Sulfonamide- and Coumarin-Tethered Acylhydrazones: Synthesis and Human Carbonic Anhydrases Inhibitory Activity

Carbonic anhydrases (CAs) are zinc metalloenzymes catalyzing the reversible hydration of CO₂. Among the 15 human isoforms, hCA IX and hCA XII are transmembrane, tumor-associated enzymes overexpressed under hypoxic conditions. These isoforms promote extracellular acidification in the tumor microenvironment, facilitating cell survival, invasion, and metastasis, and have thus been validated as antitumor drug targets[1]. However, achieving selectivity for these isoforms over the cytosolic hCA I and hCA II remains a major challenge.

Sulfonamide derivatives constitute classical CA inhibitors (CAIs), with the deprotonated sulfonamide nitrogen coordinating the Zn⁺² ion in the active site. Coumarins act as prodrug CAIs, with their hydrolysis products binding at the variable active-site entrance and conferring isoform selectivity toward hCA IX and hCA XII.[2]

Herein, we report the synthesis of two novel acylhydrazone families. The synthetic route began with the esterification of five aromatic carboxylic acids with methanol; followed by treatment of the resulting methyl esters with hydrazine hydrate to afford the corresponding acylhydrazides. Condensation of four aromatic acylhydrazides with coumarin-6-carboxaldehyde and five with 4-sulfamoylbenzaldehyde afforded nine novel acylhydrazones, which were characterized and evaluated for their inhibitory activity against hCA I, II, IX, and XII using a stopped-flow CO₂ hydration assay.  All compounds showed potent inhibition of the four evaluated hCA isoforms, with Ki values in the micromolar to nanomolar range, whereas the coumarin-derived acylhydrazones displayed pronounced selectivity for the tumor-associated CA IX (>1000) and XII (>1300).

[1] Neri, D.; Supuran, C. T. Interfering with pH regulation in tumours as a therapeutic strategy. Nat. Rev. Drug Discov. 2011, 10, 767–778.

[2] Supuran, C. T. Coumarin carbonic anhydrase inhibitors from natural sources. J. Enzyme Inhib. Med. Chem. 2020, 35, 1462–1472.