Beta and Gamma Amino Acid-Substituted Benzenesulfonamides as Inhibitors of Human Carbonic Anhydrases

A series of novel benzenesulfonamide derivatives were synthesized bearing para-N β,γ-amino acid or para-N β-amino acid and thiazole moieties and their binding to the human carbonic anhydrase (CA) isozymes determined. These enzymes are involved in various illnesses, such as glaucoma, altitude sickness, epilepsy, obesity, and even cancer. There are numerous compounds that are inhibitors of CA and used as pharmaceuticals. However, most of them bind to most CA isozymes with little selectivity. The design of high affinity and selectivity towards one CA isozyme remains a significant challenge. The beta and gamma amino acid-substituted compound affinities were determined by the fluorescent thermal shift assay and isothermal titration calorimetry for all 12 catalytically active human carbonic anhydrase isozymes, showing the full affinity and selectivity profile. The structures of several compounds were determined by X-ray crystallography, and the binding mode in the active site of CA enzyme was shown.


Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO 2 to bicarbonate and maintain the acid-base balance in tissues and blood and, thus, play a crucial physiological role [1][2][3][4][5]. Human CAs belong to the α-family, which consists of 12 catalytically active (CAI, CAII, CAIII, CAIV, CAVA, CAVB, CAVI, CAVII, CAIX, CAXII, CAXIII, CAXIV) and three non-catalytic isozymes (CAVIII, CAX, and CAXI) that are also called carbonic anhydrase-related proteins (CARPs) [6,7]. Due to their different distribution in the body, the functions performed vary widely [8]. Therefore, disruption of normal function results in illnesses associated with only one or several CAs [9]. Thus, CAs are drug targets for a variety of disorders. There is still a significant challenge to design compounds possessing both high affinity and high selectivity for a specific isozyme, due to the structural similarity of the active sites of CAs [10][11][12]. Sulfonamides were discovered in 1940 as CA inhibitors by Mann and Keilin [13], who also reported some side effects of the newly discovered 4-aminobenzenesulfonamide. This was  showing the atom-numbering scheme. The thermal displacement ellipsoids are drawn at the 50% probability level and hydrogen atoms are shown as small spheres of arbitrary radii.
Hydrazides 2a,b were used for the synthesis of a series of azole derivatives (Scheme 2). Pyrrole or pyrazole derivatives can be synthesized by the condensation reaction of carboxylic acid hydrazides with aliphatic diketones [33,34]. Pyrroles 19a,b were obtained from hydrazides 2a,b by the reactions with hexane-2,5-dione in propan-2-ol at the reflux temperature of the reaction mixture. For instance, in the 1 H NMR spectrum for 19b, singlets at 1.91, 1.94, and 1.98 ppm were assigned to the protons of CH 3 groups, and each of the singlets at 5.61 and 5.63 ppm integrated for two protons of CH groups. These proton resonances have confirmed the presence of 2,5-dimethylpyrrole moiety. The singlets at 10.72 and 10.87 ppm have been ascribed to protons of NH groups. Similar signals are found in the compound 19a 1 H NMR spectrum as well. Furthermore, there was an attempt to synthesize 3,5-dimethylpyrrazoles 20 and 20a using the same method. Unfortunately, only one of the desired products (compound 20) was obtained. In the 1 H NMR spectrum for 20, four singlets integrated for 12 protons of CH 3 groups at 2.18, 2.25, 2.35, and 2.43 ppm, and two singlets assigned to the protons in the CH groups at 6.16 and 6.24 ppm have proven the presence of 3,5-dimethylpyrazole moiety in the molecule. Moreover, the triplet at 6.52 ppm assigned to the proton of NH group proved an open 2-pyrrolidone ring structure of compound 20. To obtain compound 20 non-chlorinated analog, previously synthesized pyrazole 21 [30] was isolated from the reaction mixture. The 1 H and 13 C NMR spectra data for 21 were very similar, as was described in previous work [30], which confirmed a closed 2-pyrrolidone ring structure in 21. We have proposed a hypothesis that, due to the acidic properties of pentane-2,4-dione and high temperature, the 2-pyrrolidone ring closed during the reaction. These conditions were suitable for compound 20 synthesis. We proposed that some steric hindrance, which occurs due to the chlorine substitute at C-2 of the phenyl ring, prevents the closure of the 2-pyrrolidone ring. In order to synthesize the desired product 20a, we have introduced several changes in reaction conditions. When the reaction was carried out at room temperature with NaOAc (to reduce the acidity of pentane-2,4-dione), we failed to detect new products by TLC after 24 h. Then we raised the reaction temperature to 50 • C, and after that we observed compound 21 by TLC.

