Azetidin-2-one-based small molecules as dual hHDAC6/HDAC8 inhibitors: Investigation of their mechanism of action and impact of dual inhibition profile on cell viability

The search of new therapeutic tools for the treatment of cancer is being a challenge for medicinal chemists. Dueto their role in different pathological conditions, histone deacetylase (HDAC) enzymes are considered valuabletherapeutic targets. HDAC6 is a well-investigated HDAC-class IIb enzyme mainly characterized by a cytoplasmiclocalization; HDAC8 is an epigenetic eraser, unique HDAC-class I member that displays some aminoacidicsimilarity to HDAC6. New polypharmacological agents for cancer treatment, based on a dual hHDAC6/hHDAC8inhibition profile were developed. The dual inhibitor design investigated the diphenyl-azetidin-2-one scaffold,typified in three different structural families, that, combined to a slender benzyl linker (6c, 6i, and 6j), displaysnanomolar inhibition potency against hHDAC6 and hHDAC8 isoforms. Notably, their selective action was alsocorroborated by measuring their low inhibitory potency towards hHDAC1 and hHDAC10. Selectivity of thesecompounds was further demonstrated in human cell-based western blots experiments, by testing the acetylationof the non-histone substrates alpha-tubulin and SMC3. Furthermore, the compounds reduced the proliferation ofcolorectal HCT116 and leukemia U937 cells, after 48 h of treatment. The toxicity of the compounds was evaluatedin rat perfused heart and in zebrafish embryos. In this latter model we also validated the efficacy of thedual hHDAC6/hHDAC8 inhibitors against their common target acetylated-alpha tubulin. Finally, the metabolicstability was verified in rat, mouse, and human liver microsomes.