Benzamide-4-Sulfonamides Are Effective Human Carbonic Anhydrase I, II, VII, and IX Inhibitors

A series of benzamides incorporating 4-sulfamoyl moieties were obtained by reacting 4-sulfamoyl benzoic acid with primary and secondary amines and amino acids. These sulfonamides were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The human (h) isoforms hCA II, VII, and IX were inhibited in the low nanomolar or subnanomolar ranges, whereas hCA I was slightly less sensitive to inhibition (KIs of 5.3–334 nM). The β- and γ-class CAs from pathogenic bacteria and fungi, such as Vibrio cholerae and Malassezia globosa, were inhibited in the micromolar range by the sulfonamides reported in the paper. The benzamide-4-sulfonamides are a promising class of highly effective CA inhibitors.

Apart from the inhibition of human (h) or other vertebrate CA isoforms, the interest in inhibiting such enzymes present in various pathogenic organisms (bacteria, fungi, protozoa, or worms) has presented the possibility of designing anti-infective agents with a novel mechanism of action [40][41][42][43][44][45][46][47][48][49][50][51]. Thus, in this paper, we explored novel CAIs belonging to the sulfonamide class, incorporating benzamide moieties similar to compounds reported earlier, but that were investigated for the inhibition of isoforms involved in important diseases, such as glaucoma (hCA II), neuropathic pain (hCA VII), or tumors (hCA IX), and ubiquitous off target isoform hCA I. Furthermore, we investigated whether this chemotype shows inhibitory effects against βand γ-class CAs from pathogenic bacteria (Vibrio cholerae) or fungi (Malassezia globosa).
Apart from the inhibition of human (h) or other vertebrate CA isoforms, the interest in inhibiting such enzymes present in various pathogenic organisms (bacteria, fungi, protozoa, or worms) has presented the possibility of designing anti-infective agents with a novel mechanism of action [40][41][42][43][44][45][46][47][48][49][50][51]. Thus, in this paper, we explored novel CAIs belonging to the sulfonamide class, incorporating benzamide moieties similar to compounds reported earlier, but that were investigated for the inhibition of isoforms involved in important diseases, such as glaucoma (hCA II), neuropathic pain (hCA VII), or tumors (hCA IX), and ubiquitous off target isoform hCA I. Furthermore, we investigated whether this chemotype shows inhibitory effects against β-and γ-class CAs from pathogenic bacteria (Vibrio cholerae) or fungi (Malassezia globosa).

Chemistry
The classical coupling of carboxylic acid 1 with amines, in the presence of carbodiimides (EDCI) and hydroxybenzotriazole has been used for synthesis, as reported previously [1,2] (Scheme 1). Compound 1 was condensed with compounds 3a-e that possess primary or secondary amines as well amino acid derivatives 3f-l in the presence of EDCI and 1-hydroxy-7-azabenzotriazole (HOAT) to obtain their corresponding amides (Scheme 1). By choosing variously substituted amines and amino acids, incorporating both simple aliphatic and heterocyclic scaffolds (for the amine) and aliphatic and aromatic amino acids, the physico-chemical properties and enzyme inhibitory Scheme 1. Synthesis of compounds 3a-l.
Compound 1 was condensed with compounds 3a-e that possess primary or secondary amines as well amino acid derivatives 3f-l in the presence of EDCI and 1-hydroxy-7-azabenzotriazole (HOAT) to obtain their corresponding amides (Scheme 1). By choosing variously substituted amines and amino acids, incorporating both simple aliphatic and heterocyclic scaffolds (for the amine) and aliphatic and aromatic amino acids, the physico-chemical properties and enzyme inhibitory properties of the new compounds could be modulated. For example, the amino acid derivatives 3f, 3g, 3h, 3j, and 3l may form sodium salts leading to water soluble CAIs.

