Carbonic Anhydrase Inhibition with Sulfonamides Incorporating Pyrazole- and Pyridazinecarboxamide Moieties Provides Examples of Isoform-Selective Inhibitors

A series of benzenesulfonamides incorporating pyrazole- and pyridazinecarboxamides decorated with several bulky moieties has been obtained by original procedures. The new derivatives were investigated for the inhibition of four physiologically crucial human carbonic anhydrase (hCA, EC 4.2.2.1.1) isoforms, hCA I and II (cytosolic enzymes) as well as hCA IX and XII (transmembrane, tumor-associated isoforms). Examples of isoform-selective inhibitors were obtained for all four enzymes investigated here, and a computational approach was employed for explaining the observed selectivity, which may be useful in drug design approaches for obtaining inhibitors with pharmacological applications useful as antiglaucoma, diuretic, antitumor or anti-cerebral ischemia drugs.


Introduction
The proper function of physiological processes in the human body depends on the preservation of an adequate acid-base balance. Indeed, the normal intracellular pH range is between 7.35 and 7.45, but when the pH deviates from this range, pathological conditions are commonly observed [1]. CO 2 is generated and used in many metabolic reactions, and one of the most important buffer systems used by cells is the homeostatic HCO 3 − /CO 2 mechanism. Because of the slow reaction between CO 2 and H 2 O, enzymes are fundamental to speed up the process; for example, carbonic anhydrases (CAs, EC 4.2.1.1) are efficient catalysts for the reversible reaction between CO 2 and HCO 3 − [2]. To date this class of enzymes has been divided into eight distinct and genetically unrelated families [3][4][5][6]. In humans, there are 15 isoforms, and their overexpression is often related to different diseases. Indeed, the abnormal expression of CA I, IV, IX, and XII isoforms in serum and synovium specimens are related to rheumatoid arthritis, and their overexpression also has been demonstrated to negatively affect cellular immunity processes and to enhance associated symptoms [7][8][9]. In addition, the overexpression of hCA IX and XII was observed in several cancer diseases, as well as in patients suffering from cerebral ischemia [3,[10][11][12].
In this context, the pyrazole scaffold is a versatile molecule that has attracted attention due to its wide range of diverse pharmacological activities, which make it a versatile lead Our aim was to further support our previous studies on hCAs as valid and robust pharmacological targets for the treatment of different pathological conditions that are characterized by the overexpression of different human CA isoforms such as CA IX and XII over the ubiquitous hCA I and hCA II in order to decrease the side effects due to their inhibition, such as with the clinically approved drug acetazolamide (AAZ) depicted in Figure 2.

Chemistry
The synthesis of target compounds 4 and 5 is presented in Scheme 1. Starting 4-oxo-4H-chromene-2-carboxylic acids 1 [32,33] were initially converted into substituted amides Our aim was to further support our previous studies on hCAs as valid and robust pharmacological targets for the treatment of different pathological conditions that are characterized by the overexpression of different human CA isoforms such as CA IX and XII over the ubiquitous hCA I and hCA II in order to decrease the side effects due to their inhibition, such as with the clinically approved drug acetazolamide (AAZ) depicted in Figure 2.
Molecules 2021, 26, x FOR PEER REVIEW 2 of 19 In this context, the pyrazole scaffold is a versatile molecule that has attracted attention due to its wide range of diverse pharmacological activities, which make it a versatile lead molecule in several drug molecules such as celecoxib, ramifenazone, lonazolac, and rimonabant, drugs approved as COX-2 inhibitors [13][14][15][16], crizotinib [17] and paropanib [17] as anticancer drugs, sildenafil [17] (Viagra) PDE5 inhibitor, zometapine [18] as antidepressant, ocinaplon [19] as anxiolytic (Figure 1), and many others. Additionally, in recent years, their derivatives have been reported to possess antimicrobial activity [20][21][22] as well as antiviral [23,24], antidiabetic [25,26], anti-Alzheimer [27,28], antitubercular [29,30], and antileishmanial properties [31]. Our aim was to further support our previous studies on hCAs as valid and robust pharmacological targets for the treatment of different pathological conditions that are characterized by the overexpression of different human CA isoforms such as CA IX and XII over the ubiquitous hCA I and hCA II in order to decrease the side effects due to their inhibition, such as with the clinically approved drug acetazolamide (AAZ) depicted in Figure 2.

