Comparative Investigation of Cytotoxic Effects of Structurally Diverse Small Molecules and In Silico Analysis of 1‐Acetyl‐4‐(4‐Hydroxyphenyl)piperazine

ABSTRACT This study assessed the anticancer activity of boron‐containing and structurally diverse small molecules in 4T1 breast cancer and Caco‐2 colon adenocarcinoma cells. Initial screening showed that five boronic acids lacked significant cytotoxicity, underscoring the structural specificity required for boron‐mediated bioactivity. Similarly, reference compounds, including fumaric acid, caffeic acid, ferulic acid, dimethyl malonate and N‐(tert‐butoxycarbonyl)‐L‐alanine, showed no cytotoxic effect under identical conditions. Among the tested agents, 1‐acetyl‐4‐(4‐hydroxyphenyl)piperazine (1A4HP) displayed the most potent cytotoxicity, with IC 50 values of 149.7 μM in 4T1 and 825 μM in Caco‐2 cells. For comparison, the clinically investigated antimetastatic agent tasquinimod showed moderate activity in 4T1 cells (IC 50 = 180.7 μM), serving as a pharmacological benchmark. Mechanistic assays revealed that 1A4HP induced apoptosis and significantly impaired 4T1 cell migration, suggesting combined antiproliferative and antimetastatic effects. Computational analyses further supported 1A4HP's drug‐like potential by predicting favourable physicochemical properties, including balanced lipophilicity and high solubility. Molecular docking studies indicated a strong binding affinity to oestrogen receptor alpha (ERα), surpassing that of tamoxifen. Notably, despite 4T1's ER‐negative status, 1A4HP suppressed cell growth, suggesting possible ER‐independent or off‐target mechanisms, similar to tamoxifen's secondary effects. Collectively, these results identify 1A4HP as a promising lead compound for further exploration in breast cancers.