Scheme 2. Synthesis of compounds 19-25.
Reactions of hydrazides 2a,b with methyl isothiocyanate or its phenyl analog in DMF led to the formation of carbothioamides 22a,b and carbothioamides 23a,b, respectively (Scheme 2). Further condensation reactions of 22a,b and 23a,b in the alkaline medium resulted in the formation of methyl triazolethiones 24a,b and phenyl triazolethiones 25a,b correspondingly. In the 1 H NMR spectra for 24a and 24b, singlets at 3.33, 3.39 ppm (24a) and 3.36, 3.40 ppm (24b) have been assigned to the protons of CH 3 groups. In the 13 C NMR spectrum for 24a, two signals at 166. 49 and 166.74 ppm have been attributed to C=S carbon in triazolethione ring. Similar carbon resonances were observed in compounds 24b and 25a,b 13 C NMR spectra.
In the next stage of this work, thioureido acid 26 was synthesized by the procedure [31] and used for the synthesis of new functionalized aminothiazoles. Compound 26 was treated with an excess of chloroacetone in water, and sodium acetate was used as a base to obtain thiazole 27 (Scheme 3). In the 1 H NMR spectra for 27, singlets at 2.19 and 6.44 ppm have been assigned to the protons of CH 3 and CH group, respectively. These proton resonances have confirmed the presence of 4-methylthiazol moiety. Later, ester 28 was synthesized by an esterification reaction of thiazole 27 with an excess of methanol at reflux in the presence of sulfuric acid as a catalyst. Hydrazine hydrate reaction with ester 28 in propan-2-ol at reflux temperature yielded hydrazide 29, which was treated with benzaldehyde in propan-2-ol at reflux to afford hydrazone 30. The 1 H NMR spectra 30 display double sets of resonances for the CO-NH and N=CH group protons due to the restricted rotation around the amide bond. The splitting of the proton resonances is also observed for carbon chain protons-two triplets at 2.58 and 3.00 ppm were assigned to the protons of CH 2 group, while the multiplet at 4.06-4.36 ppm region to protons of NCH 2 group. This proton splitting indicates that in DMSO-d 6 solution hydrazone 30 exists as a mixture of Z/E isomers, and, in most cases, the Z isomer predominates [33]. The reaction of carboxylic acid 26 with ethyl 2-chloroacetoacetate in water at reflux gave thiazole 31. Sodium acetate was used as a base (Scheme 3). In the 1 H NMR spectra for 31, a triplet at 2.19 and a singlet at 2.52 ppm have been assigned to the protons of CH 3 groups. In the 13 C NMR spectrum for 31, two signals at 14.23 and 17.31 ppm have been attributed to CH 3 group carbons and the signal at 60.22 to the carbon of ester CH 2 group. These proton resonances have confirmed the formation of desired product 31. Furthermore, thiazole 31 was treated with an excess of methanol in the presence of sulfuric acid as a catalyst to afford ester 32, which was then used in the reaction with hydrazine hydrate to obtain hydrazide 33. The reaction of sulfonamide 33 with benzaldehyde in propan-2-ol at reflux yielded hydrazone 34. Double sets of resonances are also observed in the 1 H NMR spectra for 34 similar to those in the spectra for 30. Therefore, this indicates that hydrazone 34 also exists as a mixture of Z/E isomers in DMSO-d 6 solution.
Previously synthesized compound 35 [31] was obtained by stirring thioureido acid 26 with 4-chloro-2 -bromoacetophenone in acetone at reflux temperature. Chlorine was introduced into the phenyl ring at the C-3 position of the benzenesulfonamide moiety to determine the halogenation effect on the binding affinity toward carbonic anhydrases. Thiazole 35 was treated with N-chlorosuccinimide in DMF at 0 • C. In the 1 H NMR spectra for 36, the doublets at 7.54 and 7.72 ppm were attributed to the protons of the aromatic ring of 4-sulfamoylphenyl moiety. Moreover, there was no signal observed at the 7.38 ppm region as was described in previous work [31], which was assigned to the proton of CH group in the thiazole ring. This data confirmed that electrophilic substitution only occurred in the thiazole ring, which led to the formation of 5-chlorothiazole 36 but not the desired product 37. To synthesize compound 37, some changes were made to the reaction conditions. Performing the reaction at room temperature in DMF with NClS and monitoring the course of the reaction by TLC, after 1 h the formation of compound 36 was observed. It was then decided to raise the temperature to 80 • C [35], and the reaction was reperformed, but after the 20 h the only compound observed by TLC was thiazole 36. Furthermore, chlorination of compound 35 was attempted in 6 M aqueous HCl solution with hydrogen peroxide overnight [29], although the only product isolated from the reaction mixture was 5-chlorothiazole 36.

