Benzenesulfonamides Incorporating Hydantoin Moieties Effectively Inhibit Eukaryoticand Human Carbonic Anhydrases

A series of novel 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives were synthesized and evaluated for the inhibition of eukaryotic and human carbonic anhydrases (CAs, EC 4.2.1.1). The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two β-CAs from fungal pathogens. The best inhibition was observed against hCA II and VII as well as Candida glabrata enzyme CgNce103. hCA I and Malassezia globosa MgCA enzymes were, on the other hand, less effectively inhibited by these compounds. The inhibitory potency of these compounds against CAs was found to be dependent on the electronic and steric effects of substituent groups on the N3-position of the hydantoin ring, which included alkyl, alkenyl and substituted benzyl moieties. The interesting results against CgNce103 make the compounds of interest for investigations in vivo as potential antifungals.


Compounds Design and Synthesis
Considering the fact that hydantoins already possess CA inhibitory effects [30], the drug design strategy that we propose in this paper is to incorporate in the same molecule both a zinc binder fragment of the benzene-sulfonamide type [4][5][6][7][8][9][16][17][18] as well as the tail based on the 3-substituted-hydantoin motif.
The synthesis of the target 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives is shown in Scheme 1. The synthesis started from sulfanilamide 1,which was converted to 4-thioureidobenzenesulfonamide (2)via reaction with KSCN in aqueous, acidic medium [33]. The key intermediate, potassium cyano(4-sulfamoylphenyl)amide 4, was prepared by the selective S-methylation of thiourea 2 via treatment with 1 equiv. of MeI, followed by elimination of metheylthiolate from the formed methyl (4-sulfamoylphenyl)carbamimidothioate (3) by treatment with K2CO3 at elevated temperature. Subsequently, intermediate 4 was treated with ethyl 2-bromoacetate, leading to 5, which was treated with hydrochloric acid at an elevated temperature, thus affording 4-(2,4-dioxoimidazolidin-1-yl)benzenesulfonamide (6). In the final step, the selective N-alkylation/benzylation of the NH hydantoin moiety with various alkyl/allyl/benzyl-halides (7a-n) provided the desired compounds (8a-n) in acceptable to good yield. 1 H NMR, 13 C NMR, and HRMS techniques were used to confirm the chemical structure of all of the synthesized compounds. All the analyzed compounds were >95% HPLC pure.

Compounds Design and Synthesis
Considering the fact that hydantoins already possess CA inhibitory effects [30], the drug design strategy that we propose in this paper is to incorporate in the same molecule both a zinc binder fragment of the benzene-sulfonamide type [4][5][6][7][8][9][16][17][18] as well as the tail based on the 3-substituted-hydantoin motif.
The synthesis of the target 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives is shown in Scheme 1. The synthesis started from sulfanilamide 1, which was converted to 4-thioureidobenzenesulfonamide (2)via reaction with KSCN in aqueous, acidic medium [33]. The key intermediate, potassium cyano(4-sulfamoylphenyl)amide 4, was prepared by the selective S-methylation of thiourea 2 via treatment with 1 equiv. of MeI, followed by elimination of metheylthiolate from the formed methyl (4-sulfamoylphenyl) carbamimidothioate (3) by treatment with K 2 CO 3 at elevated temperature. Subsequently, intermediate 4 was treated with ethyl 2-bromoacetate, leading to 5, which was treated with hydrochloric acid at an elevated temperature, thus affording 4-(2,4-dioxoimidazolidin-1yl)benzenesulfonamide (6). In the final step, the selective N-alkylation/benzylation of the NH hydantoin moiety with various alkyl/allyl/benzyl-halides (7a-n) provided the desired compounds (8a-n) in acceptable to good yield. 1 H NMR, 13 C NMR, and HRMS techniques were used to confirm the chemical structure of all of the synthesized compounds. All the analyzed compounds were >95% HPLC pure.

Carbonic Anhydrase Inhibition
The new compounds designed here were tested as inhibitors of three human enzymes, i.e., isoforms hCA I, II, and VII (all cytosolic ones) [4][5][6][7][8][9][16][17][18], as well as two fungal β-CAs from pathogenic organisms: MgCA from Malassezia globosa, one of the fungi involved in dandruff formation [58][59][60][61]; and CgNce103 from Candida glabrata, a species known for its virulence and resistance to many classes of antifungal drugs in clinical use [62][63][64][65][66]. The classical sulfonamide CAI acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ) was used as standard in the measurements reported in Table 1. Data of Table 1 show the following structure-activity relationship (SAR) for the inhibition of these enzymes with hydantoin-substituted benzene-sulfonamides: (i) hCA I, an abundant cytosolic isoform in many tissues and organs [4][5][6][7][8][9], was moderately inhibited by compounds 6 and 8 investigated here, with K I ranging between 233.8 and 8789 nM. Some of the best hCA I inhibitors are as active as AAZ, the standard drug (Table 1). (ii) hCA II; the dominant cytosolic isoform [4][5][6][7][8][9] was, on the other hand, potently inhibited by most new sulfonamides reported here, with K I ranging between 1.2 and 91.2 nM. The best inhibitor 8l incorporates the 2-fluorobenzyl moiety in position 3 of the hydantoin ring, whereas the unsubstituted benzyl derivative 8d was also a highly effective inhibitor (K I of 8.7 nM). The alkyl or alkenyl substituted derivatives 8a-8c were slightly less effective (but still potent CAIs), whereas the position and nature of the substituent eventually present on the benzyl fragment in the remaining derivatives seemed to be the factor that strongly influenced the inhibition potency. Indeed, 4-CN, 4-nitro and 2-fluorobenzyl fragments were those associated with the best inhibitory action, whereas 3-methyl, pentafluoro, 4-CF 3 and 4-Cl led to less effective inhibitors. (iii) The SAR is rather different for the inhibition of CA VII. The unsubstituted hydantoin 6 and the alky-substituted ones, 8a and 8b, were moderately active (K I of 30.8-187.2 nM). The alkeyl and benzylsubstituted hydantoins (except 8m) were, on the other hand, effective hCA VII inhibitors, with K I ranging between 3.0-19.5 nM.
The best hCA VII inhibitors were the unsubstituted benzyl and the 4-Me-benzyl derivatives 8d and 8e, with K I of 3.0-5.3 nM, in the same range as AAZ. (iv) MgCA was poorly inhibited by these sulfonamides, which had some activity in the high micromolar range, similarly to AAZ (Table 1). (v) CgNce103 was, on the other hand, effectively inhibited by hydantoin-substituted benzene-sulfonamides, with K I ranging between 5.9 and 83.7 nM. The SAR is again diverse from what observed for other isoforms/enzymes. The unsubstituted hydantoin 6 and the alkyl-substituted derivatives 8a-8c showed K I of 29.5-83.7 nM, whereas most benzyl-substituted derivatives (except 8l and 8m) were active in the low nanomolar range.

