Exploring new quinoxaline, quinoline, and naphthalene carboxamides as HDAC-targeted anticancer agents: Cytotoxicity, enzymatic HDAC evaluation, and in silico studies

Abstract

Histone deacetylase inhibitors (HDACis) are important epigenetic anticancer agents, but new HDAC-targeted scaffolds are still needed to improve biological profiles. Herein, quinoxaline, quinoline, and naphthalene were selected as structurally related fused-aromatic cap groups within the HDAC inhibitor pharmacophore, allowing comparison of naphthalene and its aza-analogs in a unified carboxamide series. A new series of quinoxaline, quinoline, and naphthalene carboxamides was synthesized through amidation of amino acid methyl esters, followed by hydrolysis, hydrazinolysis, and hydroxamate formation. Their structures were confirmed by spectroscopic and mass spectrometric analyses. Cytotoxicity was evaluated against A549, HeLa, and MCF-7 cancer cells and non-cancerous Vero cells using the MTT assay. Most derivatives were weakly active; however, hydroxamic acids showed improved potency. Compound 10d exhibited submicromolar cytotoxicity against A549, HeLa, and MCF-7 cells (IC₅₀ = 0.22, 0.13, and 0.21 µM, respectively), although it was also toxic to Vero cells. Compound 7d showed selective cytotoxicity toward HeLa cells (IC₅₀ = 6.61 µM) with low Vero toxicity (IC₅₀ > 100 µM). Enzymatic evaluation demonstrated that 10d strongly inhibited HDAC activity in HeLa nuclear extract and showed potent HDAC2 inhibition, whereas 7d showed weak total HDAC and low HDAC2 inhibition. Western blot analysis showed that 7d increased Ac-H3 levels, suggesting possible epigenetic modulation. Apoptosis and cell-cycle analyses indicated strong late apoptosis and SubG1 accumulation by 10d, whereas 7d induced moderate late apopotosis and G2/M arrest. Molecular docking of 10d with HDAC2 supported its binding within the catalytic pocket, and ADMET prediction suggested drug-like properties with potential liabilities requiring validation.