Chemistry
The synthesis of target compounds 4 and 5 is presented in Scheme 1. Starting 4-oxo-4H-chromene-2-carboxylic acids 1 [32,33] were initially converted into substituted amides 2 and 3, which without further purification were used for the preparation of pyrazole derivatives 4a-d,f and 5a,b,d-f. Some examples of such transformations of 4-oxo-4Hchromene-2-carboxamides to substituted pyrazoles were also described earlier [34]. 2 and 3, which without further purification were used for the preparation of pyrazole derivatives 4a-d,f and 5a,b,d-f. Some examples of such transformations of 4-oxo-4Hchromene-2-carboxamides to substituted pyrazoles were also described earlier [34]. The structure and composition of compounds 4a-d,f and 5a,b,d-f were confirmed by 1 H, 13 C NMR spectroscopy and elemental analysis. The signals of all observed in the 1 H NMR spectra protons appear in usual spectral regions that unambiguously correspond to the structure of the synthesized substances. For compound 5a 1 H NMR, spectra were recorded in solutions of DMSO-d6, CF3COOD and DMSO-d6 + 5% (mass) CF3SO3H. It should be noted that, obviously, compound 5a predominantly exists as two tautomeric forms in the solution of DMSO-d6. In this case, a double set of signals of the OH group (13.78 s, 0.35H and 13.17 s, 0.65H), NH pyrazole fragment (10.39 s, 0.35H and 10.20 s, 0.65H) and NH carboxamide group (8.69 s, 0.35H and 8.13 s, 0.65H) was observed, while the signals of aromatic protons of hydroxyaryl substituents, 4-H pyrazole moiety and aliphatic groups appeared as broad multiplets and the signals of the arylsulfamide moiety were clearly separated and resolved at 7.28 (s, 2H, NH2).
In the 1 H NMR spectra of compound 5a, measured in CF3COOD, OH and NH proton signals were not detected; at the same time, the aromatic and aliphatic signals were more clearly separated and resolved, although some of their broadening was also observed. In the spectrum of compound 5a measured in DMSO-d6 + 5% (mass) CF3SO3H, signals of protons of NH-pyrazole, hydroxy and aminosulfonyl groups were not detected, whereas the signals of aromatic and aliphatic protons were more clearly resolved and separated. Signals of aromatic protons of the arylsulfamide fragment were at 7.73 (d, J = 8.0 Hz, 2H, Ar) and 7.41 (d, J = 8.0 Hz, 2H, Ar). The signal of 4-pyrazole was superimposed with the signal of the orthohydroxyphenyl substituent at 7.24-7.11 ppm. At the same time, the NH signal of the carboxamide group of 5a (8.45 s, 1H, NH) was clearly observable. In the NMR 1 H spectra of compounds 4a-d,f and 5b,d-f recorded in DMSO-d6 and DMSO-d6 + 5% (mass) CF3SO3H, there was a set of signals similar to the corresponding set of signals in the NMR 1 H spectra of compound 5a measured in the same solvents. Measurement of the 13 C NMR spectra of compound 5a in DMSO-d6 or CF3COOD gave low-resolved or undefined peaks, while in spectra recorded in the solution of DMSO-d6 + 5% (mass) CF3SO3H, all the signals of carbon atoms (the number of corresponding to the number of carbon atoms in their structure) appeared as well-defined and highly resolved peaks.
Probably, the addition of a small amount of trifluoromethanesulfonic acid (as one of the strongest acids) is sufficient for the protonation of the pyrazole ring, which leads to an Scheme 1. Synthesis of 3-(2-hydroxyaryl)-1H-pyrazole derivatives.