Crystal Structure of Compound 4a
The structure of 4a was examined in more detail. Figure 1 shows a perspective view of molecule 4a with thermal ellipsoids and the atom-numbering scheme followed in the text. The newly obtained 2,5-dioxopyrrolidine ring is planar and forms with the CH 2 (C10) group a dihedral angle of 104.6(7). In the molecular structure, the 2,5-dioxopyrrolidine is almost perpendicular to both the hydrazone fragment (dihedral angle is 115.0(8)) and the benzenesulfonamide system (dihedral angle is equal to 113.3(8)).

Compound Binding to Carbonic Anhydrases (CA)
The dissociation constants of all synthesized compounds were measured for five CA isozymes, CAI, CAII, CAIX, CAXII, and CAXIII (Table 1), by the fluorescent thermal shift assay (FTSA, also referred to as TSA and DSF-differential scanning fluorimetry [36][37][38][39]) ( Figure 2). In addition, the binding strength of several compounds was confirmed by isothermal titration calorimetry [41]. The binding data of compounds 28, 31, and 36 to CA II and compound 32 binding to CA VB were in good agreement; the K d _ obs values measured by both methods differed by less than three-fold. Structural characterization of three compounds' (28, 31, and 36) binding was performed by determining their X-ray crystal structures in complexes with CAII ( Figure 5). , raw data only of CAVB) and isothermal titration calorimetry (ITC) data of compound 32 binding to CAVB (panels (C,D)). Dissociation constant, K d_obs , values at 37 • C and pH 7.0 in 50 mM sodium phosphate buffer containing 100 mM NaCl are shown near the corresponding graphs in panels (A,C). To calculate the intrinsic dissociation constants, the pK a values of compound sulfonamide group were measured spectrophotometrically at 37 • C (panels (E,F)). The normalized absorbances at selected wavelengths were plotted as a function of pH and fitted to the Henderson-Hasselbalch equation. Table 2. The observed dissociation constants of all 12 catalytically active CAs with tested compounds at pH 7.0 and 37 • C by the fluorescent thermal shift assay. The value ≥ 200,000 is the upper limit of K d_obs detection; ND-K d_obs value was not determined. The standard deviation of the FTSA measurements is ± 1.6-fold in K d_obs . The ITC data are shown in the brackets (pH 7.0, 37 • C).

