Pyrazolo[4,3-c]pyridine Sulfonamides as Carbonic Anhydrase Inhibitors: Synthesis, Biological and In Silico Studies

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the essential reaction of CO₂ hydration in all living organisms, being actively involved in the regulation of a plethora of patho-/physiological conditions. A series of chromene-based sulfonamides were synthesized and tested as possible CA inhibitors. On the other hand, in microorganisms, the β- and γ- classes are expressed in addition to the α- class, showing substantial structural differences to the human isoforms. In this scenario, not only human but also bacterial CAs are of particular interest as new antibacterial agents with an alternative mechanism of action for fighting the emerging problem of extensive drug resistance afflicting most countries worldwide. Pyrazolo[4,3-c]pyridine sulfonamides were synthesized using methods of organic chemistry. Their inhibitory activity, assessed against the cytosolic human isoforms hCA I and hCA II, the transmembrane hCA IX and XII, and β- and γ-CAs from three different bacterial strains, was evaluated by a stopped-flow CO₂ hydrase assay. Several of the investigated derivatives showed interesting inhibition activity towards the cytosolic associate isoforms hCA I and hCA II, as well as the 3β- and 3γ-CAs. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds within the active site of hCA IX. Four compounds (1f, 1g, 1h and 1k) were more potent than AAZ against hCA I. Furthermore, compound 1f also showed better activity than AAZ against the hCA II isoform. Moreover, ten compounds out of eleven appeared to be very potent against the γ-CA from E.coli, with a Ki much lower than that of the reference drug. Most of the compounds showed better activity than AAZ against hCA I as well as the γ-CA from E.coli and the β-CA from Burkholderia pseudomallei (BpsCAβ). Compounds 1f and 1k showed a good selectivity index against hCA I and hCA XII, while 1b was selective against all 3β-CA isoforms from E.coli, BpsCA, and VhCA and all 3γ-CA isoforms from E.coli, BpsCA and PgiCA.

Keywords: 3β and 3γCAs; CA inhibitors; carbonic anhydrases; cytotoxicity; docking.

Figure 1

Known CA inhibitors.

Figure 2

Structure of drugs bearing the pyrazole moiety.

Figure 3

Structure of approved drug with pyrazolopyridine scaffold.

Scheme 1

Synthesis of compounds 1a–f.

Scheme 2

Synthesis of compounds 1g,h.

Scheme 3

Synthesis of compound 1i.

Scheme 4

Synthesis of compound 1j.

Scheme 5

Synthesis of compound 1k.

Scheme 6

Probable mechanism of synthesis of 1k.

Figure 4

(A) Superposition of compound 1k (green) bound to hCA II in comparison with compound 1c (light blue) bound to hCA II, with specific residues labeled. (B) 2D interaction diagram of compound 1k docking pose interactions with the key amino acids in hCA II. (C) 2D interaction diagram of compound 1c docking pose interactions with the key amino acids in hCA II. Active-site zinc shown as blue sphere, red dotted arrows indicate H-bond, and yellow spheres are hydrophobic interactions.

Figure 5

Superposition of compound 1f bound to hCA I (red) in comparison to hCA II (magenta), with specific residues labeled. Active-site zinc shown as blue sphere, red dotted and green arrows indicate H-bonds, and yellow spheres are hydrophobic interactions.

Figure 6

2D interaction diagram of compound 1f docking pose interactions with the key amino acids in (A) hCA I and (B) hCA II.

Figure 7

2D interaction diagram of compound 1g docking pose interactions with the key amino acids in (A) hCA IX and (B) hCA I. (C) Superposition of compound 1g bound to hCA I (yellow) in comparison to hCA IX (grey). Active-site zinc shown as blue sphere, red dotted arrows indicate H-bonds, and yellow spheres are hydrophobic interactions.

Figure 8

(A) Docking pose of compound 1j in E. coli β-CA enzyme. (B) 2D interaction diagram of compound 1j. (C) Superposition of compound 1j bound to E. coli β-CA enzyme (magenta) in comparison to AAZ (blue). Active-site zinc shown as blue sphere, red dotted arrows indicate H-bonds, and yellow spheres are hydrophobic interactions.

Figure 9

(A) Docking pose of compound 1b in γ-CA enzyme. (B) 2D interaction diagram of compound 1b. Active site zinc shown as blue sphere, water molecule shown as red sphere, red dotted arrows indicate H-bonds, and yellow spheres hydrophobic are interactions.