Inhibitory, Anticancer Efficacy and Structural Insights of a Novel Ethyl 1‐Hydroxy‐3‐Oxo‐1‐Phenylisoindoline‐2‐Sulfonate Compound Selectively Targeting Human CA IX and CA XII Carbonic Anhydrase, Ferroptosis‐Mediated Cell Death

ABSTRACT Carbonic anhydrase (CA) isoforms IX and XII are critical in anticancer drug development due to their essential roles in regulating pH and hypoxic conditions. In this study, the inhibitory potential of ethyl‐1‐hydroxy‐3‐oxo‐1‐phenylisoindoline‐2‐sulfonate (1), synthesized via click chemistry, was evaluated against hCA IX and hCA XII. The cellular effects of compound on human breast cancer (MCF‐7 cells) were investigated using a combination of viability, oxidative stress, and molecular level. Compound exhibited stronger inhibitory activity than the acetazolamide (AAZ), with K i values of 11.48 ± 4.18 nM for hCA IX and 9.32 ± 2.35 nM for hCA XII. The compound exhibited a higher binding affinity to hCA IX with −8.8 kcal/mol docking score. While no cytotoxic effect was observed on MCF‐10A cells, a significant dose‐dependent reduction was observed in cell viability, confirmed loss of membrane integrity in cancer cells. ROS levels were decreased at higher concentrations, indicating that the toxicity was independent of ROS production. Caspase‐3 and LC3B expression showed strong positivity at moderate concentrations, suggesting activation of apoptotic and autophagic pathways. GPX4 and TFRC fluorescence intensities were markedly decreased that disruption of redox homeostasis and iron metabolism. These findings indicate that compound is a promising lead molecule for the development of next‐generation anticancer agents targeting tumor‐associated CA isoforms.