Cmpd.
Lab. Name   For some compounds, the pK a values for the sulfonamide group were measured and used to calculate the intrinsic, pH-independent dissociation constants or changes in Gibbs energy [40] (Figure 4). In addition, the binding strength of several compounds was confirmed by isothermal titration calorimetry [41]. The binding data of compounds 28, 31, and 36 to CA II and compound 32 binding to CA VB were in good agreement; the K d _ obs values measured by both methods differed by less than three-fold. Structural characterization of three compounds' (28, 31, and 36) binding was performed by determining their X-ray crystal structures in complexes with CAII ( Figure 5).  Table 1), with on average a 40fold increase in affinity for CAI and CAXIII, 15-20-fold for CAII, CAVII, and CAXIV, and marginally increased affinity toward CAIII, CAIV, CAIX, and CAXII.
Compounds 2a and 2b, which are precursors to compounds 3a,b-25a,b, exhibited weak interaction with CAI, CAII, CAIX, CAXII, and CAXIII (K d_obs 0.83-130 µM). Compound 2b had a moderately strong interaction with CAIX (K d_obs = 0.83 µM). The size and hydrophobicity of substituents increases in the direction 3a→6a and 3b→6b, resulting in the increment of affinity toward CAI, CAII, CAIX, CAXII, and CAXIII. However, observed affinity for CAXII of compounds 5a→6a decreased 50 times. Ligand flexibility could also play a role; ligands 5a and 5b having an extra rotatable bond might allow better interaction of the benzene ring with hydrophobic amino acids of CAXII, compared to 6a and 6b. Compound 6b was also tested with all other catalytically active CA isozymes (Table 2); it exhibited weak interaction with CAIII, CAIV, CAVA, and CAXII (K d_obs ≥ 200,000 nM, 4200 nM, 20,000 nM, and 7900 nM, respectively), moderately strong interaction with CAI and CAVI, and strong interaction with CAII, CAVB, CAVII, CAIX, CAXIII, and CAXIV (K d_obs in the range of 100-290 nM). Although compounds 4a and 4b contain closed dioxopyrrolidine ring structure (Figure 3), the affinities for CAI, CAII, CAIX, CAXII, and CAXIII did not differ from the other compounds described in this section. No remarkable selectivity toward any CA isozyme was observed for compounds 2a,b, 3a,b, 5a,b, 6a,b, nor even 4a,b, but usually there was an increase in observed affinity (up to 10-100 times) for CAIX compared to other tested CAs.
Ligands 7-18a and 7-18b are structurally similar to 6a and 6b, only differing by symmetric substituents on the phenyl ring. Small substituents such as -OH, -Cl, -F, -OCH 3 , -COOH, and -NO 2 as well as their positions in the phenyl ring did not significantly affect the binding affinity towards CAs. Compounds 7-18a exhibited low affinity for tested CA isozymes, with no significant selectivity for a particular CA. The K d ranges for CAI were 5.0-36 µM, CAII-1.3-5.0 µM, CAIX-0.5-3.3 µM, CAXII-20-100 µM, and for CAXIII-1.7-23 µM. Chlorinated compounds 7-18b proved to be of more interest, with a generally increased observed affinity toward CAII, CAIX, and CAXIII compared to analogous non-chlorinated compounds 7-18a ( Figure 3). Compounds 7-9b, having -OH substituent in, respectively, ortho-, meta-, and para-positions on the benzene ring, interacted similarly with CAI, II, IX, XII, and XIII as compound 6b. Compound 14b, having OCH 3 substitute in para-position on the benzene ring, exhibited the strongest interaction with CAII and CAXIII (K d_obs 59 nM and 66 nM, respectively), although binding was not selective, due to moderately strong interaction with CAI and CAIX (K d_obs 510 nM and 290 nM, respectively).
