Harnessing Nitric Oxide-Donating Benzofuroxans for Targeted Inhibition of Carbonic Anhydrase IX in Cancer

We describe here the design and antitumor evaluation of benzofuroxan-based nitric oxide (NO)-donor hybrid derivatives targeting human carbonic anhydrases (hCAs) IX and XII. The most effective compounds, <b>27</b> and <b>28</b>, demonstrated potent dual action, exhibiting low nanomolar inhibition constants against hCA IX and significant NO release. Notably, compound <b>27</b> showed significant antiproliferative effects against various cancer cell lines, particularly renal carcinoma A-498 cells. In these cells, it significantly reduced the expression of CA IX and iron-regulatory proteins, inducing apoptosis via mitochondrial caspase activity and ferroptosis pathways, as evidenced by increases in ROS, nitrite, and down-regulated expression of ferritin-encoding genes. In three-dimensional tumor models, compound <b>27</b> effectively reduced spheroid size and viability. <i>In vivo</i> toxicity studies in mice indicated that the compounds were well-tolerated, with no significant alterations in kidney function. These findings underscore the potential of benzofuroxan-based CA inhibitors for further preclinical evaluations as therapeutic agents targeting renal cell carcinoma.