Investigation on Hydrazonobenzenesulfonamides as Human Carbonic Anhydrase I, II, IX and XII Inhibitors

A small series of hydrazonobenzenesulfonamides was designed, synthesized and studied for their human carbonic anhydrase (hCA) inhibitory activity. The synthesized compounds were evaluated against hCA I, II, IX and XII isoforms using acetazolamide (AAZ) as the standard inhibitor. Various hydrazonosulfonamide derivatives showed inhibitory activity at low nanomolar levels with selectivity against the cytosolic hCA II isoform, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The most potent and selective hydrazones 8, 9, 10, 11, 19 and 24 were docked into isoforms I, II, IX and XII to better understand their activity and selectivity for the different CA isoforms.


Introduction
Human carbonic anhydrases (hCAs) belong to the alpha CA class and are zinccontaining enzymes which catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton (CO 2 + H 2 O HCO 3 − + H + ). To date, 15 diverse isoforms have been identified, which differ in molecular structure, catalytic properties, organ and tissue distribution as well as subcellular localization, and ability to interact with different inhibitor classes [1]. Different physiological and pathological functions are regulated by hCAs, such as electrolyte secretion and pH regulation, gluconeogenesis, osteoclast functionality and tumorigenicity [1,2]. Thus, hCA inhibitors can be used for the treatment of glaucoma, obesity, neuropathic pain, arthritis, Alzheimer's disease and cancer. The cytosolic isoforms hCA I and hCA II are widely expressed in erythrocytes, eyes, gastrointestinal tract, osteoclasts and kidney cells [2]. Some hCA II inhibitors are currently used as anti-glaucoma drugs and as mild diuretics [3]. However, the hCA I and II isoforms are considered off-targets in the development of inhibitors for the transmembrane, tumor-associated hCA IX and XII isoforms [4]. Despite the prominent role of hCA II in the regulation of intraocular pressure, anti-glaucoma agents also decrease bicarbonate and aqueous humor secretion by inhibition of hCA I, IV and XII [5][6][7]. hCA II activity has been also associated with neurodegeneration and aging processes [8] and altered hCA II expression constitutes a biomarker of carcinogenesis in some human tumors [9]. The transmembrane hCA IX and XII, among others, are involved in tumorigenesis (hCA IX and XII) and glaucoma (hCA II and XII). Under physiological conditions, the expression of hCA isoform IX is restricted to the basolateral membrane of epithelial cells of the gastric, intestinal and gallbladder mucosa [10] as well as in specialized cells of the ovaries and testes, in some sites of the peripheral and central nervous systems and in mesodermal cells of the placenta and cartilaginous tissues of joints [11,12]. A very low expression of hCA XII is present in tissues such as kidney, pancreas, colon, prostate, ovary, testis, endometrium, lung, brain and colon [13][14][15][16] and is associated with rapid tumor growth, metastasis and infiltration into surrounding normal tissues [17][18][19].
As a continuation of our efforts in the design and synthesis of new potent and selective hCA Is [37][38][39][40][41], this study presents a small library of hydrazonobenzenesulfonamidebased compounds. The target compounds are endowed with a benzenesulfonamide-type zinc-binding group (ZBG) connected by a carbonyl bridge to a piperidine ring, which provides an appropriate orientation and connects the two tails with both the hydrophilic and hydrophobic halves of the active site. The hydrazone moiety can also improve the flexibility and hydrophilicity of the designed compounds to improve hCA inhibitory activity through favorable interactions with the specific residues in the hydrophilic part of the active site.
Molecules 2022, 27, x FOR PEER REVIEW 2 of 20 mucosa [10] as well as in specialized cells of the ovaries and testes, in some sites of the peripheral and central nervous systems and in mesodermal cells of the placenta and cartilaginous tissues of joints [11,12]. A very low expression of hCA XII is present in tissues such as kidney, pancreas, colon, prostate, ovary, testis, endometrium, lung, brain and colon [13][14][15][16] and is associated with rapid tumor growth, metastasis and infiltration into surrounding normal tissues [17][18][19].
As a continuation of our efforts in the design and synthesis of new potent and selective hCA Is [37][38][39][40][41], this study presents a small library of hydrazonobenzenesulfonamidebased compounds. The target compounds are endowed with a benzenesulfonamide-type zinc-binding group (ZBG) connected by a carbonyl bridge to a piperidine ring, which provides an appropriate orientation and connects the two tails with both the hydrophilic and hydrophobic halves of the active site. The hydrazone moiety can also improve the flexibility and hydrophilicity of the designed compounds to improve hCA inhibitory activity through favorable interactions with the specific residues in the hydrophilic part of the active site.

