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β-Carboline alkaloids, such as harmine (1), have demonstrated notable anticancer properties, making them attractive candidates for anticancer drug development. This study evaluated the antiproliferative activity of compound 1 and thirty-three N⁹-substituted derivatives across a panel of cancer cell lines representing various histotypes. Among these, derivative 6, a harmine analog bearing a 3,5-dimethylbenzyl substituent, was the most potent, showing enhanced cytotoxicity and selectivity toward cancer cells. Compound 6 exhibited IC₅₀ values below 10 µM in all tested cancer cell lines, while its IC₅₀ in non-cancerous cells exceeded 100 µM. Viability assays and xCELLigence real-time monitoring confirmed a concentration-dependent inhibition of cancer cell growth with minimal effects on non-malignant cells. Flow cytometry demonstrated G1 phase arrest in MOLT-4 cells, supported by Western blot data showing reduced phosphorylated Rb and increased p27 protein levels. Apoptosis induction was confirmed through Annexin V/PI staining, TUNEL assays, and caspase activation studies. These revealed the involvement of both intrinsic (caspase-9) and extrinsic (caspase-8) apoptotic pathways, along with activation of caspases 3/7. Western blot analysis also showed a concentration-dependent increase in the Bax/Bcl-2 ratio. Immunofluorescence microscopy visualization indicated DNA damage through elevated levels of PAR and γH2AX, consistent with single- and double-strand DNA breaks. Importantly, compound 6 exhibited low inhibitory activity against monoamine oxidase A (MAO-A) and did not promote reactive oxygen species (ROS) generation, minimizing potential off-target effects. Together, these findings support the potential of compound 6 as a selective and effective candidate for antileukemia therapy.