Design, synthesis and biological evaluation of a phenyl butyric acid derivative, N-(4-chlorophenyl)-4-phenylbutanamide: A HDAC6 inhibitor with anti-proliferative activity on cervix cancer and leukemia cells

Background: The epigenetic regulation of genes in cancer could be targeted by inhibiting Histone deacetylase 6 (HDAC6), an enzyme involved in several types of cancer such as lymphoma, leukemia, ovarian cancer, etc. Objective: Through in silico methods, a set of Phenyl butyric acid derivatives with possible HDAC6 inhibitory activity were designed, rendering monophenylamides and biphenylamides using tubacin (HDAC6 selective inhibitor) as reference. Method: The target compounds were submitted to theoretical ADMET analyses and their binding properties on different HDAC6 conformers were evaluated through docking calculations. Results: These in silico studies allowed us to identify a compound named B-R2B. In order to have more information about the B-R2B binding recognition properties on HDAC6, the B-R2B-HDAC6 complex was submitted through 100 ns-long Molecular Dynamics (MD) simulation coupled to MMGBSA approach, revealing that B-R2B is located at the entrance of HDAC6 active pocket, blocking the passage of the substrate without reaching the HDAC6 binding site. Based on these results, B-R2B was synthesized, characterized and biologically tested. The HDAC6 fluorometric drug discovery kit Fluor-de-Lys (ENZO Life Sciences Inc.) was used to determine the HDAC6 human inhibitory activity (IC₅₀ value) of B-R2B compound. In addition, B-R2B show IC₅₀ values on cancer cell lines (HeLa; IC₅₀ = 72.6 µM), acute myeloid leukemia (THP-1; IC₅₀ = 16.5 µM), human mast leukemia (HMC; IC₅₀ = 79.29 µM) and chronic myelogenous leukemia (Kasumi; IC₅₀ = 101 µM). Conclusion: These results show that B-R2B is a HDAC6 inhibitor, specifically a non-competitive type in a similar way that tubacin does, according to MD simulations.