Chemistry
Reagents, starting materials and solvents were obtained from commercial sources and used as received. Thin-layer chromatography was performed on silica gel, spots were visualized with UV light (254 and 365 nm). NMR spectra were recorded on Bruker 300 spectrometer with chemical shifts values (δ) in ppm relative to TMS using the residual DMSO-d 6 signal ( 1 H 2.50; 13 C 39.52) see also Supplementary Materials. High-resolution mass spectra (HRMS) were recorded on a mass spectrometer with a Q-TOF micro mass analyzer using the ESI technique. (2) 4-Aminobenzensulfonamide (1) (30 g, 174.3 mmol) was dissolved in aqueous HCl (3.5 M, 180 mL) at 70 • C. After cooling to room temperature, KSCN (16.94 g, 174.3 mmol) was added, and the mixture was refluxed for 3 h. After cooling to room temperature, the reaction mixture was poured onto ice/cold water, and the formed precipitate was collected by filtration, washed with water, and air dried to afford 2 (12.49 g, 31%) as a white powder. 1  was added, and the mixture was refluxed for 3 h. After cooling to room temperature, the reaction mixture was poured onto ice/cold water, and the formed precipitate was collected by filtration, washed with water, and air dried to afford 2 (12.49 g, 31%) as a white powder. 1 (3) To a solution of 4-thioureidobenzenesulfonamide (2) (300 mg, 1.3 mmol) in DMF (4 mL),MeI (0.08 mL, 1.3 mmol) was added, and the mixture was heated at 40 °C for 2.5 h. After cooling to room temperature, the reaction mixture was extracted with EtOAc (3 × 20 mL). Organic layer was washed with aq. sat. NaHCO3 (2 × 20 mL) and then aq. sat. NH4Cl (1 × 20 mL), and dried over Na2SO4. Solvent removal in vacuum resulted in 3 (223 mg, 70%) as a white powder. 1

CA Inhibition Assay
An applied photophysics stopped-flow instrument was used for assaying the CA catalysed CO2 hydration activity [67]. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer for α-CAs or 20 mM TRIS (pH 8.4) as buffer for β-CAs, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10-100 s. The CO2 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 uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6 h at room temperature prior to assay in order to allow for the formation of the E-I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier [68][69][70][71][72][73][74], and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier [25,[58][59][60][61]66,75], and their concentrations in the assay system ranged between 9-12 nM.

Conclusions
Starting from commercially available inexpensive 4-aminobenzenesulfonamide, a library of novel hydantoin-based benzenesulfonamides were synthesized, and the structures of all derivatives were confirmed by 1 H NMR, 13 C NMR, and HRMS spectral techniques. The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two β-CAs from fungal pathogens. The best inhibition was observed against hCA II and VII, as well as Candida glabrata enzyme CgNce103. hCA I and MgCA were, on the other hand, less effectively inhibited by these compounds. The interesting results against CgNce103 make the compounds of interest for investigations in vivo as potential antifungals.

CA Inhibition Assay
An applied photophysics stopped-flow instrument was used for assaying the CA catalysed CO 2 hydration activity [67]. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer for α-CAs or 20 mM TRIS (pH 8.4) as buffer for β-CAs, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. 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 uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water, and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 6 h at room temperature prior to assay in order to allow for the formation of the E-I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier [68][69][70][71][72][73][74], and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier [25,[58][59][60][61]66,75], and their concentrations in the assay system ranged between 9-12 nM.

Conclusions
Starting from commercially available inexpensive 4-aminobenzenesulfonamide, a library of novel hydantoin-based benzenesulfonamides were synthesized, and the structures of all derivatives were confirmed by 1 H NMR, 13 C NMR, and HRMS spectral techniques. The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two β-CAs from fungal pathogens. The best inhibition was observed against hCA II and VII, as well as Candida glabrata enzyme CgNce103. hCA I and MgCA were, on the other hand, less effectively inhibited by these compounds. The interesting results against CgNce103 make the compounds of interest for investigations in vivo as potential antifungals.