Synthesis, In Silico, and Biological Evaluation of Non‐Hydroxamate Benzoic Acid–Based Derivatives as Potential Histone Deacetylase Inhibitors (HDACi)

Unregulated epigenetic modifications, including histone acetylation/deacetylation mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), contribute to cancer progression. HDACs, often overexpressed in cancer, downregulate tumor suppressor genes, making them crucial targets for treatment. This work aimed to develop non-hydroxamate benzoic acid-based HDAC inhibitors (HDACi) with comparable effect to the currently four FDA-approved HDACi, which are known for their poor solubility, poor distribution, and significant side effects. All compounds were structurally verified using FTIR, ¹HNMR, ¹³CNMR, and mass spectrometry. In silico analysis showed that compound A3bn (3-chloro-4-((2-(2-(4-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)amino)benzoic acid) has strong binding affinity towards HDAC2, HDAC6, and HDAC8 and exhibits molecular similarity to trichostatin and SAHA (HDACi). A3bn achieved IC₅₀ values comparable to SAHA against MCF-7 (20.3 vs. 39.2 µM) and K562 (42.0 vs. 36.1 µM) cancer cells. Western blot analysis confirmed that A3bn inhibited H3 and H4 deacetylation. Additionally, A3bn induced the extrinsic apoptotic pathway via caspase 8 activation, leading to cell death. Its enhanced activity across HDAC isoforms may result from its hydrophilic linker, facilitating zinc coordination. In conclusion, A3bn demonstrated efficacy similar to FDA-approved HDACi and represents a promising candidate for further optimization. Future studies will focus on structural modifications to enhance potency and selectivity at lower concentrations.