Synthesis, characterization and in vitro inhibition of metal complexes of pyrazole based sulfonamide on human erythrocyte carbonic anhydrase isozymes I and II

Abstract Sulfonamides represent an important class of biologically active compounds. A sulfonamide possessing carbonic anhydrase (CA) inhibitory properties obtained from a pyrazole based sulfonamide, ethyl 1-(3-nitrophenyl)-5-phenyl-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)-1H-pyrazole-4-carboxylate (1), and its metal complexes with the Ni(II) for (2), Cu(II) for (3) and Zn(II) for (4) have been synthesized. The structures of metal complexes (2–4) were established on the basis of their elemental analysis, 1H NMR, IR, UV–Vis and MS spectral data. The inhibition of two human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes I and II, with 1 and synthesized complexes (2–4) and acetazolamide (AAZ) as a control compound was investigated in vitro by using the hydratase and esterase assays. The complexes 2, 3 and 4 showed inhibition constant in the range 0.1460–0.3930 µM for hCA-I and 0.0740–0.0980 µM for hCA-II, and they had effective more inhibitory activity on hCA-I and hCA-II than corresponding free ligand 1 and than AAZ.


Introduction
Sulfonamides represent a significant class of biologically active compounds that inhibit carbonic anhydrase (CA, EC 4.2.1.1) isoenzymes involved in different pathological and physiological events. Carbonic anhydrases are a super family of metallo enzymes that catalyze the interconversion of carbondioxide (CO 2 ) and water (H 2 O) to bicarbonate (HCO 3 À ) and proton (H þ ) 1-6 . The a-CA is the best known group in the six genetically distinct CA families (a-, b-, c-, d-, e-, fand g-) [7][8][9][10][11][12][13] . A total of 16 isoenzymes have been previously described as members of the a-CA family, and classified according to their subcellular localization. CAs I, II, III, VII and XIII are cytosolic isoenzymes [14][15][16] . CAs VA and VB are localized in the mitochondria [17][18][19] , CA VI is a unique secreted isoenzyme 18,19 CAs IX, XII and XIV are transmembrane proteins 20,21 and CAs IV and XV are GPI-anchored to the cell membrane. 22 1,3,4-Thiadiazole-2-sulfonamide derivatives 23-28 played a critical role in the development of several important classes of pharmacological agents, such as the diuretics with saluretic action, 29,30 benzothiadiazine 31 and high-ceiling diuretics, 32 or the antiglaucoma drugs with CA inhibitory action, among others. 33,34 The coordination chemistry of pyrazoles and other heterocyclic compounds containing nitrogen has attracted much attention because of their interesting structural properties and application in diverse areas. 35 The ease of synthesis of various substituted pyrazoles is the most interesting feature in the incorporation of pyrazole groups in the design of new ligands and offers the opportunity of both electronic and steric control of the properties of the metal complexes.
Generally, metal complexes of heterocyclic sulfonamides are 10-100 times more active as CA inhibitors than the sulfonamides from which they were obtained, assuring affinities for the receptor in the 10 10 to 10 12 range. [36][37][38][39][40][41] Recently, we have reported the metal complexes of some pyrazole-based 1,3,4-thiadiazole-2-sulfonamides show very strong CA inhibitory properties. 42,43 In this work, three new air stable complexes 2, 3 and 4 have been reported, and they can be seen as candidates for further in vivo studies for the treatment of glaucoma which were synthesized by the reaction of M(II) (Ni(II) (2), Cu(II) (3) and Zn(II) (4)) with 1 ( Figure 1). The newly obtained complexes 2, 3 and 4 were characterized by standard procedures in order to assign their structures, and were assayed as CA inhibitors against hCA-I and hCA-II isoenzymes.  (4)) (2.20 Â 10 À4 mol) in 10 mL ethanol was added dropwise with continuous stirring to 20 mL of an ethanolic solution containing 1 (1.1 Â 10 À4 mol). The pH of the reaction mixture was adjusted by addition of 0.1 M aqueous NaOH solution in the range between 6.0 and 7.0. The reaction mixture was stirred overnight at room temperature to give an air stable solid of titled complexes 2, 3 and 4.

Purification of isoenzymes hCA-I and hCA-II from human erythrocytes
In order to purify hCA-I and hCA-II isoenzymes, first, human blood was centrifuged at 1500 rpm for 20 min, and after the removal of the plasma, the erythrocytes were washed with an isotonic solution (0.9% NaCl). After that, the erythrocytes were lysed with 1.5 volume of ice-cold water. The lysate was centrifuged at 20 000 rpm for 30 min to remove cell membranes and non-lysed cells. The pH of the supernatant was adjusted to 8.7 with tris and was then loaded onto an affinity column containing Sepharose-4B-L-tyrosine-p-aminobenzene sulfonamide as the binding group. After extensive washing with 25 mM tris-HCl/22 mM Na 2 SO 4 (pH 8.7), the hCA-I and hCA-II isoenzymes were eluted with 1.0 M NaCl/25 mM Na 2 HPO 4 (pH 6.3) and 0.1 M CH 3 COONa/0.5 M NaClO 4 (pH 5.6). 45,46 The amount of purified protein was estimated by the Bradford method 47 and SDS-PAGE was carried out to determine whether the elute containing the enzyme 48 (Supplemental Figure 1).

