Pro-Apoptotic Activity of 1-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)propan-2-one, an Intracellular Inhibitor of PIM-1 Kinase in Acute Lymphoblastic Leukemia and Breast Cancer Cells

Inhibition of CK2 and/or PIM-1 kinases has been shown to induce apoptosis in a variety of cancer cell lines, underscoring their potential as valuable targets in anti-cancer drug development. In this study, a series of <i>N</i>-substituted derivatives of 4,5,6,7-tetrabromo-1<i>H</i>-benzimidazole, including 2-oxopropyl/2-oxobutyl substituents and their respective hydroxyl analogues, were synthesized and evaluated for anti-cancer activity. The compounds' ability to inhibit CK2α and PIM-1 kinases was assessed through enzymatic assays, complemented by comprehensive in silico enzyme-substrate docking analyses. Cytotoxicity was evaluated using the MTT assay in human cancer cell lines-including acute lymphoblastic leukemia (CCRF-CEM) and breast cancer (MCF-7, MDA-MB-231)-as well as in normal Vero cells. Apoptosis induction in the two most responsive cell lines (CCRF-CEM and MCF-7) was further examined using flow cytometry-based assays, including annexin V binding, mitochondrial membrane potential disruption, caspase-3 activation, and cell cycle analysis. Intracellular inhibition of CK2 and PIM-1 kinases was confirmed in CCRF-CEM and MCF-7 cells using Western blot and phospho-flow cytometry. Among the synthesized compounds, we identified a novel TBBi derivative exhibiting pronounced pro-apoptotic activity and the ability to inhibit PIM-1 kinase intracellularly. These findings support the hypothesis that PIM-1 kinase represents a promising molecular target for the treatment of leukemia.