Computational Design, Synthesis, and In Vitro Evaluation of Novel Piperazine-Based HDAC Inhibitors as Potential Breast Cancer Therapeutics

INTRODUCTION: Limited treatment options and poor prognosis create a need for new therapies for triplenegative breast cancer. Modulating lysine acetylation of histone and non-histone proteins via histone deacetylase inhibitors is a promising strategy in cancer therapy. This study aimed to design, synthesize, and test novel panHDAC inhibitors in vitro, building on our previous research. METHODS: Compound design was based on a previously validated diphenylmethyl piperazine scaffold, used as a template for further modifications. In silico studies included molecular docking and pharmacokinetic profiling for absorption, distribution, metabolism, excretion, and toxicity. Compounds identified from these analyses were synthesized, tested for in vitro enzymatic inhibition, and evaluated for cytotoxicity on cancer cell lines using the MTT assay. RESULTS: Compound 8o had the strongest HDAC inhibitory profile. It was highly potent against HDAC6 (IC50 = 18.5 nM) and active against HDAC1 and HDAC8 (IC50 = 430 nM and 1620 nM, respectively). Compound 8p also inhibited HDAC6 (IC50 = 54 nM) but was less potent against the nuclear isoforms. Both compounds were less active than trichostatin A. In the cytotoxicity assay, 8o and 8p reduced the viability of triple-negative breast cancer cells in a dosedependent manner. Compound 8o was the most active (IC50 = 6.74 μM on MDA-MB-231), exceeding the effect of tubastatin A. Moderate activity was seen on MDA-MB-468 cells. DISCUSSION: In vitro enzymatic assays showed that compound 8o strongly inhibited HDAC6, similar to the reference compound 8b. However, its cellular activity did not exceed 8b, suggesting that factors other than target engagement may limit its cell efficacy. These findings show the need to combine biochemical and physicochemical data when optimising HDAC inhibitors for triple-negative breast cancer. CONCLUSION: Compounds 8o and 8p were identified as potent panHDAC inhibitors. They demonstrated cytotoxicity against triple-negative breast cancer cells. This provides a promising foundation for future structural optimization and preclinical development.