Synthesis and Biological Investigation of Phenothiazine-Based Benzhydroxamic Acids as Selective Histone Deacetylase 6 Inhibitors

J Med Chem. 2019 Feb 14;62(3):1138-1166. doi: 10.1021/acs.jmedchem.8b01090. Epub 2019 Feb 1.


The phenothiazine system was identified as a favorable cap group for potent and selective histone deacetylase 6 (HDAC6) inhibitors. Here, we report the preparation and systematic variation of phenothiazines and their analogues containing a benzhydroxamic acid moiety as the zinc-binding group. We evaluated their ability to selectively inhibit HDAC6 by a recombinant HDAC enzyme assay, by determining the protein acetylation levels in cells by western blotting (tubulin vs histone acetylation), and by assessing their effects on various cancer cell lines. Structure-activity relationship studies revealed that incorporation of a nitrogen atom into the phenothiazine framework results in increased potency and selectivity for HDAC6 (more than 500-fold selectivity relative to the inhibition of HDAC1, HDAC4, and HDAC8), as rationalized by molecular modeling and docking studies. The binding mode was confirmed by co-crystallization of the potent azaphenothiazine inhibitor with catalytic domain 2 from Danio rerio HDAC6.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetylation
  • Animals
  • Catalytic Domain
  • Cells, Cultured
  • Crystallography, X-Ray
  • HL-60 Cells
  • Histone Deacetylase 6 / antagonists & inhibitors*
  • Histone Deacetylase 6 / metabolism
  • Histone Deacetylase Inhibitors / chemical synthesis*
  • Histone Deacetylase Inhibitors / chemistry
  • Histone Deacetylase Inhibitors / pharmacology*
  • Humans
  • Hydroxamic Acids / chemistry*
  • In Vitro Techniques
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Microsomes, Liver / metabolism
  • Molecular Docking Simulation
  • Molecular Structure
  • Phenothiazines / chemistry*
  • Structure-Activity Relationship
  • Zebrafish


  • Histone Deacetylase Inhibitors
  • Hydroxamic Acids
  • Phenothiazines
  • HDAC6 protein, human
  • Histone Deacetylase 6
  • phenothiazine
  • benzohydroxamic acid