The structure and composition of compounds 4a-d,f and 5a,b,d-f were confirmed by 1 H, 13 C NMR spectroscopy and elemental analysis (see Supplementary Materials). The signals of all observed in the 1 H NMR spectra protons appear in usual spectral regions that unambiguously correspond to the structure of the synthesized substances. For compound 5a 1 H NMR, spectra were recorded in solutions of DMSO-d 6 , CF 3 COOD and DMSO-d 6 + 5% (mass) CF 3 SO 3 H. It should be noted that, obviously, compound 5a predominantly exists as two tautomeric forms in the solution of DMSO-d 6 . In this case, a double set of signals of the OH group (13.78 s, 0.35H and 13.17 s, 0.65H), NH pyrazole fragment (10.39 s, 0.35H and 10.20 s, 0.65H) and NH carboxamide group (8.69 s, 0.35H and 8.13 s, 0.65H) was observed, while the signals of aromatic protons of hydroxyaryl substituents, 4-H pyrazole moiety and aliphatic groups appeared as broad multiplets and the signals of the arylsulfamide moiety were clearly separated and resolved at 7.28 (s, 2H, NH 2 ).
In the 1 H NMR spectra of compound 5a, measured in CF 3 COOD, OH and NH proton signals were not detected; at the same time, the aromatic and aliphatic signals were more clearly separated and resolved, although some of their broadening was also observed. In the spectrum of compound 5a measured in DMSO-d 6 + 5% (mass) CF 3 SO 3 H, signals of protons of NH-pyrazole, hydroxy and aminosulfonyl groups were not detected, whereas the signals of aromatic and aliphatic protons were more clearly resolved and separated. Signals of aromatic protons of the arylsulfamide fragment were at 7.73 (d, J = 8.0 Hz, 2H, Ar) and 7.41 (d, J = 8.0 Hz, 2H, Ar). The signal of 4-pyrazole was superimposed with the signal of the orthohydroxyphenyl substituent at 7.24-7.11 ppm. At the same time, the NH signal of the carboxamide group of 5a (8.45 s, 1H, NH) was clearly observable. In the NMR 1 H spectra of compounds 4a-d,f and 5b,d-f recorded in DMSO-d 6 and DMSO-d 6 + 5% (mass) CF 3 SO 3 H, there was a set of signals similar to the corresponding set of signals in the NMR 1 H spectra of compound 5a measured in the same solvents. Measurement of the 13 C NMR spectra of compound 5a in DMSO-d 6 or CF 3 COOD gave low-resolved or undefined peaks, while in spectra recorded in the solution of DMSO-d 6 + 5% (mass) CF 3 SO 3 H, all the signals of carbon atoms (the number of corresponding to the number of carbon atoms in their structure) appeared as well-defined and highly resolved peaks.
It should be mentioned that N-[4-(aminosulfonyl)phenyl]-1-(4-chlorophenyl)-4-oxo-1,4-dihydropyridazine-3-carboxamide 10d was obtained earlier by another method [38]. The proton signal data in the 1 H NMR spectra of obtained compound 10d did not match the data described in [38], while all 1 H NMR spectral data of the acids 8a-d and amides 10a-d correlated with 1 H NMR spectral data of the similar compounds described in [35][36][37]39,40]. Due to this, probably, the structure of 10d presented in [38] is not correct. The number of signals in the 13  The structure and composition of compounds 8a-d and 10a-d were confirmed by 1 H, 13 C NMR spectroscopy and elemental analysis. The signals of the 1-aryl-4-oxo-1,4dihydropyridazine moiety of compounds 8 and 10 were observed at: H-5 (d, J = 7.9 Hz) 6.90-7.05 ppm; and H-6 (d, J = 7.9 Hz) 7.95-8.10 ppm. The carboxamide group signals of compounds 10a-d appeared as a singlet at 11.90-12.15 ppm, whereas the signals of the sulfonamide groups appeared as a singlet at 7.30-7.35 ppm.

