Novel Glycosylpyrazoles as Tumour-Associated Carbonic Anhydrase Inhibitors: Key Roles of Sulfonamide and Methoxyaryl Groups in Isoform Selectivity

Hypoxic tumour microenvironments overexpress transmembrane human carbonic anhydrases IX and XII (hCA IX and XII), metalloenzymes that feature a Zn²⁺ ion at their active sites and play key roles in pH regulation, tumour survival, and metastasis. Selective inhibition of these tumour-associated isoforms over widespread cytosolic isoforms hCA I and II represents an attractive strategy for targeted cancer therapy.

Herein, we report the rational design and synthesis of a novel family of sulfonamide-functionalized glycosyl pyrazoles incorporating D-glucose and D-galactose scaffolds. The synthetic route involves constructing key glycosyl isoxazolines intermediates via 1,3-dipolar cycloaddition, followed by ring opening and oxidation to afford glycosyl-β-diketones, which are subsequently cyclized with 4-hydrazinobenzenesulfonamide to yield 1,5-disubstituted pyrazoles. Inhibitory activities (Ki) against hCA I, II, IX and XII were evaluated using a stopped-flow CO₂ hydration assay.

The synthetised glycoconjugates displayed potent nanomolar inhibition against tumour-associated isoforms hCA IX (Ki values as low as 54.6 nM) and hCA XII (Ki values as low as 32.4 nM). Comparative assays confirmed that the sulfonamide group is essential for Zn²⁺ coordination and enzyme inhibition. Incorporation of an aromatic methoxy substituent substantially decreased affinity for cytosolic hCA I, thereby significantly improving isoform selectivity, with selectivity ratios of up to 165-fold for hCA I over hCA XII.

These findings demonstrate that these novel glycosyl pyrazoles represent promising scaffolds for the development of targeted anticancer agents. Ongoing work is focused on evaluating their cellular activity in osteosarcoma cell lines under normoxic and hypoxic conditions, aiming to further validate their efficacy in 3D spheroid models.