Functionalization of Fluorinated Benzenesulfonamides and Their Inhibitory Properties toward Carbonic Anhydrases

Abstract

Substituted tri- and tetrafluorobenzenesulfonamides were designed, synthesized, and evaluated as high-affinity and isoform-selective carbonic anhydrase (CA) inhibitors. Their binding affinities for recombinant human CA I, II, VA, VI, VII, XII, and XIII catalytic domains were determined by fluorescent thermal shift assay, isothermal titration calorimetry, and a stopped-flow CO2 hydration assay. Variation of the substituents at the 2-, 3-, and 4-positions yielded compounds with a broad range of binding affinities and isoform selectivities. Several 2,4-substituted-3,5,6-trifluorobenzenesulfonamides were effective CA XIII inhibitors with high selectivity over off-target CA I and CA II. 3,4-Disubstituted-2,5,6-trifluorobenzenesulfonamides bound CAs with higher affinity than 2,4-disubstituted-3,5,6-trifluorobenzenesulfonamides. Many such fluorinated benzenesulfonamides were found to be nanomolar inhibitors of CA II, CA VII, tumor-associated CA IX and CA XII, and CA XIII. X-ray crystal structures of inhibitors bound in the active sites of several CA isoforms provide structure-activity relationship information for inhibitor binding affinities and selectivity.

Intrinsic Thermodynamics and Structures of 2,4‐ and 3,4‐Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases

“…The observed binding affinities toward the CA IX isoform measured by fluorescence thermal shift assay (FTSA) are presented in Table S1 in the Supporting Information. The binding of the substituted fluorinated benzenesulfonamides to CA I, CA II, CA VII, CA XII, and CA XIII has been measured previously by FTSA (compound numbers are the same as those used in reference ), and the results are presented in Table S1 in the Supporting Information to compare the affinities for CA IX with those for the off‐target isoforms. The chemical nature of the ortho and meta substituents influenced both the affinity and selectivity toward CA IX.…”

“…The crystal structures of CA I– 10 h , CA XIII– 10 h , and CA II– 2 m contained two subunits in the asymmetric unit, whereas the structures of CA II– 1 b , CA II– 10 n , and CA II– 10 d contained one protein chain in the asymmetric unit. The benzene ring of the fluorinated benzenesulfonamides in the crystal structures with CA II and CA XII can be found in two positions, as previously described . The benzene ring can be located in the same plane as the nitrogen atom of the sulfonamide group, and such orientation will be referred to as the “in‐plane” orientation (Figure S3 A in the Supporting Information).…”

“…These two variations of the ligand–protein interaction may have different thermodynamic binding profiles. Two conformations of the ligand were found in the crystal structures of CA II with meta ‐substituted compounds 10 h and 10 d , as shown in Figure B and C (this study and references ). Different positions of the fluorobenzene ring were also found in complexes of 10 h with CA I (Figure ).…”

“…Note the similar positioning of the ortho substituents. B) Different binding modes of ligand 10 d from PDB ID 5EHE (green) and PDB ID 4QTL (magenta) crystal structures. C) Alternative binding modes of compound 10 h from the PDB ID 4PYY crystal structure .…”

“…The first ring of 10 is positioned in the crystal structure (PDB ID: 4HU1) in an “in‐plane” orientation. In the active site of CA XIII, there is a hydrophobic valine side chain instead of the T200 residue in CA II and this might prevent the alternative orientation of the fluorobenzene ring in the active site of CA XIII . Compound 10 h is found in the “in‐plane” orientation as well, with the first ring position nearly identical to that for 10 and the cyclooctyl ring located in the hydrophilic environment.…”