Chemistry
The synthesis of hydrazonobenzenesulfonamide derivatives 5-29 reported here was performed as highlighted in Scheme 1, starting from 4-sulfamoylbenzoic acid 1. By using our previously reported synthetic procedure [42], the coupling of sulfanilamide 1 with ethyl piperidine-4-carboxylate 2 in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) in a dry acetonitrile solution (MeCN) produced ethyl 1-(4-sulfamoylbenzoyl)piperidine-4-carboxylate 3. This ester was treated with hydrazine hydrate in absolute ethanol (EtOH) to obtain the corresponding 4-(4-(hydrazinecarbonyl)piperidine-1-carbonyl)benzenesulfonamide 4. Treatment of the hydrazide 4 with an equimolecular amount of appropriately substituted aldehydes in EtOH solution furnished the hydrazones 5-29 with yields of 52-98%. The spectral data are consistent with the assigned structure.  According to the literature, the presence of a downfield resonating (10.89-11.73 ppm) CH=N signal can exclusively account for the formation of E isomers [43]. The signal is split due to hindered amide rotation around the C-N bond of the acyl fragment that generates two conformers [43].

Carbonic Anhydrase Inhibition
Sulfonamidohydrazones 5-29 were tested for their ability to inhibit four human (h) CA isoforms involved in crucial physiologic/pathologic processes: the cytosolic hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Table 1 shows the inhibition data of the derivatives reported here and the sulfonamide acetazolamide (AAZ) as the standard inhibitor tested using a stopped-flow CO2 hydrase assay [44]. According to the literature, the presence of a downfield resonating (10.89-11.73 ppm) CH=N signal can exclusively account for the formation of E isomers [43]. The signal is split due to hindered amide rotation around the C-N bond of the acyl fragment that generates two conformers [43].

Carbonic Anhydrase Inhibition
Sulfonamidohydrazones 5-29 were tested for their ability to inhibit four human (h) CA isoforms involved in crucial physiologic/pathologic processes: the cytosolic hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Table 1 shows the inhibition data of the derivatives reported here and the sulfonamide acetazolamide (AAZ) as the standard inhibitor tested using a stopped-flow CO2 hydrase assay [44]. According to the literature, the presence of a downfield resonating (10.89-11.73 ppm) CH=N signal can exclusively account for the formation of E isomers [43]. The signal is split due to hindered amide rotation around the C-N bond of the acyl fragment that generates two conformers [43].

Carbonic Anhydrase Inhibition
Sulfonamidohydrazones 5-29 were tested for their ability to inhibit four human (h) CA isoforms involved in crucial physiologic/pathologic processes: the cytosolic hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Table 1 shows the inhibition data of the derivatives reported here and the sulfonamide acetazolamide (AAZ) as the standard inhibitor tested using a stopped-flow CO2 hydrase assay [44]. According to the literature, the presence of a downfield resonating (10.89-11.73 ppm) CH=N signal can exclusively account for the formation of E isomers [43]. The signal is split due to hindered amide rotation around the C-N bond of the acyl fragment that generates two conformers [43].

Carbonic Anhydrase Inhibition
Sulfonamidohydrazones 5-29 were tested for their ability to inhibit four human (h) CA isoforms involved in crucial physiologic/pathologic processes: the cytosolic hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Table 1 shows the inhibition data of the derivatives reported here and the sulfonamide acetazolamide (AAZ) as the standard inhibitor tested using a stopped-flow CO2 hydrase assay [44]. According to the literature, the presence of a downfield resonating (10.89-11.73 ppm) CH=N signal can exclusively account for the formation of E isomers [43]. The signal is split due to hindered amide rotation around the C-N bond of the acyl fragment that generates two conformers [43].

