Complexes With Biologically Active Ligands. Part 111. Synthesis and Carbonic Anhydrase Inhibitory Activity of Metal Complexes of 4,5-Disubstituted-3-Mercapto-1,2,4-Triazole Derivatives

Complexes containing five 4,5-disubstituted-3-mercapto-1,2,4-triazoles and Zn(II), Hg(II) and Cu(I) were synthesized and characterized by standard procedures (elemental analysis; IR, electronic and NMR spectroscopy, conductimetry and TG analysis). Both the thione as well as the thiolate forms of the ligands were evidenced to interact with the metal ions in the prepared complexes. The original mercaptans and their metal complexes behave as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II and IV, but did not lower intraocular pressure in rabbits in animal models of glaucoma.


Introduction
Carbonic anhydrase (CA, EC 4.2.1.1), an enzyme playing a central role to both transport and metabolic processes involving CO2 and bicarbonate, is present in a variety of tissues of higher vertebrates in the form of eight isozymes [2][3][4]. By catalyzing the reversible interconversion between the two chemical species mentioned above, in metabolically active tissues (such as the muscle), cytoplasmic (CA I-III) and sarcolemmal (CA III) isozymes facilitate CO2 transport out of the cell [3]. The only membrane-bound isozyme known (CA IV), wlfich is higlfly abundant in the kidneys and lungs, lms been shown to possess an extracellular orientation of the active site, and to be critical in acidifying file outer boundary layer through the protons formed by CO2 hydration [5,6], a process that then facilitates among others cellular anunonia transport by providing the I-V ion for the protonation of NH3 and maintaining thus the trans-membrane anunonia gradient [3,5].
The mitochondrial isozyme (CA V) is known to supply bicarbonate/CO2 for the initial reaction of gluconeogenesis and ureagenesis in many m,'unmalian tissues [7,8], as well as for tile pyruvate carboxylation in tile de novo lipogenesis in adipocytes [9].

Materials and Methods
IR spectra were recorded on a Perkin-Ehner 16PC FTIR instrmnent, in the range 200-4000 cm1, in KBr pellets. Solution electronic spectra were recorded with a Cary 3 spectrophotometer interfaced with a PC. Conductimetric measurements were done in DMF solutions, at 25C (concentrations of mM of complex) with a Fisher conductimeter. Magnetic susceptibility measurenents were carried out at room temperature with a fully automated AZTEC DSM8 pendulmn-type susceptometer. Mercury(II) tetrakis-(thiocyanato)cobaltate(II) was used as a susceptibility standard. Corrections for the di,'unagnetism were estimated from Pascal's constants [37]. Elemental analyses were done by combustion for C, H, N with an automated Carlo Erba analyzer, and gravimetrically for the metal ions, and were + 0.4% of the theoretical values. NMR spectra were recorded in DMSO-d6 as solvent with a Bruker CPX-200 instrument.
Thennogravimetric measurements were done in air, at a heating rate of 10C/rain., with a Perkin Elmer 3600 thennobalance.
Mercaptans 9-13 were prepared as described in the literature [38]. Metal salts (Cu(I) chloride, Hg(II) chloride and Zn(II) sulfate pentahydrate), triethylamine and solvents were from E. Merck (analytical grade) and were used without additional purification.
