vitro for their ability to inhibit HDAC6, and a selection of five potent compounds was further screened toward all HDAC isoforms (HDAC1-11). The capability of these Tubathian analogs to inhibit α-tubulin deacetylation was assessed as well, and ADME/Tox data were collected. This thorough SAR evaluation revealed that the oxidized, para-substituted hydroxamic acids can be recognized as valuable lead structures in the pursuit of novel potent and selective HDAC6 inhibitors.