Harnessing Nitric\nOxide-Donating Benzofuroxans for\nTargeted Inhibition of Carbonic Anhydrase IX in Cancer

We\ndescribe here the design and antitumor evaluation of benzofuroxan-based\nnitric oxide (NO)-donor hybrid derivatives targeting human carbonic\nanhydrases (hCAs) IX and XII. The most effective compounds, <b>27</b> and <b>28</b>, demonstrated potent dual action, exhibiting\nlow nanomolar inhibition constants against hCA IX and significant\nNO release. Notably, compound <b>27</b> showed significant antiproliferative\neffects against various cancer cell lines, particularly renal carcinoma\nA-498 cells. In these cells, it significantly reduced the expression\nof CA IX and iron-regulatory proteins, inducing apoptosis via mitochondrial\ncaspase activity and ferroptosis pathways, as evidenced by increases\nin ROS, nitrite, and down-regulated expression of ferritin-encoding\ngenes. 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,\nwith no significant alterations in kidney function. These findings\nunderscore the potential of benzofuroxan-based CA inhibitors for further\npreclinical evaluations as therapeutic agents targeting renal cell\ncarcinoma.