Development of potent and selective tetrahydro-β-carboline-based HDAC6 inhibitors with promising activity against triple-negative breast cancer

Overexpression of histone deacetylase 6 (HDAC6) is implicated in tumorigenesis, invasion, migration, survival, apoptosis, and growth of various malignancies, making it a promising target for cancer treatment. Building on our previous work, we report a novel series of tetrahydro-β-carboline-piperazinedione derivatives as HDAC6 inhibitors. Structural modifications were introduced at the 6-aryl group, with the <i>m</i>-bromophenyl derivative (9c) emerging as the most potent HDAC6 inhibitor, exhibiting an IC₅₀ of 7 nM. Compound 9c demonstrated robust growth inhibitory activity across 60 cancer cell lines from the NCI panel, with a mean GI₅₀ of 2.64 μM and a GI₅₀ below 5 μM for nearly all tested lines, while exhibiting significantly lower cytotoxicity towards non-tumor cell lines. The triple-negative breast cancer cell line MDA-MB-231 was selected for further investigation of 9c's cellular effects. 9c selectively increased the acetylation of non-histone α-tubulin in MDA-MB-231 cells, confirming its HDAC6 selectivity. Furthermore, 9c effectively induced apoptosis, caused apoptotic sub-G1 phase accumulation, upregulated pro-apoptotic caspase-3, and downregulated anti-apoptotic Bcl-2. Notably, 9c reduced the expression of programmed death-ligand 1 (PD-L1), a key immune checkpoint protein that enables tumor cells to evade immune surveillance, highlighting its potential role in enhancing anti-tumor immunity. In addition, 9c inhibited phosphorylated extracellular signal-regulated kinase (ERK)1/2, a central signaling pathway that drives cell proliferation, survival, and migration, further highlighting its significance in suppressing tumor progression and growth. In migration assays, 9c impaired cell motility, achieving 80% gap closure inhibition in a wound-healing assay. Collectively, these findings underline compound 9c as a highly promising candidate for the treatment of triple-negative breast cancer, with the added benefits of PD-L1 and ERK inhibition for potential synergy in enhancing anti-tumor immunity and reducing tumor cell proliferation.