Discussion
The following structure-activity relationship (SAR) were determined from the data of Table 1, in which the standard sulfonamide inhibitor acetazolamide (AAZ) was also included for comparison.
The slow cytosolic isoform hCAI, involved in some ocular diseases (not glaucoma) [3][4][5][6][7], was inhibited by sulfonamides 3a-l reported here with K I s in the range of 5.3 to 334 nM. The ethyl-(3a) derivative was the weakest inhibitor, whereas 3c, 3f, 3i, and 3j showed medium potency inhibitory action, with a K I s in the range of 57.8 to 85.3. These compounds incorporate propargyl, valyl, aspartyl, and alanyl moieties. The remaining derivatives, 3b, 3d, 3e, 3g, 3h, 3k, and 3l showed very effective hCA I inhibitory properties, with a K I s in the range of 5.3 to 29.7 nM, being CAIs an order of magnitude better compared to acetazolamide (Table 1). Small changes in the scaffold (compare 3a and 3b) led to dramatic changes in the hCA I inhibitory effects, with the propyl derivative 3b being 40.7 times more effective an inhibitor compared with the ethyl derivative 3a.
All sulfonamides 3a-l reported here were excellent hCA II inhibitors, with a K I s in the range of 1.9 to 7.0 nM, thus being more effective than AAZ (Table 1). With this highly effective inhibition and small range in the variation of the K I s, the SAR is flat and the only conclusion is that all the explored substitution patterns led to highly effective hCA II inhibitors. This is also the dominant cytosolic isoform, involved in glaucoma, diuresis, respiration, and electrolyte secretion in a multitude of tissues [3][4][5][6][7][8][9][10][11][12], meaning these results are highly significant.
The third cytosolic isoform investigated here, hCA VII, predominantly found in the brain and involved in epileptogenesis and neuropathic pain [16][17][18][19][20][21][22][23][24], was also effectively inhibited by sulfonamides 3a-l, which showed a K I s in the range of 0.4 to 26.7 nM. Most of these compounds were sub-nanomolar hCA VII inhibitors (e. g., 3b-3d, 3g-3i, 3k, 3l), being more effective by an order of magnitude compared with the standard AAZ, whereas few of them showed the same potency as AAZ (3e, 3f, 3j) and only the ethyl derivative 3a was a less effective inhibitor compared to AAZ, with a K I of 26.7 nM. Overall, the SAR is extremely simple, and except for the ethyl derivative mentioned above, all the substitution patterns from derivatives 3b-3l indicated all compounds are highly effective hCA VII inhibitors.
The tumor-associated, hypoxia-inducible isoform hCA IX was effectively inhibited by sulfonamides 3a-l, with a K I s in the range of 8.0 to 26. 0 nMh. AAZ has an inhibition constant of 25.8 nM against this isoform. The most effective inhibitors, 3h and 3k, with a K I s of 8.0-9.3 nM, incorporated amino acyl moieties, but all substitution patterns present in compound 3, of the amine or amino acid type, led to highly effective hCA IX inhibition.
Conversely, the βand γ-CAs from pathogenic organisms investigated here were poorly inhibited by these compounds, which showed activity in the micromolar range, with few exceptions (Table 1). Thus, for VchCAβ, the K I s was in the range of 0.41 to 8.58 µM; for MgCA, in the range of 87.3 nM to 7.67 µM; and for VchCAγ, in the range of 0.27 to 4.45 µM. Notably, 3j compounds, which incorporate the alanyl moiety, showed a good inhibitory effect against the Malassezia enzyme, one of the causative agents of dandruff. Acetazolamide is a highly ineffective MgCA inhibitor, and most other sulfonamides investigated here, although less effective than 3j, showed a better activity compared with the standard sulfonamide CAI. Overall, βand γ-CAs are less sensitive to inhibition with sulfonamides compared with α-CAs [3][4][5][6][7][8][9][10][11][12][13][14].

Chemistry
Amines, 4-sulfamoyl-benzoic acid, buffers, solvents, and acetazolamide (AAZ) were commercially available, obtained as highest purity reagents from Sigma-Aldrich/Merck, Milan, Italy. Nuclear magnetic resonance ( 1 H NMR, 13 C NMR) spectra were recorded using a Bruker Avance III 400 MHz spectrometer (Bruker, Billerica, MA, USA) in dimethyl sulfoxide (DMSO-d 6I ). Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns were designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet; and dd, double of doubles. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D 2 O. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230-400 mesh ASTM) as the stationary phase and MeOH/DCM were used as eluents.

CA Enzyme Inhibition Assay
An Sx.18Mv-R Applied Photophysics (Oxford, U.K.) stopped-flow instrument was used to assay he catalytic activity of various CA isozymes for CO 2 hydration reaction [60]. Phenol red, at a concentration of 0.2 mM, was used as an indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5, for α-CAs) or TRIS (pH 8.3, for βand γ-CAs) as buffers, 0.1 M sodium sulfate (Na 2 SO 4 ) (for maintaining constant ionic strength), following the CA-catalyzed CO 2 hydration reaction for a period of 10 s at 25 • C. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5-10% of the reaction were used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionized water. Dilutions up to 1 nM were performed thereafter with the assay buffer. Enzyme and inhibitor solutions were pre-incubated together for 15 min (standard assay at room temperature) prior to assay, to allow for the formation of the enzyme-inhibitor complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier [62][63][64][65][66][67][68][69][70][71][72][73][74][75]. All CAs were recombinant proteins produced as reported earlier by our groups .

Conclusions
We report a series of benzamides incorporating 4-sulfamoyl moieties, which were obtained by reacting 4-sulfamoyl benzoic acid with primary and secondary amines and amino acids. These sulfonamides were investigated as inhibitors of several enzymes, including the human (h) isoforms hCA II, VII, and IX, involved in severe pathologies, such as glaucoma, epilepsy, neuropathic pain and cancer; and βand γ-class CAs from pathogenic bacteria and fungi. hCA II, VII, and IX were inhibited in the low nanomolar or subnanomolar ranges by all investigated sulfonamides, whereas hCA I was slightly less sensitive to inhibition (K I s of 5.3-334 nM). The Vibrio cholerae and Malassezia globosa CAs were generally inhibited in the micromolar range by the sulfonamides reported in the paper. The benzamide-4-sulfonamides constitute a promising class of highly effective CA inhibitors. Further investigations will focus on extending the series of sulfanilamide possessing aliphatic tails with carbamide linkers, such as cyclic and aliphatic and aromatic, to investigate and obtain isoform selective inhibitors for their profiling and possible in vivo applications.