Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations

Abstract

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (KIs of 73-131 nM), effective hCA II inhibition (KIs of 9.1-36 nM) and less effective hCA IX and XII inhibition (KIs of 55-128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with KIs of 5.9-14.2 nM, although it was less effective as hCA I and II inhibitor (KIs of 36-120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (KI of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various alpha-CAs found in mammals or parasites, such as Plasmodium falciparum.

Figure 1

Stick representation compound 1 bound in the active site of hCA II. Atoms are coloured; zinc grey; sulfur, orange; oxygen, red; nitrogen, blue; and carbon, yellow. Solvent are depicted as red spheres. Amino acids are as labeled. The electron density is represented by a 2σ-weighted 2Fo - Fc Fourier map (grey mesh). Figure made using PyMOL.

Figure 2

Schematic of hCA II – compound 1 interactions. Hydrophobic contacts are indicated by red hash marks and H-bonds by black dashed lines. Atoms are coloured; zinc grey; sulfur, yellow; oxygen, red; nitrogen, blue; and carbon, black. Amino acids are as labeled. Figure made using Ligplot.

Figure 3

Overall (a) and zoom-in (b) view of hCA II – sulfonamide 1 adduct, superposed onto acetazolamide (AZA) one (PDB accession code 3hs4). Note the two compounds follow the same trajectory out of the active site with the extended coumarin heterocycle ring at an angle to the benzene ring with an edge-to-face interaction with the side chain of Phe131. hCA II is depicted as a surface representation (bulk solvent accessible area, light pink; hydrophilic and hydrophobic regions of the active site, blue and orange respectively). Compound 1 and AZA are represented as sticks. Compounds 1 (grey) and AZA (yellow) are represented as sticks. Non-carbon atoms of both compounds are colored as in Figure 1. Figure made using PyMOL (DeLano Scientific).

Scheme 1

Formation of 2-hydroxy-cinnamic acids A1 and B1 by the CA-mediated hydrolysis of coumarins A and B.