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In this research paper, we report the design and synthesis of novel substituted biscarbazole derivatives which were characterized by ¹H and ¹³C NMR, high resolution mass spectroscopy (HRMS). The SAR study of the compounds is reported based on different substituents and their positions in the biscarbazole scaffold. In vitro cytotoxicity of the compounds was evaluated against human glioma U87 MG cell line by MTT assay for 24 h. The IC₅₀ values of the compounds (30-35, 48-53 and 54-62) were calculated at the concentration range from 1.00 µM to 500 µM. The compound 34 showed the most significant in vitro cytotoxicity (IC₅₀ = 3.9 µM) against human glioma U87 MG cell line and was found to be better than standard drugs used for the treatment of brain tumors such as temozolomide (IC₅₀ = 100 µM) and carmustine (IC₅₀ = 18.2 µM) respectively. To determine the mode of binding of compound 34 with CT …