Thiourea derivatives 22-25a,b ( Figure 3) exhibited similar binding patterns to CAI, CAII, CAIX, CAXII, and CAXIII, showing increases in observed binding affinity going from CAI to CAIX. Compounds 22a-25a had a trend of decreasing affinity toward CA isozymes going from CAIX to CAXIII. A similar trend was not observed for their chlorinated analogs 22-25b, which showed a stronger affinity for CAIX but did not have significant differences in binding strength for CAXII and CAXIII (Table 1). Compounds 22a,b, 23b, and 25b were also tested with all catalytically active CA isozymes, and no selectivity was observed. Compound 22a exhibited weak binding to all tested CA isozymes (micromolar K d_obs values). This is consistent with a trend of non-chlorinated compounds having weaker observed binding affinity to CAs than chlorinated analogs. On the other hand, 22a interacted with a stronger affinity with CAIX (K d_obs 330 nM). Compound 22b bound CA isozymes more than three times stronger than 22a and showed the highest affinities for CAIX and CAXIV (K d_obs values of 100 nM and 130 nM, respectively). The K d_obs decreased in the direction 22b→23b→25b for most CAs, especially for CAII (400 nM→50 nM→25 nM), CAIX (100 nM→33 nM→25 nM), and CAXIV (from 130 nM→50 nM→25 nM). Compound 25b also had an unusually strong interaction with CAIII (K d_obs 5000 nM, Table 2), despite having large substituents, which usually contribute to steric hindrance and weak binding to CAIII. (27)(28)(29)(30)(31)(32)(33)(34)(35)(36) Binding to CA Compounds 27-6 had greater asymmetry than compounds 2-25a,b ( Figure 3). Compounds 27-34 and 36 were tested with all catalytically active CA isozymes. In general, compounds from this group exhibited weak interaction with CAIII and CAVA (Table 2), with the K d_obs values ranging from 17 µM to ≥200 µM (200 µM being the weakest detectable K d_obs value). Compounds 27-30 differed by substitutes on β-aminoacid carbonyl group, increasing in size (-OH, -OCH 3 , -NHNH 2 , -NHN=CPh). Carboxylic acid 27 strongly bound to CAI and CAIX (K d_obs 67 nM and 48 nM, respectively) but also had moderately strong interactions with CAII, CAVII, CAXII, and CAXIV (K d_obs in the ranges 170-250 nM). Esterification of thiazole in 27 to compound 28 increased affinity toward CAII, CAVA, CAVB, and CAXIV (K d_obs values of 45 nM, 50 µM, 46 nM, 29 nM of compound 28, respectively). Of special interest was an increase in affinity toward CAVB in the direction 27→28, with K d_obs value decreasing from 560 nM to 46 nM ( Table 2). The hydrazide 29 interacted with all CAs slightly weaker than 28. Hydrazone 30, having by far the largest, hydrophobic, and inflexible -NHN=CPh group, showed a significantly decreased binding strength to all tested CA isozymes (K d s in the micromolar range). Compounds 31-34 were analogous to compounds 27-30 but had an additional ester substitute on the thiazole ring, which made compounds larger and less flexible than their counterparts ( Figure 3). Ligands 31-33 showed similar or significantly weaker interactions with most CA isozymes than their analogous compounds without ethylester group on thiazole ring, 27-29. However, of all tested compounds, the strongest interaction was measured between 32 and CA VB (K d = 30 nM). Binding affinities of compound 34 towards CAII, CAVB, and CAIX were in the range of 100 nM. Compounds 35 and 36 showed the strongest affinities to CAI (K d_obs 25 nM (Table 1) and 20 nM (Table 2), respectively). Furthermore, compound 36 interacted strongly with CAII, CAVII, CAIX, and CAXIII (K d_obs in the range of 23-40 nM), showing no selectivity toward CAs.