Carbonic Anhydrase Inhibition
Sulfonamidohydrazones 5-29 were tested for their ability to inhibit four human (h) CA isoforms involved in crucial physiologic/pathologic processes: the cytosolic hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Table 1 shows the inhibition data of the derivatives reported here and the sulfonamide acetazolamide (AAZ) as the standard inhibitor tested using a stopped-flow CO2 hydrase assay [44].  The following structure activity relationship (SAR) may be noted regarding the inhibition data in Table 1.
hCA IX was generally effectively inhibited by the new hydrazones reported here. The best inhibitory effects were observed using compounds 10, 11, 17, 19 and 23, which had The following structure activity relationship (SAR) may be noted regarding the inhibition data in Table 1.
hCA IX was generally effectively inhibited by the new hydrazones reported here. The best inhibitory effects were observed using compounds 10, 11, 17, 19 and 23, which had inhibition constants ranging between 15.4 and 23.4 nM (better than the reference inhibitor AAZ, which has a KI of 25 nM against this isoform). These hydrazones contain the following benzylidene moieties: 2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl, 2-methoxynaphthyl, 3-fluorophenyl, 2-chlorophenyl 2,4,5-trimetoxyphenyl, 2,6-dichlorobenzyl and 4-(dimethy lamino)phenyl benzylidene moieties. Another group of derivatives, As for hCA I and hCA II, small structural changes between hydrazones led to marked differences in their inhibitory activity. For example, a large difference in inhibitory potency was observed for the two isomeric methoxyphenyl-substituted compounds 8 and 9, with the 3-methoxyphenyl derivative being an 8-fold better inhibitor than its isomer with the 4-methoxyphenyl group.
A rather similar situation as was noted above for hCA IX has also been observed for the inhibition of the second transmembrane isoform investigated here, hCA XII. Almost all hydrazone derivatives showed efficient inhibition of this isoform (KIs in the range of 8.05-68.7 nM). The most active hydrazones incorporate the following benzylidene moieties: 2-methoxyphenyl, 2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl, naphthyl and 3-fluorophenyl. The shift of the 2-methoxy group in compound 7 to the 4-position to produce hydrazone 9 results in a approx. 12-fold decrease in activity.
The unsubstituted hydrazone 5 showed no significant difference in activity towards the four tested hCA isoforms. The introduction of a 4-methyl group gave the highly hCA II-selective hydrazone 6. The replacement of the methyl by a 4-methoxy group produced a reduction in hCA II selectivity (compound 9). The shift of the methoxy group to the 2-position caused a reduction of hCA II selectivity and enhancement of hCA XII selectivity. The comparison of compound 8 and 9 activities indicated that the shift of the methoxy group to the 3-position resulted in an approx. 10-and 5-fold increase in activity against hCA IX and hCA XII, respectively, as well as an approx. 10-fold reduction in hCA II activity. The 2,4-dimethoxybenzilidene (compound 10) and the 3,4-dimethoxybenzilidene (compound 11) moieties produced high activity and selectivity against the tumor-associated hCA IX and hCA XII isoforms with hydrazone 10 as the most selective of the series towards hCA XII. The introduction of a 2-bromine atom to compound 11 (hydrazone 15) produced a slight decrease in activity on all the tested hCA isoforms. The introduction of a second methoxy group to the 5-position on compound 7 to give hydrazone 12 led to a reduction in hCA XII activity and to an improvement in hCA II activity as compared to 7. Similar inhibition data were shown by hydrazone 13 bearing a third methoxy group at the 4-position. The shift of the methoxy group to the 3-position (hydrazone 14) increased the selectivity towards hCA IX and hCA XII. The naphthyl derivative 16 showed about a 420-fold hCA XII/hCA I and 22-fold hCA XII/CA II increase in selectivity. The introduction of a methoxy group to produce hydrazone 17 increased hCA IX in activity and reduced hCA XII selectivity as compared with 16. The chlorine-substituted hydrazones displayed high hCA II selectivity except for the 3-chlorobenzylidene derivative 24 that showed about a 3-fold hCA XII/hCA II and 20-fold hCA XII/hCA I increase in selectivity.
The replacement of the methyl group on compound 6 by a 4-trifluoromethyl group reduced hCA I and hCA II selectivity (compound 18). The replacement of the 4-trifluoromethyl with a 4-fluorine atom (hydrazone 20) caused a further reduction in hCA I activity and increase of inhibitory activity against the other three hCA isoforms. The shift of the fluorine into the 3-position (hydrazone 19) produced a large increase in both activity and selectivity towards hCA IX and hCA XII. The 3-nitro-substituted hydrazone 21 showed a reduction in activity against hCA I, hCA IX and hCA XII as compared to the 3-fluorine-substituted hydrozone, while the high hCA II selectivity was maintained.