Hydratase activity assay
Carbonic anhydrase activity was assayed by following the hydration of CO 2 according to the method described by Wilbur and Anderson. 49 CO 2 -hydratase activity as an enzyme unit (EU) was calculated by using the equation ((t 0 Àt c )/t c ) where t 0 and t c are the times for pH change of the nonenzymatic and the enzymatic reactions, respectively. IC 50 values (the concentration of inhibitor producing a 50% inhibition of CA activity) have been obtained as in vitro for the free ligand (1), newly synthesized compounds (2)(3)(4) and AAZ as the control compound.

Esterase activity assay
Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25 C using a spectrophotometer (CHEBIOS UV-VIS, Roma, Italy) according to the method described in the literature. 50

Determination of K i values
The method for determination of K i values is described elsewhere. [52][53][54][55][56] In the first part of this study, IC 50 values have been obtained as in vitro. IC 50 of the free ligand (1), synthesized complexes (2)(3)(4) and AAZ as the control compound were assayed by the hydrolysis of p-nitrophenylacetate on esterase activities of CA isoenzymes in the presence of various inhibitor concentrations. The absorbance was determined at 348 nm after 3 min. 52 Regression analysis graphs were drawn by plotting inhibitor concentrations versus enzyme activity by using Microsoft Excel Program.
In the second part of the study, enzyme activity was measured in the presence of five different substrate concentrations at each of these inhibitor concentrations (30%, 50% and 70%), and the data were linearized with Lineweaver-Burk plot in order to obtain K i values.

H NMR spectrum of Zn(II) complex (4)
Due to its diamagnetic nature, the Zn(II) complex (4) was studied by NMR spectroscopy and its 1 H NMR spectrum is given in Supplemental Figure 2. The comparison of the 1 H NMR spectrum of the complex (4) with the spectrum of free ligand 44 gave valuable information regarding the coordination mode of ligand during complexation. The peak corresponds to CONH proton found in the spectrum of free ligand 44 at d 12.95 ppm was absent in the spectrum of 4 indicating that the deprotonated CONH group of nitrogen atom participates in the coordination. In free ligand, SO 2 NH 2 protons give a signal at d 8.32 ppm which is shifted to d 8.06 ppm in Zn(II) complex. In the NMR sectrum of complex 4, the signals due to aromatic protons are observed at d 7.99-7.25 ppm range. Upfield shifts of these protons in comparison of the ligand 44 (d 8.11-7.29 ppm) confirm the coordination of the free ligand to zinc atom. The signals correspond to CH 3 and CH 2 are observed at d 1.05 and 4.11 ppm, respectively. The singlet with an intensity of four is found at d 1.07 ppm which assigned as coordinated oxygen atoms of two water molecules to zinc ion.

FT-IR measurements
The significant frequencies in the IR spectra of the free ligand 44 (1) and the complexes (2-4) are given in Table 1. The broad absorption bands at 3504, 3600 and 3441 cm À1 are attributed to (O-H) of water in compounds 2, 3 and 4 respectively. Two stretching frequencies were obtained for the asymmetric (3390 cm À1 ) and symmetric (3235 cm À1 ) vibrations of the sulfonamido NH 2 moiety for compound 2. These asymmetric and symmetric bands were observed at 3412 and 3241 cm À1 , respectively, for compound 3 and at 3397 and 3276 cm À1 , respectively, for compound 4.
The N-H stretching vibration was observed at 3227 cm À1 for 1.
The band corresponding to the amide m(C¼O) vibration of the pyrazole carboxamide (1659 cm À1 ) of 1 is observed as a single band shifted to lower frequencies (about 1624-1606 cm À1 ) in the complexes (2)(3)(4). This fact could be attributed to the bonding of the metal ions with the amide nitrogen of the ligand. 57 Absorptions at 1523, 1478 and 1431 cm À1 assigned to the (C¼C/C¼N) vibrations of 1 are slightly shifted from those found in 2 (1536 and 1491 cm À1 ), 3 (1534, 1489 cm À1 ) and 4 (1549, 1473 cm À1 ). These data of complexes 2, 3 and 4 are also attributed to the coordination occurring from nitrogen atom of pyrazole ring of 1. Weak bands at 591 and 484 cm À1 for 2, 589 and 472 cm À1 for 3 and 586 and 478 cm À1 for 4 are corresponded with M-O and M-N vibrations, respectively.