Biological Evaluation
All synthesized compounds were evaluated for their inhibitory activity against four human CA isoforms, which are: hCA I, hCA II, hCA IX and hCA XII (Table 1).

Biological Evaluation
All synthesized compounds were evaluated for their inhibitory activity against four human CA isoforms, which are: hCA I, hCA II, hCA IX and hCA XII (Table 1). .

Biological Evaluation
All synthesized compounds were evaluated for their inhibitory activity against four human CA isoforms, which are: hCA I, hCA II, hCA IX and hCA XII (Table 1).  4a-f, 5a, b, d-f, 10a- achieved for compound 10d with K i 6.2 nM, followed by compound 5e (K i 71.4nM). The lowest activity was shown by compound 4a with K i at 3822 nM. The order of activity of these compounds against hCA I can be presented as follows: According to structure-activity relationships, the presence of [(4-sulfamoylphenyl)am ino]carbonyl substitute at position 3 of 1-(4-chlorophenyl)pyridazin-4(1H)-one (10d) is beneficial for hCA I inhibitory activity. The order of activity of phenyl-substituted pyridazine-4(1H)-one derivatives can be presented as 10d > 10a > 10b > 10c. Thus, replacement of 4-chlorobenzene by 3-tolyl resulted in compound 10a with much lower activity than 10d, followed by 4-fluoro-(10b) and 3-chlorobenzene derivatives (10c). The replacement of pyridazine derivative 10d by 3-(2-hydroxy-3,5-dimethylphenyl)-N-(4-sulfamoylphenethyl)-1H-pyrazole-5-carboxamide (5e) decreased some activity. Removing both methyl groups from 5e led to less-active compound (5a) compared with the previous one. Introduction of 2hydroxy-4-methylbenzene to position 3 of pyrazole moiety as well as [(4-sulfamoylphenyl) amino]carbonyl substitute at position 5 (4b) further decreased the activity against hCA I, but this was still among the active compounds. The presence of 2-hydroxyphenyl substituent at position 3 of pyrazole ring and [N-(4-aminosulfonyl)phenyl]-5-carboxamide (4a) appeared to be detrimental. It is interesting to notice that for [4-(aminosulfonyl)phenyl]ethyl-1H-pyrazole carboxamides (compounds 5a-5f), the presence of 2-OH and 3,5 dimethyl substitution of phenyl ring, respectively, is very important for hCA I inhibitory activity, while for [4-(aminosulfonyl)phenyl]-1H-pyrazole-5-carboxamides (4a-4f) a positive role is played by the presence of 2-OH, 4-Me substitution of phenyl ring. The K i values against cytosolic hCA II isoform were in the range of 3.3 to 866.7 nM, and the order of activity was . Compound 15 displayed the highest activity among others against hCA II, with Ki at 3.3 nM compared to AAZ (Ki at 12.1 nM). Furthermore, this compound was the most selective one, with a selectivity index (SI) of 219.9 towards hCA I, 2.0 compared to hCA IX and 24.4 towards hCA XII isoforms. Four compounds displayed higher activity against cytosolic hCA II isoform than reference drug AAZ.

Molecular Docking Studies
In order to predict the possible mechanism of inhibition of the tested compounds, molecular docking studies were performed on the most active compounds 5d, 5e, 10d and 15 as representative of the whole set of compounds. Human CAs isoforms have analogous active sites containing His94, His96 and His119 as conserved residues, which act as zinc ligands, and conserved residues Thr199 and Glu105, which act as ''gate keepers'' [42][43][44][45]. Nevertheless, these isoforms differ in the residues mostly in the middle and to the exit of the active site cavity. Table 2 presents the results of the molecular docking studies of the tested compounds on hCA I, II, IX and XII isoforms. According to the docking results, all tested compounds bind the enzymes, chelating the Zn (II) ion, in a deprotonated form, as anions (negative nitrogen of the sulfonamide group) [46].