Molecular Docking
Due to their potent activity profile, the binding mode of compounds 8, 9, 10, 11, 19 and 24 to hCA isoforms II, IX and XII was investigated by docking simulations in order to rationalize the hCA isoform selectivity observed for these derivatives. All the compounds occupied the active sites of the studied hCA isoforms with the NHand C=O groups of the sulfonamide moiety involved in the coordination of the Zn ion and in H-bonds with Thr199 or 200, respectively. As discussed above, derivative 9 showed the best selectivity profile towards hCA II which decreased when the 4-methoxy group was shifted to the 3-position as in compound 8, for which an increase in affinity towards hCA IX and hCA XII was observed. Both compounds might occupy the hCA II-binding pocket by engaging hydrophobic interactions with Phe131 and a H-bond with Asn67 ( Figure 1A). The main differences between the two binding poses rely on the different orientations assumed by the benzensulfonamide moiety and the tail of the molecules. More specifically, in compound 8 the former could be involved in hydrophobic contacts with Val121 while no interactions were observed for the tail portion. In the case of inhibitor 9, the arylsulfonamide moiety might be stabilized by π-stacking interactions with His94, while the tail is oriented towards Leu57 which establishes hydrophobic contacts with the methoxy group and might contribute to further stabilization of the binding to the hCA II active site. When bound to hCA IX ( Figure 1B), hydrazone 9 might form hydrophobic contacts with Pro199 through the phenyl ring of the tail, while additional interactions were observed for the 3-methoxy-substituted analogue 8 which might elicit several hydrophobic contacts with Leu198, Val121, Pro202, Gln2 and Val19 and a H-bond with Trp5, thus explaining its higher affinity towards hCA IX compared to compound 9. Despite these two inhibitors showing similar binding modes to the hCA XII active site ( Figure 1C), the 3-methoxy group of hydrazone 8 allowed the formation of a convenient H-bond with His64 that was not observed for hydrazone 9 and this might be the reason for the higher inhibitory activity of derivative 8 towards hCA XII.
Hydrazones 10, 11, 19 and 24 displayed a good selectivity towards the tumor-associated isoforms hCA IX and hCA XII over hCA II. As shown in Figure 2, these hydrazones might bind the hCA II active site by establishing hydrophobic interactions with Phe131 through the piperidine ring, which might also be stabilized by additional contacts with Pro202 and Ile91 in the case of compounds 11 and 24, respectively. The main differences between the two binding poses rely on the different orientations assumed by the benzensulfonamide moiety and the tail of the molecules. More specifically, in compound 8 the former could be involved in hydrophobic contacts with Val121 while no interactions were observed for the tail portion. In the case of inhibitor 9, the arylsulfonamide moiety might be stabilized by π-stacking interactions with His94, while the tail is oriented towards Leu57 which establishes hydrophobic contacts with the methoxy group and might contribute to further stabilization of the binding to the hCA II active site. When bound to hCA IX ( Figure 1B), hydrazone 9 might form hydrophobic contacts with Pro199 through the phenyl ring of the tail, while additional interactions were observed for the 3-methoxysubstituted analogue 8 which might elicit several hydrophobic contacts with Leu198, Val121, Pro202, Gln2 and Val19 and a H-bond with Trp5, thus explaining its higher affinity towards hCA IX compared to compound 9. Despite these two inhibitors showing similar binding modes to the hCA XII active site ( Figure 1C), the 3-methoxy group of hydrazone 8 allowed the formation of a convenient H-bond with His64 that was not observed for hydrazone 9 and this might be the reason for the higher inhibitory activity of derivative 8 towards hCA XII.