UV/vis spectra and magnetic susceptibility
The electronic spectra of compounds 1-4 were recorded in DMSO solutions at a 1 Â 10 À3 M concentration at room temperature ( Table 2). The electronic spectrum of 2 exhibits three intense absorption bands at 290, 301 and 304 nm that were assigned to p-p Ã and the broad absorption band at 784 nm was assigned to d-d transitions of a 6-coordinate octahedral geometry. 58 Two bands for compound 3 (290 and 301 nm) were assigned to p-p Ã transitions similarly to other sulfonamide derivatives containing 1,3,4-thiadiazole-2-sulfonamide ring. 59 In addition, the electronic spectrum of 3 also exhibiting a broad band at 780 nm is attributable to d-d transition. The Zn(II) complex (4) did not show any d-d transition and its spectrum was dominated only by the p-p Ã transition bands at 282 and 323 nm. The magnetic susceptibility measurements were carried out on powdered samples at room temperature. The magnetic moment value for Ni(II) complex (2) is 3.64 B.M. which is in good agreement with a d 8 system in an octahedral environment. The magnetic moments of the complexes at room temperature lie in the range of 3.53-4.26 B.M. for mono nuclear octahedral Ni(II) centers. 60 The measured magnetic moment of 3 is 1.87 B.M. which is consistent with the expected spin-only magnetic moment of d 9 Cu(II) systems. The spectral data and magnetic moment value indicate an octahedral geometry around Cu(II) ion. 61,62 As expected, the room temperature magnetic moment value of Zn(II) complex (4) is found to be diamagnetic due to non-availability of unpaired electrons.

MS spectral studies of 2, 3 and 4
The MS spectra give additional structural information about the chemical structure of the studied complexes 2, 3 and 4. None of the MS spectra of complexes showed a molecular ion [M] þ peak. The fragment [MC 42 H 32 N 14 O 14 S 4 ] þ which suggests the monomeric nature of the complexes is observed at m/z 1142.9 for 2 and Table 1. IR spectral data (cm À1 ) of the free ligand (1) and complexes 2, 3 and 4. ] þ , respectively. These data confirmed the proposed formula of the complexes (Figure 1).

In vitro inhibition studies
Inhibition effects on hCA-I and hCA-II isoenzymes of the newly synthesized compounds (2)(3)(4) and acetazolamide (AAZ) as a control compound were studied by hydratase and esterase activity methods and K i values were determined for each compound and compared to inhibition effect of the free ligand (1) ( Table 3). According to the in vitro studies, the IC 50 values of hydratase activities of newly synthesized compounds 3 and 4 (0.0600 and 0.0520 mM for hCA-I and 0.0340 and 0.0420 mM for hCA-II, respectively) are lower than the IC 50 values of 1 (1.4000 and 0.9000 mM for hCA-I and hCA-II, respectively) and of AAZ (3.3000 and 2.4000 mM for hCA-I and II, respectively). The IC 50 values of hydratase activities of 2 2.4000 mM for hCA-I is lower than AAZ but higher than 1 and 0.0625 mM for hCA-II lower than both 1 and AAZ. The IC 50 values of esterase activities of compounds 2, 3 and 4 (0.0820, 0.1450 and 0.3400 mM for hCA-I and 0.0190, 0.0900 and 0.0920 for hCA-II) are all lower than the IC 50 values of esterase activities for free ligand (1) (2.8000 and 5.6000 mM for hCA-I and hCA-II, respectively) and AAZ (4.6000 and 3.9000 mM for hCA-II, respectively).
In relation to the esterase activities, the inhibition equilibrium constants (K i ) were also determined. The pyrazole carboxamide group of 1 forms a hydrogen bond with the histidine residue in the active site of CA to inhibit the isozymes (K i : 1.1000 and 5.3000 mM for hCA-I and hCA-II, respectively), resulting in less movement of the carbonic acid toward CO 2 production. 63 The coordination compounds (2, 3 and 4) show remarkable inhibition on hCA-I and hCA-II (K i : 0.1460, 0.1480 and 0.3930 mM and 0.0960, 0.0724 and 0.0980 mM, respectively), having a higher inhibition as compared to 1 as well as to the control compound AAZ (2.8000 and 2.1000 mM for hCA I and hCA-II, respectively), which is probably due to complexation of metal(II) ions to ligand. 64 In summary, these new compounds (2-4) have quite a lot higher potential inhibitory effects than their parent inhibitors 1 and AAZ. Thus, the complexes (2-4) might be good candidates for further clinical studies of glaucoma treatment. Especially, compounds 3 and 4 have shown remarkable inhibition against hCA-II and hCA-I isoenzymes for hydratase activities, and compound 2 has shown remarkable inhibition against hCA-I and hCA-II isoenzymes for esterase activities. The order of the metal ions for the inhibition of hCA-I for hydratase activity is Zn > Cu > Ni, and of hCA-I for esterase activity is Ni > Cu > Zn, and of hCA-II for hydratase activity is Cu > Zn > Ni, and of hCA-II for esterase activity is Ni > Cu > Zn.