Molecular Docking Studies
In order to predict the possible mechanism of inhibition of the tested compounds, molecular docking studies were performed on the most active compounds 5d, 5e, 10d and 15 as representative of the whole set of compounds. Human CAs isoforms have analogous active sites containing His94, His96 and His119 as conserved residues, which act as zinc ligands, and conserved residues Thr199 and Glu105, which act as ''gate keepers" [42][43][44][45]. Nevertheless, these isoforms differ in the residues mostly in the middle and to the exit of the active site cavity. Table 2 presents the results of the molecular docking studies of the tested compounds on hCA I, II, IX and XII isoforms. According to the docking results, all tested compounds bind the enzymes, chelating the Zn (II) ion, in a deprotonated form, as anions (negative nitrogen of the sulfonamide group) [46]. Docking studies revealed that the selectivity profile as well as the inhibition mode of some compounds to each isoform depend on the variances in the active sites of the enzymes. More precisely, the nature of the amino acids of the active site of the enzymes affects the inhibition profile of the compounds because they play an important role in the final conformation adopted and interactions formed by compounds within the enzyme active site. For example, compound 15, with K i value for hCA I enzyme of 725.6 nM and K i value for hCA II of 3.3 nM, adopts a different conformation when binding both hCAs. This is probably due to the presence of the hydrophobic residue Phe131 in hCA II enzyme in contrast to the minor residue Leu131 in hCA I. Despite the fact that this smaller residue in the hCA I enzyme allows ligands to freely enter the active site of the enzyme in compound 15 with a bulky part, it is not favorable. On the other hand, residue Phe131 interacts hydrophobically with compound 15, increasing the enzyme-ligand interactions and consequently the inhibition and selectivity of the compound to this isoform ( Figure 4A). Furthermore, compound 15 forms a hydrogen bond between the sulfonamide and the backbone of Thr200 to both isoforms and another H-bond between N-atom and residue Gln92 to isoform hCA II, which further stabilizes the complex and explains the high inhibition potency ( Figure 4B,C). Docking studies revealed that the selectivity profile as well as the inhibition mode of some compounds to each isoform depend on the variances in the active sites of the enzymes. More precisely, the nature of the amino acids of the active site of the enzymes affects the inhibition profile of the compounds because they play an important role in the final conformation adopted and interactions formed by compounds within the enzyme active site. For example, compound 15, with Ki value for hCA I enzyme of 725.6 nM and Ki value for hCA II of 3.3 nM, adopts a different conformation when binding both hCAs. This is probably due to the presence of the hydrophobic residue Phe131 in hCA II enzyme in contrast to the minor residue Leu131 in hCA I. Despite the fact that this smaller residue in the hCA I enzyme allows ligands to freely enter the active site of the enzyme in compound 15 with a bulky part, it is not favorable. On the other hand, residue Phe131 interacts hydrophobically with compound 15, increasing the enzyme-ligand interactions and consequently the inhibition and selectivity of the compound to this isoform ( Figure 4A). Furthermore, compound 15 forms a hydrogen bond between the sulfonamide and the backbone of Thr200 to both isoforms and another H-bond between N-atom and residue Gln92 to isoform hCA II, which further stabilizes the complex and explains the high inhibition potency ( Figure 4B, 4C).  On the other hand, compound 5d differs from compound 15 by the presence of an ethyl -longer chain. This longer chain provides flexibility to the compound, and enables it to avoid the steric hindrance of the bulky residue Phe131 hCA II isoform, increasing the inhibition potency. This is illustrated in Figure 5 where compound 5d in both hCA II and hCA XII isoforms adopts a conformation that favors the interactions with both active sites of the isoforms, increasing the stability of the complex and the inhibition potency ( Figure 5C,D). In both structures, the negative nitrogen of the sulphonamide group chelates the Zn (II) ion and forms hydrogen bonds. In isoform hCA I, the one oxygen atom of the sulphonamide group forms a hydrogen bond with residue Thr199, while in isoform hCA XII, it forms two hydrogen bonds with residues Thr200 and Thr199. Moreover, in isoform hCA XII, the N atom of heterocycle ring forms another H-bond with residue Ser135. Additionally, the benzene moiety interacts hydrophobically with residues Val121 and Leu198 ( Figure 5A,B). inhibition potency. This is illustrated in Figure 5 where compound 5d in both hCA II and hCA XII isoforms adopts a conformation that favors the interactions with both active sites of the isoforms, increasing the stability of the complex and the inhibition potency ( Figure  5C, 5D). In both structures, the negative nitrogen of the sulphonamide group chelates the Zn (II) ion and forms hydrogen bonds. In isoform hCA I, the one oxygen atom of the sulphonamide group forms a hydrogen bond with residue Thr199, while in isoform hCA XII, it forms two hydrogen bonds with residues Thr200 and Thr199. Moreover, in isoform hCA XII, the N atom of heterocycle ring forms another H-bond with residue Ser135. Additionally, the benzene moiety interacts hydrophobically with residues Val121 and Leu198 (Figure 5A, 5B). The docking pose of compound 10d into the active site of hCA I isoform revealed the probable reason for its high inhibition profile. As illustrated in Figure 6, compound 10d binds hCA I in the same manner as AAZ, with the negative nitrogen of the sulphonamide group chelating the Zn (II) ion. However, the benzene moiety of the compound additionally interacts hydrophobically with residues His200 and Tyr204, increasing the stability of the enzyme-compound complex, probably explaining its lower Ki value in accordance with that of reference drug AAZ. The docking pose of compound 10d into the active site of hCA I isoform revealed the probable reason for its high inhibition profile. As illustrated in Figure 6, compound 10d binds hCA I in the same manner as AAZ, with the negative nitrogen of the sulphonamide group chelating the Zn (II) ion. However, the benzene moiety of the compound additionally interacts hydrophobically with residues His200 and Tyr204, increasing the stability of the enzyme-compound complex, probably explaining its lower K i value in accordance with that of reference drug AAZ.

Materials and Methods
All used solvents were of analytical grade. The 1 H and 13 C spectra were recorded at 298 K on a Bruker AVANCE DRX-500 spectrometer (Rheinstetten, Germany) (at 500 and 125 MHz) in solutions of (TMS as internal reference): DMSO-d6, CF3COOD and DMSO-d6 + 5%(mass)CF3SO3H. Chemical shifts (δ) are reported in ppm, and coupling constants (J) in Hz. Chemical ionization at atmospheric pressure mass spectra (APCI) were measured with an Agilent 1200 LC/MSD SL system (Waldbronn, Germany) equipped with DAD/ELSD/LSMS-6120 diode matrix and mass-selective detector, scan range m/z 80-1000. Melting points were determined in a Fischer-Johns melting point apparatus (Pittsburgh,