Hydrazones 10, 11, 19 and 24 displayed a good selectivity towards the tumor-associated isoforms hCA IX and hCA XII over hCA II. As shown in Figure 2, these hydrazones might bind the hCA II active site by establishing hydrophobic interactions with Phe131 through the piperidine ring, which might also be stabilized by additional contacts with Pro202 and Ile91 in the case of compounds 11 and 24, respectively. Other hydrophobic interactions were observed between the benzensulfonamide moiety of hydrazone 11 and Leu198 and Val121. Furthermore, hydrazones 10, 19 and 24 might engage a H-bond between the side chain of Asn67 and the amide carbonyl group. No relevant interactions were observed between the tails of derivatives 10, 11, 19 and 24 and the rim of the hCA II active site. Conversely, a major stabilization of these molecules' tails was observed in the docking simulations performed with the hCA IX and hCA XII binding pockets, which can explain the major affinity of these compounds towards the tumor-associated isoforms. Specifically, the four inhibitors might assume similar binding orientations within the hCA IX active site (Figure 3), with the benzensulfonamide moiety and the piperidine ring engaging hydrophobic contacts with Val121, Leu198 and Val131, while the tails occupy a region lined by Gln2, Ser3, Trp5, Val19, Ser20, Pro198 and Pro199, which are involved in hydrophobic interactions with these inhibitors. Moreover, hydrazones 10 and 11 could form a H-bond with Gln92 through the amide carbonyl group ( Figure 3A and 3B), while the fluorine atom of compound 19 might elicit halogen bonds with Val19 and Gln2 ( Figure 3C). These additional interactions exerted by hydrazones 10, 11 and 19 might explain their high potency towards hCA IX compared to derivative 24. Other hydrophobic interactions were observed between the benzensulfonamide moiety of hydrazone 11 and Leu198 and Val121. Furthermore, hydrazones 10, 19 and 24 might engage a H-bond between the side chain of Asn67 and the amide carbonyl group. No relevant interactions were observed between the tails of derivatives 10, 11, 19 and 24 and the rim of the hCA II active site. Conversely, a major stabilization of these molecules' tails was observed in the docking simulations performed with the hCA IX and hCA XII binding pockets, which can explain the major affinity of these compounds towards the tumor-associated isoforms. Specifically, the four inhibitors might assume similar binding orientations within the hCA IX active site (Figure 3), with the benzensulfonamide moiety and the piperidine ring engaging hydrophobic contacts with Val121, Leu198 and Val131, while the tails occupy a region lined by Gln2, Ser3, Trp5, Val19, Ser20, Pro198 and Pro199, which are involved in hydrophobic interactions with these inhibitors. Moreover, hydrazones 10 and 11 could form a H-bond with Gln92 through the amide carbonyl group ( Figure 3A,B), while the fluorine atom of compound 19 might elicit halogen bonds with Val19 and Gln2 ( Figure 3C). These additional interactions exerted by hydrazones 10, 11 and 19 might explain their high potency towards hCA IX compared to derivative 24.  Regarding hCA XII (Figure 4), compounds 10, 11, 19 and 24 could establish hydrophobic contacts with Leu198 and Val121 through their phenyl moiety and with Ala131 through the piperidine ring. Moreover, derivatives 11 and 24 might elicit H-bonds with Thr91 and Ser132 through one of their carbonyl group and hydrazone nitrogen ( Figure 4B and 4D), while compound 10 might engage H-bonds with Trp5 and Tyr20 through its two methoxy groups ( Figure 4A). Conversely, hydrazone 19 interacts with Gln92 and Ser135 by forming hydrogen bonds with its two carbonyl groups ( Figure 4C). Finally, hydrophobic interactions were detected between: i) His64 and the 2-methoxy group of compound 10; ii) Tyr123 and the 3-methoxy group of hydrazone 11; and iii) Pro202 and the 3-fluorophenyl ring of compound 19. Regarding hCA XII (Figure 4), compounds 10, 11, 19 and 24 could establish hydrophobic contacts with Leu198 and Val121 through their phenyl moiety and with Ala131 through the piperidine ring. Moreover, derivatives 11 and 24 might elicit H-bonds with Thr91 and Ser132 through one of their carbonyl group and hydrazone nitrogen ( Figure 4B,D), while compound 10 might engage H-bonds with Trp5 and Tyr20 through its two methoxy groups ( Figure 4A). Conversely, hydrazone 19 interacts with Gln92 and Ser135 by forming hydrogen bonds with its two carbonyl groups ( Figure 4C). Finally, hydrophobic interactions were detected between: (i) His64 and the 2-methoxy group of compound 10; (ii) Tyr123 and the 3-methoxy group of hydrazone 11; and (iii) Pro202 and the 3-fluorophenyl ring of compound 19.