Intrinsic Thermodynamics of Compounds 27-36 Binding to CAs
We calculated the intrinsic dissociation constants (K d_int , Table 3) and intrinsic Gibbs energy (∆G int , Table S1) values of binding between CA isozymes and compounds 27-29, 31-33, and 36 and constructed a compound structure-affinity correlation map ( Figure 4) which demonstrates how small changes in functional groups influence compound capability to bind a particular CA isozyme. The intrinsic binding parameters represent the actual binding reaction between the deprotonated sulfonamide and the Zn-bound water form of CA. To calculate the intrinsic binding parameters by Equations (4) and (5), the pK a s of tested sulfonamides were measured spectrophotometrically by measuring absorption at different pH values. The average pK a values of compounds 27-29, 31, 32, 35, and 36 were determined to be 10.1. Calculation of the intrinsic values has a significant advantage over the observed values, because the intrinsic values eliminate the pH and buffer effects that may be misleading in the structural origins of the altered affinity [5,42,43]. Table 3. The intrinsic dissociation constants (K d_int ) for compounds binding to CAs at 37 • C. ND-not determined. *-the pK a value was not determined and is an average of all measured compounds. Experimental data and statistics of pK a values are provided in Supplementary Materials.

Cmpd.
Lab All compounds exhibited relatively weak binding to CAIII (K d_int at limit of detection for compounds 28-34 and K d_int values of 6 and 2.6 nM, respectively, for compounds 27 and 36) and CAVA (K d_int at limit of detection for all tested compounds except compound 28, which binds with K d_int of 13 nM (Table 3)). Figure 4 depicts some compounds in which minor structural changes had a significant effect on affinity for carbonic anhydrases. Esterification of carboxyl group (directions 27→28 and 31→32) (Figure 4) was favorable or had negligible effect on the Gibbs energy for all CAs except for CAXII, which showed a slight decrease in affinity. Addition of methoxy group leads to a large gain in ∆G int for CAVB (∆∆G int (27→28) = -4.6 and ∆∆G int (31→32) = −14.4 kJ/mol). Further modification of the methoxy with hydrazide group (directions 28→29 and 32→33) decreased the Gibbs energy of binding to all CAs but did not change that to CAVB (∆∆G int (28→29) = 0.1 and ∆∆G int (32→33) = 0.7 kJ/mol). Interestingly, addition of ethoxycarbonyl group on the thiazole ring (directions 27→31, 28→32, and 29→33) resulted in the decrease of ∆G int or had negligible effect for all CAs, with the largest reduction of affinity for CAXII (∆∆G int (28→32) = 11.2 kJ/mol). In summary, the intrinsic binding data showed that esterification increased the binding affinity for the majority of CA isozymes (Table 3). Thus, it would be very interesting to investigate the influence of small and hydrophobic substitutes on the carboxyl group with the perspectives of finding possible new lead compounds for use as inhibitors for CAVB while binding weakly to CAVA isozyme and possibly having increased selectivity for CAVB isozyme.

X-ray Crystallography of Human CAII Complexes with Inhibitors
The crystal structures of compounds 28, 31, and 36 in the complex with CAII were determined by X-ray crystallography ( Figure 5). The electron density maps of the ligands bound in the active site of CAII are shown in Figure S2 in Supplementary Materials. The data collection and refinement statistics are presented in Table S2. In all three structures, the benzenesulfonamide ring occupies a similar position ( Figure 5) due to the interaction with Leu198; only the positions of the substituents differed. The carboxy group of compounds 31 and 36 faces the hydrophilic side (colored cyan in Figure 5) of the active site of CAII and forms hydrogen bonds with Asn62, Asn67, or water molecules, while the methyl ester in compound 28 adheres to a more hydrophobic surface (colored brown in Figure 5). The 2D schemes of the interactions are shown as LigPlot schemes based on X-ray crystallography in Figure S3. The thiazole-based substituent interacts with Phe131 and was found on either side of the Phe131 side chain depending on the other substituents ( Figure 5). Thus, the smallest thiazole substituent of compound 28 occupied the "right side" of the hydrophobic pocket, while the larger thiazole-based substituents of compounds 31 and 36 occupied the more spacious "left side" of the pocket, because they would not fit in the pocket on the "right side" without changing the position of the benzenesulfonamide due to steric hindrance.

Organic Synthesis
Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The melting points were determined on a MEL-TEMP (Electrothermal, Bibby Scientific Company, Burlington, NJ, USA) melting point apparatus and were uncorrected. The IR spectra (ν, cm −1 ) were recorded on a Perkin-Elmer Spectrum BX FT-IR spectrometer using KBr pellets. The 1 H and 13 C NMR spectra were recorded in DMSO-d 6 medium on Brucker Avance III (400, 101 MHz) spectrometer (see Supplementary Materials, Figures S4-S103). The chemical shifts (δ) were reported in parts per million (ppm), calibrated from TMS (0 ppm) as an internal standard for 1 H NMR and DMSO-d 6 (39.43 ppm) for 13 C NMR. Mass spectra were obtained on a Bruker maXis UHR-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with ESI ionization. Elemental analysis was performed on a CE-440 elemental analyzer (Exeter Analytical Inc., North Chelmsford, MA, USA). The reaction course and purity of the synthesized compounds were monitored by TLC using aluminium plates pre-coated with the silica gel 60 F 254 (MerckKGaA, Darmstadt, Germany). Single crystals of 4a were investigated on a Rigaku, XtaLAB Synergy, Dualflex, HyPix diffractometer. The crystal was kept at 170.0(1) K during data collection. Using Olex2 [44], the structure was solved with the SIR2014 [45] structure solution program using Direct Methods and refined with the olex2.refine [46] refinement package using Gauss-Newton minimization.