Human CA and CA II cDNAs were expressed in Escherichia coli strain BL21 (DE3) from the plasmids pACA/HCA ,and pACMHCA II (the two plasmids were a gift from Prof. Sven Lindskog, Uxnea University, Sweden). Cell growth conditions were those described by Lindskog's group [39] and enzymes were purified by affinity chromatography according to the method of Khalifah et al. [40]. Enzyme concentrations were determined spectrophotometric,'flly at 280 nm, using a molar absorptivity of 49 mM-1. cm -1 for CA I and 54 mM-l.cm -1 for CA II, respectively, based on Mr 28.85 kDa for CA I, and 29.3 kDa for CA II, respectively [41]. CA IV was isolated from bovine lung nficrosomes [42]. hfitial rates of 4-nitrophenyl acetate hydrolysis were mofitored spectrophotometfically, at 400 nm and 25C, with a Cary 3 apparatus interfaced with an IBM compatible PC by the method of Pocker and Stone [43]. Solutions of substrate were prepared in anhydrous acetonitrile; the substrate concentrations varied between 10 .2 and 10 .6 M. A molar absorption coefficient 18,400 Ml.cm -1 was used for the 4nitrophenolate formed by hydrolysis, in flae conditions of the experiments (pH 7.80), as reported by Pocker and Stone [43]. Non-enzymatic hydrolysis rates were always subtracted from flae observed rates. Duplicate experiments were done for each inlfibitor, ,and file values reported throughout the paper ,are the averages of such results. ICso represents the molarity of ilthibitor producing a 50% decrease of enzyme catalyzed hydrolysis of 4-nitrophenyl acetate.
General procedure for the preparation of the Zn(II) and Hg(II) complexes 14-23 4 mMol of mercaptan 9-13 were suspended in 25 mL MeOH mad 4mMol of Et3N were added in order to deprotonate the ligand. Tlfis was treated thereafter with a methanolic solution of the metal salt (Zn(II) sulfate and Hg(II) clfloride, respectively), working at file molar ratios M 2+ mercapt,'m of 1:2. A yellowish-white precipitate formed inunediately. The obtained reaction mixture was heated on a ste,-un bath for 2 hours, then the precipitated complexes were filtered, thorouglfly washed with cold alcohol and ,air dried. Crystallization was not done as the only solvents in which the complexes possessed good solubility were DMSO and DMF. The obtained powders of complexes 14-23 melted with decomposition at temperatures lfigher than 300 C.
Preparation of the Cu(1) complexes [24][25][26][27][28] An ,'unount of 6 mMol of the ligand 9-13 in 50 mL of absolute ethanol was added to 0.300 g CuC1 (3 mMol) dissolved in 30 mL of,'ufllydrous acetonitrile. The gelatinous cream-like precipitate was filtrated with suction and air dried under reduced pressure over silica gel at room temperature. Basically this method used by us is the stone as the one described by  for the preparation of Cu(I) complexes of imi&azole thiones and related lig,'mds.

Results and Discussion
The heterocyclic mercaptans 9-13 used for file preparation of metal complexes, were deprotonated in the presence of triethylanine prior to complexation with Zn(II) ,and Hg(II),or were used as neutral ligands in In the solid state and in neutral solution, the tlfione forms of type B ,are the dominant tautomers, with the tlfione sulphur atom as the favoured donor site [44][45][46]. Deprotonation of these derivatives in a variety of conditions generates thionate mions in which both the tlfionate sulphur as well as the endocyclic nitrogen atoms are available, either singly or collectively, for coordination. In this work we have used both these possibilities for preparing metal complexes of derivatives 9-13. Thus, in the case of the Zn(II) and Hg(II) derivatives, the metal complexes were prepared using the deprotonated derivatives 9-13, whereas the Cu(I) complexes were obtained from the neutral (thione) form of the ligands, as in the classical studies of Raper's group [44][45][46].
