hCAIX/XII-IN-5 (Coumarin 9a) is an inhibitor of carbonic anhydrase (CA) with excellent selectivity for hCA IX/XII, displaying Kis of 93.3 nM and 85.7 nM over hCA I and hCA II, respectively. It exhibits anti-proliferative activities in cancer cells, delaying the cell cycle and inducing apoptosis [1].

Treatment with hCAIX/XII-IN-5 (2.36-32.17 μM) has demonstrated anti-proliferative effects on MCF-7 and PANC-1 cell lines under both normoxic and hypoxic conditions. At a specific concentration of 2.69 μM over 24 hours, this compound significantly increases the accumulation of MCF-7 cells in the Sub-G1 phase and induces arrest in the G2-M phase, indicating a halt in cell cycle progression. Furthermore, the same treatment markedly elevates the proportion of annexin V-FITC-positive apoptotic MCF-7 cells, encompassing both early and late stages of apoptosis (UR+LR), which suggests an enhancement of programmed cell death. The results from Cell Proliferation and Apoptosis Analysis clearly demonstrate the compound's potential in inhibiting cell growth and inducing apoptosis in cancerous cells, with a particular efficacy noted under varying oxygen levels, as seen in the differential IC50 values under normoxic versus hypoxic conditions for both tested cell lines.