Fluorescent Thermal Shift Assay (FTSA)
The fluorescent thermal shift assay was performed to obtain the observed binding constants (inversely proportional to the observed dissociation constants (K d_obs s)) between CA isozymes and synthesized compounds. The QIAGEN Rotor-Gene Q was used to carry out the experiments, with a blue channel used for excitation (365 nm) and detection (460 nm). FTSA in this case relies on an extrinsic probe, 8-anilino-1-naphthalenesulfonate (ANS), to detect the fluorescence increase during protein denaturation, from which protein melting temperature (T m ) can be determined. Since the binding compounds usually thermally stabilize a protein, an increase in T m is observed when increasing the compound concentration. The observed affinity of ligands for CAs was determined by plotting the T m as a function of ligand concentration and then fitting a curve as described previously [48]. Samples consisted of constant concentration of CAs (5 µM for all proteins except 7.5 or 10 µM of CAIV) and varied concentrations of ligand ((0-200) µM) in 50 mM sodium phosphate buffer at pH 7.0 containing 100 mM NaCl, 50 µM ANS, and 2% (v/v) DMSO. Samples were heated from 25 • C to 99 • C at the speed of 1 • C/min. An example of an experiment is given in Figure 2.

Isothermal Titration Calorimetry (ITC)
ITC determined the observed standard enthalpy changes upon binding as well as the K d_obs values [41] that were compared to ones obtained by FTSA. The ITC experiments were performed by adding the proteins to the calorimeter cell and the compound solutions to the syringe at 37 ºC. The CAII binding with compounds 28, 31, and 36 and CAVB with compound 32 ( Figure S104) were determined using a MicroCal PEAQ-ITC calorimeter (Northampton, MA, USA). Experiments consisted of 19 injections, the first injection of 0.4 µL over 0.8 s, with the remaining injections of 2 µL over 4 s with spacing of 150 s between injections, at temperature 37 • C, reference power 10 µcal/s, and stirring speed of 750 rpm. The protein concentrations for CAII and CAVB were 10 µM and 8 µM, respectively; the compound concentration was 10 times higher than the protein (100 µM for compounds tested with CAII, 80 µM for compound tested with CAVB). Proteins and compounds were diluted in sodium phosphate 50 mM buffer at pH 7.0, containing 100 mM NaCl and 2% (v/v) DMSO. Data were analyzed using NITPIC (version 1.2.7) [49,50] and Sedphat (v. 14.0) [51] software; the heat of dilution was subtracted from the titration curves.

Determination of Compound Sulfonamide Group pK a
To calculate the intrinsic thermodynamic parameters of compound binding to CA isozymes, the pK a values of the sulfonamide group and the water molecule bound to the Zn(II) ion at the active site of CA proteins had to be determined. The pK a values for CA isozyme-bound water molecules were taken from [40], CAI-8.1; CAII-6.9; CAIII-6.5; CAIV-6.6; CAVA-6.7; CAVB-7.0; CAVI-6.0; CAVII-6.8; CAIX-6.6; CAXII-6.8; CAXIII-8.0; CAXIV-6.1. Compound pK a values were measured by obtaining UV-Vis spectra at 37 • C at different pH values (from 7.0 to 12.0 with 0.5 pH increment) using BMG Labtech CLARIOstar Plus plate reading spectrophotometer. Compounds were diluted to a constant concentration of 50-200 µM (concentration was chosen depending on compound solubility and absorbance peak) in a universal buffer, consisting of 50 mM sodium phosphate, 50 mM sodium acetate, 25 mM sodium borate, and 50 mM sodium chloride, and also contained 2% (v/v) DMSO. The pK a values were calculated as described previously in [28,52] by normalizing the absorbance, plotting it as a function of pH, then fitting to the Henderson-Hasselbalch equation using the least square method ( Figure S105).
The pK a s of compounds 27, 28, 29, 31, 32, 35, and 36 were measured, with an average pK a value of 10.1 for all compounds. Since the pK a values were only able to be measured for compound group 26-36, due to universal buffer pH limitations, intrinsic thermodynamic parameters were not calculated for compounds 2(a,b)-25(a,b), only for compounds 26-36 using experimentally obtained pK a values when possible and using the average pK a value mentioned above for compounds not measured.

