Identification and Development of Pyrano[2,3- c ]Pyrazole-Based Scaffold for Selective Human Carbonic Anhydrase IX Inhibition

Abstract The high structural similarity among the human Carbonic Anhydrase (hCA) isoforms represents a major challenge for developing selective inhibitors for targeted anticancer therapies. Herein, we report the design, synthesis, biological assessment, and mechanistic investigation of a series of pyrano[2,3-c]pyrazole derivatives as hCA inhibitors. The most active compounds displayed remarkable inhibitory potency and selectivity toward hCAIX, with compound 12 emerging as the most promising derivative, whose binding mode was further rationalized through computational studies. Notably, compound 12, in combination with the GPX4 inhibitor RSL3, demonstrated a synergistic effect in pancreatic cancer cell lines. Moreover, this lead exhibited a potential role in regulating cell migration and ultrastructural studies suggested changes in the expression pattern of markers related to autophagy, metabolism, and EMT associated processes. Overall, these findings identify pyrano[2,3-c]pyrazoles as promising hCAIX-selective chemotypes for targeting hypoxia-associated tumor adaptation and combination anticancer strategies aimed at limiting tumor progression and metastatic dissemination.