The new complexes 14-28 reported in the present work and their elemental ,analysis data are shown in Table I. The most important IR bands in the spectra of compounds 9-28 are shown in Table II. Several important features of flese spectra should be mentioned: (i) the two SO2 vibrations appear mchanged in the IR spectra of the ligands 9-13 and their met,'fl complexes 14-28, proving flat these moieties do not interact with fle metal ions (data not shown); (ii) major modifications in the spectra of the complexes as compared to lhose of the corresponding ligands, regard fle thioamide vibrations (Table II). Thus, with the exception of the thioanide III band, generally appearing at the same wavelength in the spectra of the lig,'mds and those of the complexes, fle other tlvee flfioaxnide b,'mds are perturbed by the presence of the metal ions. In the spectra of complexes, the tlfioamide II and IV bands appeared with 5 45 cmat lower wavelength as compared to fle corresponding band of the ligand, whereas fle thio,-unide band appeared wifl 20-30 cmat lfigher wavelength for the Zn(II) and Hg(II) complexes, and were unchanged for the Cu(I) derivatives. The most perturbed was the thiomnide IV band, wlfich was generally splitted in two or tlu'ee intense bands in the spectra of all metal complexes, whereas in the spectra of the ligands it was presented as a single band. This behaviour has been previusly reported for other metal complexes of heterocyclic thiones [44-46]; (iii) a clear distinction could be made regarding the tautomeric form of the ligand in the case of the Zn(II) and Hg(II) complexes on one hand, and the Cu(I) derivatives on the other hand. Thus, for the first derivatives (prepared from the deprotonated form of the ligand) no NH bands were evidenced in the IR spectra (these bands were present in the spectrum of the corresponding ligand), whereas for the Cu(I) derivatives these arre present at the stone frequencies as in the case of the ligand. The thio,-unide band is ,also different for the two groups of complexes, with the Cu(I) derivatives showing this band at the same frequency as in the case of the ligand, whereas for the Zn(II) and Hg(II) derivatives the correponding band was shifted at higher wavelength. The thioamide II band on the other hand was splitted only in the case of the Cu(I) derivatives. In the Hand 3C-MR data of the Hg(II) complexes, the following modifications have been noted as compared to the corresponding spectra of the original mercaptans 9-13: (i) the signal of the NH proton, appearing at 8.30 8.35 ppm in the H-NMR spectra of colnpounds 9-13, is absent in the spectra of the corresponding Zn(II) and Hg(II) complexes, 14-23; (ii) other signals (the arolnatic protons, the lnoiety substituting the N-4 atom) appear in the same ranges in the original mercaptans and the corresponding Zn(II) and Hg(II) coxnplexes (data not shown); (iii) in the 3C-NMR spectra, the C-3 carbon atoms show a signal at 168.2 168.6 ppm in the spectra of compounds 9-13, whereas in the spectra of the corresponding Zn(II) and Hg(II) complexes these signals appear at 165-167 ppm. This sltift is presumably due to the presence of the coordinated metal ions in the neighbourhood of these atoms; (iv) other sigqals in the C-NMR spectra of xnercaptans 9-13 and metal complexes 14-23 appear tmchanged, at the same chemical shifts (data not shown). All the prepared complexes showed no weight loss under 200C, were non-electrolytes (in DMF as solvent, at room temperature data not shown), diamagnetic (data not shown) and colorless also in the case of the Cu(I) derivatives, prompting us to propose the stnctures shown below. All these data indicate that in the case of the Zn(II) and Hg(II) derivatives the ligand acts in deprotonated form, bidentately, with the donor system probably constituted by the endocyclic xfitrogen and the mercaptide sulfur atoms. In the case of the Cu(I) derivatives, the thione form of the ligands probably acts monodentately, with the thione sulfur as donor atom, similarly as in the Cu(I) derivatives of 1-methylimidazoline-2(3H)-thione reported by Raper's group [44][45][46]. The Cu(I) derivatives are probably dimers, as the similar complexes reported by Raper's group [44][45][46].
The most efficient inhibitors were fle Hg(II) complexes, followed by the Cu (I)   Metal-Based Drugs iflfibitors were those possessing an N-Et or NH moiety in the 4 position of the heterocyclic ring, whereas the compounds possessing N-cyclohexyl such groups had a largely decreased affinity for the enzyme, presumably due to steric hindrance induced by the bulky cyclohexyl group: (iii) the susceptibility of the different CA isozymes to inhibition with tlfis class of derivatives was: CA II > CA IV > CA I, being similar to that for the aromatic/heterocyclic sulfonamides [4]. The newly prepared complexes as well as the heterocyclic mercaptans 9-13 were tested for their ability to lower intraocular pressure (IOP) in rabbits, in animal models of glaucoma, since recently it was discovered by this group that metal complexes of heterocyclic sulfonamides act as efficient IOP lowering agents [48][49]. None of these derivatives showed any effect when applyed as a 2% solution (in DMSO) directly into the rabbit eye.