1,2,3-Benzoxathiazine-2,2-dioxides - effective inhibitors of human carbonic anhydrases.

A series of 1,2,3-benzoxathiazine-2,2-dioxides possessing various substituents in the 5, 7, or 8 position was obtained from corresponding 2-hydroxybenzaldehydes in their reaction with sulfamoyl chloride. 5-, 7-, and 8-aryl substituted 1,2,3-benzoxathiazine-2,2-dioxides were prepared from aryl substituted 2-hydroxybenzaldehydes obtained from 3-, 4-, or 6-bromo-2-hydroxybenzaldehydes via two-step protocol. 1,2,3-Benzoxathiazine-2,2-dioxides were investigated for the inhibition of four human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, cytosolic hCA I and II and tumour-associated transmembrane hCA IX and XII. Twenty four derivatives of 1,2,3-benzoxathiazine 2,2-dioxide were obtained. Most of them act as nanomolar inhibitors of hCA IX and XII. Almost all compounds except 2d and 5a-e also express nanomolar inhibitory activity for hCA II. hCA I is poorly inhibited or not inhibited by 1,2,3-benzoxathiazine 2,2-dioxides. Some of the new derivatives exhibit promising selectivity towards CA IX/XII over hCA I, although none of the compounds are selective towards CA IX/XII over both hCA I and II.

Derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide as selective inhibitors of human carbonic anhydrases IX and XII over the cytosolic isoforms I and II.

A series of 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides with various substituents in 5, 6 or 7 positions was obtained from corresponding 2'-hydroxyacetophenones in their reaction with sulphamoyl chloride. 6- and 7-aryl substituted 4-methyl-1,2,3-benzoxathiazine-2,2-dioxides were obtained from aryl substituted 2'-hydroxyacetophenonesprepared from 4- or 5-bromo-2'-hydroxyacetophenones via two-step protocol. 4-Methyl-1,2,3-benzoxathiazine-2,2-dioxides were investigated as inhibitors of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, off-target cytosolic hCA I and II, and target transmembrane, tumour-associated hCA IX and XII. Twenty derivatives of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxide were obtained. With one exception (compound2a), they mostly act as nanomolar inhibitors of target hCA IX and XII. Basically, all screened compounds express none or low inhibitory properties towards off-target hCA I. hCA II is inhibited in micromolar range. Overwhelming majority of 4-methyl-1,2,3-benzoxathiazine 2,2-dioxides express excellent selectivity towards CA IX/XII over hCA I as well as very good selectivity towards CA IX/XII over hCA II.

5-Substituted-benzylsulfanyl-thiophene-2-sulfonamides with effective carbonic anhydrase inhibitory activity: Solution and crystallographic investigations.

A series of 5-substituted-benzylsulfanyl-thiophene-2-sulfonamides was prepared by reacting 5-bromo-thiophene-2-sulfonamide with 5-substituted-benzyl mercaptans. The new compounds were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The cytosolic human (h) isoforms hCA I was poorly inhibited by the new sulfonamides (KIs in the range of 683-4250nM), whereas hCA II, and the transmembrane, tumor associated isoforms hCA IX and XII were effectively inhibited in the subnanomolar-nanomolar range. A high resolution X-ray crystal structure of the adduct of hCA II with one of the new sulfonamides allowed us to rationalize the excellent inhibitory activity of these heterocyclic sulfonamides.

N-Substituted and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-associated carbonic anhydrases IX and XII.

A series of N-substituted saccharins incorporating aryl, alkyl and alkynyl moieties, as well as some ring opened derivatives were prepared and investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The widespread cytosolic isoforms CA I and II were not inhibited by these sulfonamides whereas transmembrane, tumor-associated ones were effectively inhibited, with KIs in the range of 22.1-481nM for CA IX and of 3.9-245nM for hCA XII. Although the inhibition mechanism of these tertiary/secondary sulfonamides is unknown for the moment, the good efficacy and especially selectivity for the inhibition of the tumor-associated over the cytosolic, widespread isoforms, make these derivatives of considerable interest as enzyme inhibitors with various pharmacologic applications.