Calculation of the Intrinsic Thermodynamic Parameters
The intrinsic thermodynamic parameters were calculated as described in [40]. The intrinsic reaction of inhibitor binding to CA is shown in Equation (1). The observed reaction includes the associated protonation-deprotonation reactions of protein, ligand, and buffer and thus has to be accounted for in order to make correct conclusions about compound binding affinities. It is known that CA proteins bind sulfonamides only when water molecules are coordinated to the Zn(II) ion, and sulfonamides only bind to CA proteins when their amino group is deprotonated (confirmed by neutron crystallography [53][54][55] (2) and (3)). The intrinsic dissociation constant is calculated by multiplying K d_obs value by the corresponding fractions of protein and ligand (Equation (4)).
3.2.6. X-ray Crystallography: Crystallization, Data Collection, and Structure Determination CAII was concentrated by ultrafiltration to 31 mg/mL and mixed with the crystallization solution (0.1 M sodium bicine (pH 9.0), 0.2 M ammonium sulfate, and 2 M sodium malonate (pH 7.0)) at a ratio 1:1 and crystallized by the sitting drop method. One µL of 50 mM compound solution in DMSO was combined with 50 µL of reservoir solution for crystals soaking.
X-ray diffraction data were collected at the EMBL beamline P13 at PETRA III ring of DESY synchrotron in Hamburg. Crystals were flash cooled to 100 K without additional cryo-protection. The data were processed and scaled using XDS [56], MOSFLM [57], and SCALA [58]. MOLREP [59] was used for molecular replacement, with an initial model of 4HT0. The model was refined with REFMAC [60] and fitted in the electron density map using COOT [61]. The 3D models of compounds were constructed by the AVOGADRO [62] program, while ligand parameter files were created using LIBCHECK [63,64]. The PDB IDs, data collection, and refinement statistics are listed in Table S2 in Supplementary Materials. The 2D schemes based on X-ray crystallography are shown as LigPlot+ [65] schemes in Figure S3.

Conclusions
A series of benzenesulfonamides bearing para-N β,γ-amino acid or para-N β-amino acid and thiazole moieties were synthesized, and the binding affinities for the entire family of 12 catalytically active human CA isozymes were determined by the fluorescent thermal shift assay. Selected compound affinities were also determined by isothermal titration calorimetry, while X-ray crystallography helped to determine the structural arrangement of the inhibitors in the active site of CAII. The intrinsic affinities that are independent of protonation reactions were determined according to the described model. The analysis showed that the chlorine atom in the meta-position of para-N β-amino acid bearing benzenesulfonamide derivatives increases the observed affinity for CA over non-chlorinated compounds, while the substituents located further away from this group of benzenesulfonamides have lower influence on the affinity. The most potent compound, 36, bearing para-N β-amino acid and thiazole moieties bound CAI and CAIX with 20 nM K d_obs . Correlation of the chemical structures of the compounds with the intrinsic affinities showed that small changes in the chemical structure of the compound can have significant effects on the affinity.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ph15040477/s1, Table S1: Intrinsic standard Gibbs energy change values for compounds binding to CAs; Table S2: X-ray crystallography data collection and refinement statistics; Figure S1: Determination of compound 32 binding to human CAs by the fluorescent thermal shift assay; Figure S2: The electron densities of the ligands in the active site of CAII; Figure  Acknowledgments: Access to the EMBL beamline P13 at PETRA III (DESY) has been supported by iNEXT-Discovery, project number 871037, funded by the Horizon 2020 program of the European Commission.
Conflicts of Interest: D.M., A.Z. declare that they have several patents and patent applications on carbonic anhydrase inhibitors. Other authors declare that they have no conflicts of interest, including no financial, personal, or other relationships with other people or organizations.