Chemistry
All commercially available solvents and reagents were used without further purification. 1 H NMR spectra were recorded using an Inova 500 spectrometer (Varian, Palo Alto, CA, USA). 13 C NMR spectra were recorded using a Bruker Advance III HD 600 (Bruker, Bremen, Germany) at 126 MHz. The chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane (TMS), which was used as an internal standard. The spectra were recorded in hexadeuteriodimethylsulphoxide (DMSO-d6). Infrared spectra were recorded using a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol mulls. The main bands are given in cm −1 . Positive-ion electrospray ionization (ESI) mass spectra were recorded using a double-focusing MAT 95 instrument (Finnigan, Waltham, MA, USA) with BE geometry. Melting points (mp) were determined with a SMP1 Melting Point apparatus (Stuart Scientific, Stone, UK) and are uncorrected. All products reported showed 1 H NMR spectra in agreement with the assigned structures. The purity of the tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Chemistry Department of the University of Ferrara with a MT-5 CHN recorder elemental analyzer (Yanagimoto, Kyoto, Japan) and the values found were within 0.4% of theoretical values. 4-(4-(Hydrazinecarbonyl)piperidine-1-carbonyl)benzenesulfonamide (4) was synthesized as previously reported [42]

Chemistry
All commercially available solvents and reagents were used without further purification. 1 H NMR spectra were recorded using an Inova 500 spectrometer (Varian, Palo Alto, CA, USA). 13 C NMR spectra were recorded using a Bruker Advance III HD 600 (Bruker, Bremen, Germany) at 126 MHz. The chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane (TMS), which was used as an internal standard. The spectra were recorded in hexadeuteriodimethylsulphoxide (DMSO-d 6 ). Infrared spectra were recorded using a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol mulls. The main bands are given in cm −1 . Positive-ion electrospray ionization (ESI) mass spectra were recorded using a double-focusing MAT 95 instrument (Finnigan, Waltham, MA, USA) with BE geometry. Melting points (mp) were determined with a SMP1 Melting Point apparatus (Stuart Scientific, Stone, UK) and are uncorrected. All products reported showed 1 H NMR spectra in agreement with the assigned structures. The purity of the tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Chemistry Department of the University of Ferrara with a MT-5 CHN recorder elemental analyzer (Yanagimoto, Kyoto, Japan) and the values found were within 0.4% of theoretical values. 4-(4-(Hydrazinecarbonyl)piperidine-1-carbonyl)benzenesulfonamide (4) was synthesized as previously reported [42].

Carbonic Anhydrase Inhibition
An Applied Photophysics stopped-flow instrument was used for assaying the CAcatalyzed CO 2 hydration activity [44]. Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM HEPES (pH 7.5) as the buffer, and 20 mM Na 2 SO 4 (for maintaining a constant ionic strength), following the initial rates of the CA-catalyzed 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 uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in 10% DMSO aqueous solution and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to the assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier [45][46][47] and represent the mean from at least three different determinations.
All CA isoforms were recombinant ones obtained in-house as reported earlier. Their concentrations in the assay system were 5.7-11.9 nM [41,48,49].

Molecular Docking
Molecular docking simulations were carried out by using the crystal structures of CAII (PDB ID 3HS4) [50], CAIX (PDB ID 3IAI) [51] and CAXII (PDB ID 1JD0) [52] in complex with the inhibitor AAZ that was used as a reference compound in this study. The protein structures were prepared as described elsewhere [53]. Ligand structures were submitted to a preliminary conjugate gradient minimization by using AMMP tool implemented in VEGA ZZ software [54]. The minimized structures underwent a conformational search by following a Monte Carlo procedure as implemented in VEGA ZZ. The lowest energy conformation was selected and further optimized by the PM7 semi-empirical method using MOPAC 2016 [55]. The prepared ligands were docked to the CAII, CAIX and CAXII binding pockets by means of GOLD V 5.8.1 [56]. The binding site was defined in order to include all the residues within 10 Å from the co-crystallized ligand. In considering the conserved binding motif of the sulfonamide portion, a scaffold constraint was applied to restrict the docking solutions to those in which this moiety assumes a binding orientation similar to that observed for the native ligand AAZ in the experimental structures. The option "never dock a ligand when a constraint is physically impossible" was checked. All the ligands were submitted to ten genetic algorithms runs following the docking protocols reported elsewhere [57,58] The best docking pose was chosen for the analysis and representation.

Conclusions
In the present study, we described a small library of benzenesulfonamides as potential hCA Is, endowed with a benzenesulfonamide-type zinc-binding group (ZBG) connected by a carbonyl bridge to a piperidine ring, which provide an appropriate orientation and connections of the two tails with both the hydrophilic and hydrophobic halves of the active site. The 4-methoxyphenylhydrazone 9 showed the best selectivity against hCA II that might be due the tail orientation producing hydrophobic interactions with the enzyme's active site. The dimethoxyphenylhydrazone 10 and 11 as well as the 3-halophenylhydrazones 19 and 24 showed the best selectivity against the tumor-associated hCA IX and hCA XII isoforms. The tails of these compounds established hydrophobic interactions with the active site of hCA IX and hCA XII; conversely, no interaction of the tails was possible in the hCA II binding site. Overall, these results indicate that through small structural changes of the benzylidene tail portion of these hCAI inhibitors, it is possible to modulate both their inhibitory activity and hCA isoform selectivity.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 5-29 are available from the authors.