Materials and Methods
All used solvents were of analytical grade. The 1 H and 13 C spectra were recorded at 298 K on a Bruker AVANCE DRX-500 spectrometer (Rheinstetten, Germany) (at 500 and 125 MHz) in solutions of (TMS as internal reference): DMSO-d 6 , CF 3 COOD and DMSO-d 6 + 5%(mass)CF 3 SO 3 H. Chemical shifts (δ) are reported in ppm, and coupling constants (J) in Hz. Chemical ionization at atmospheric pressure mass spectra (APCI) were measured with an Agilent 1200 LC/MSD SL system (Waldbronn, Germany) equipped with DAD/ELSD/LSMS-6120 diode matrix and mass-selective detector, scan range m/z 80-1000. Melting points were determined in a Fischer-Johns melting point apparatus (Pittsburgh, USA) and are uncorrected. Elemental analysis was carried out in the Analytical Laboratory of the Institute of Bioorganic and Petrochemistry of the National Academy of Sciences of Ukraine by manual methods: the carbon and hydrogen contents were determined using the Pregl gravimetric method, nitrogen was determined using the Duma's gasometrical micromethod, and sulfur was determined by the Scheininger titrimetric method. To a solution of 2.5 mmol of the corresponding 4-oxo-4H-chromene-2-carboxylic acid 1 in 15 mL of acetonitrile, 2.5 mmol of CDI was added, and the mixture was heated at 50 • C until completion of carbon dioxide evolution. After cooling to ambient temperature, 0.374 g (2.2 mmol) of 4-aminobenzenesulfonamide or 0.44 g (2.2 mmol) 4-(2aminoethyl)benzenesulfonamide was added to reaction mixture, which was then heated in a sealed vial for 10 h at 100 • C. After cooling to ambient temperature, the mixture was evaporated to dryness under reduced pressure. The residue was treated with 15 ml of 5% water sodium bicarbonate solution and stirred for 2 h on an ultrasonic stirrer at cooling (0-5 • C). The formed precipitate was filtered off, washed with water (10 mL) and then with ethanol (5 mL), and suspended in 15 mL of ethanol. Then, 0.63 g (6 mmol) of hydrazine hydrate was added to this suspension while stirring and the mixture was allowed to reflux for 3 h. After the cooling to ambient temperature, the resulted solution was left for 12 h, then the formed precipitate was filtered off, washed with water (10 mL), then ethanol (5 mL) and finally crystallized with DMF-ethanol mixture to yield 4a-d,f and 5a, b, d- To the suspension of 2 mmol of 8 in 20 mL of dry chloroform 2 mL of freshly distilled thionyl chloride and one drop of dry DMF was added. The mixture was heated for 1 h at 45-50 • C and then evaporated to dryness under reduced pressure. The residue was treated with 5 mL of dry toluene and the mixture was evaporated to dryness under reduced pressure again. The residues were used for the next step without any additional purification.
high concentration of inhibitor, being in the range of 16-25%. However, the background activity from the uncatalyzed reaction was always subtracted when IC50 values were obtained by using the data analysis software for the stopped-flow instrument. Enzyme concentrations ranged between 5 nM and 12 nM. 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 the inhibitor (0.1 mM) were prepared in distilled-deionized water, and dilutions up to 0.01 nM were performed thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to the assay, 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 and represented the mean from at least three different determinations. All CA isoforms were recombinant proteins obtained in-house, as reported earlier [2,51,52].

Conclusions
In conclusion, we synthesized and investigated a novel series of sulfonamides incorporating pyrazole-and pyridazinecarboxamide moieties for their effective inhibition against different and most relevant human carbonic anhydrase isoforms such as the ubiquitous hCA I, hCA II, and tumor associated isoforms hCA IX and XII, which are involved in a variety of diseases such as glaucoma, retinitis pigmentosa, epilepsy, and tumors. Compound 4c showed a good selectivity index of 26 on hCA IX compared to hCA I and two times compared to hCA II. On the other hand, compound 10f was shown to be the most active on hCA II with an SI of 236 compared to hCA I and 8.7 compared to hCA IX. These interesting features make them good candidates for preclinical evaluation in glaucoma or various tumors in which the two enzymes (hCA II and hCA IX) are involved. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds.
Supplementary Materials: The following are available online: Copies of 1 H, 13 C NMR and LCMS Spectra of Products (Figures S1A-S19C).