Structural Basis of Saccharin Derivative Inhibition of Carbonic Anhydrase IX.

This study explores the binding mechanisms of saccharin derivatives with human carbonic anhydrase IX (hCA IX), an antitumor drug target, with the aim of facilitating the design of potent and selective inhibitors. Through the use of crystallographic analysis, we investigate the structures of hCA IX-saccharin derivative complexes, unveiling their unique binding modes that exhibit both similarities to sulfonamides and distinct orientations of the ligand tail. Our comprehensive structural insights provide information regarding the crucial interactions between the ligands and the protein, shedding light on interactions that dictate inhibitor binding and selectivity. Through a comparative analysis of the binding modes observed in hCA II and hCA IX, isoform-specific interactions are identified, offering promising strategies for the development of isoform-selective inhibitors that specifically target tumor-associated hCA IX. The findings of this study significantly deepen our understanding of the binding mechanisms of hCA inhibitors, laying a solid foundation for the rational design of more effective inhibitors.

Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX.

Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regarding the expression and crystallization of the enzyme. Currently, only one baculovirus-produced CA IX structure in complex with a nonspecific CA inhibitor, acetazolamide, is available in Protein Data Bank. We have developed an efficient system for the production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily crystallized in the presence of inhibitors, as we have demonstrated for several 2-thiophene-sulfonamide compounds. We have also observed significant differences in the binding mode of chemically identical compounds to CA IX and CA II, which can be further exploited in the design of CA IX-specific inhibitors.

X-ray crystallography-promoted drug design of carbonic anhydrase inhibitors.

1-N-Alkylated-6-sulfamoyl saccharin derivatives were prepared and assayed as carbonic anhydrase inhibitors (CAIs). During X-ray crystallographic experiments an unexpected hydrolysis of the isothiazole ring was evidenced which allowed us to prepare highly potent enzyme inhibitors with selectivity for some isoforms with medical applications.

Saccharin sulfonamides as inhibitors of carbonic anhydrases I, II, VII, XII, and XIII.

A series of modified saccharin sulfonamides have been designed as carbonic anhydrase (CA) inhibitors and synthesized. Their binding to CA isoforms I, II, VII, XII, and XIII was measured by the fluorescent thermal shift assay (FTSA) and isothermal titration calorimetry (ITC). Saccharin bound the CAs weakly, exhibiting the affinities of 1-10 mM for four CAs except CA I where binding could not be detected. Several sulfonamide-bearing saccharines exhibited strong affinities of 1-10 nM towards particular CA isoforms. The functional group binding Gibbs free energy additivity maps are presented which may provide insights into the design of compounds with increased affinity towards selected CAs.

Hydrophobic substituents of the phenylmethylsulfamide moiety can be used for the development of new selective carbonic anhydrase inhibitors.

A new series of compounds containing a sulfamide moiety as zinc-binding group (ZBG) has been synthesized and tested for determining inhibitory properties against four human carbonic anhydrase (hCA) isoforms, namely, CAs I, II, IX, and XII. The X-ray structure of the cytosolic dominant isoform hCA II in complex with the best inhibitor of the series has also been determined providing further insights into sulfamide binding mechanism and confirming that such zinc-binding group, if opportunely derivatized, can be usefully exploited for obtaining new potent and selective CAIs. The analysis of the structure also suggests that for drug design purposes the but-2-yn-1-yloxy moiety tail emerges as a very interesting substituent of the phenylmethylsulfamide moiety due to its capability to establish strong van der Waals interactions with a hydrophobic cleft on the hCA II surface, delimited by residues Phe131, Val135, Pro202, and Leu204. Indeed, the complementarity of this tail with the cleft suggests that different substituents could be used to discriminate between isoforms having clefts with different sizes.