Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues, and its selective inhibition provides a potential target for the treatment of numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anticancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring was designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzenesulfonamide group. The bulky ortho substituents, such as cyclooctyl or even cyclododecyl groups, fit into the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compound's co-crystal structure with chimeric CA IX. The strongest inhibitor of recombinant human CA IX's catalytic domain in human cells achieved an affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound that showed the best balance between affinity and selectivity bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX.

Carbonic anhydrase (CA) VI is a potential drug target for cariogenesis and cancer of the salivary gland. It is the only secreted human CA isozyme which is found in saliva and milk. Here, CA VI was expressed in bacterial and mammalian cell cultures and directly affinity‐purified from human saliva. The binding of 4‐substituted‐2,3,5,6‐tetrafluorobenezenesulfonamides to the native and recombinant CA VI from these three sources was compared. Interaction between the enzyme and inhibitors was determined by fluorescent thermal shift assay and isothermal titration calorimetry. The observed dissociation constants were the same within the error margin for all three CA VI preparations. The intrinsic binding parameters for the compounds were obtained by determining and dissecting the binding‐linked protonation reactions. Intrinsic thermodynamic parameters of binding arrange the compounds in a buffer‐ and pH‐independent manner. Intrinsic binding constants of nonfluorinated compounds were significantly stronger than those of fluorinated benzenesulfonamides. An opposite result was determined for the observed binding constants. The increase in observed affinity of the fluorinated compounds was due to the fluorine effect on diminishing the pKa of the compounds but not due to direct recognition of the protein. The temperature–stability profiles for recombinant and native CA VI were compared and showed that CA VI is more stable in slightly acidic than neutral conditions.

Human carbonic anhydrases comprise a family of isoforms that form structurally similar active site and thus it is difficult to design inhibitors that would selectively inhibit the targeted isoform and leave uninhibited all remaining ones. Most common inhibitors are aromatic sulfonamides that strongly bind and inhibit CAs, but usually with limited selectivity towards a targeted isoform. However, sometimes seemingly minor changes in the inhibitor structure may cause significant increase or decrease in affinity towards a CA isoform. Here, the affinities of previously designed N‐alkylated benzimidazoles were determined to all active human CA isoforms. This combinatorial approach yielded several compounds that were highly selective for CA VA. Intrinsic affinities were determined by subtraction of the binding‐linked protonation reactions that, together with the X‐ray crystallographic structures revealing the arrangement of the inhibitors in the active site, help in the design of inhibitors exhibiting subnanomolar affinities and improved selectivities towards a particular CA isoform.

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