Complexes With Biologically Active Ligands. Part 61 Ni(II) Coordination Compounds of Hydrazine and Heterocyclic Sulfonamides as Inhibitors of the Zinc Enzyme Carbonic Anhydrase

Ternary Ni(II) complexes of hydrazine and eight heterocyclic sulfonamides possessing carbonic anhydrase (CA) inhibitory properties, were prepared and characterized by elemental analysis, spectroscopic, magnetic, thermogravimetric, and conductimetric measurements. The complexes behave as strong inhibitors for two isozymes (I and II) of carbonic anhydrase.


Introduction
Heterocyclic sulfonamides constitute a class of strong inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and have wide clinical applications in the treatment of a variety of disorders such as glaucoma, gastro-duodenal ulcers, and acid-base disequilibria among others. Compounds 1-6 are all clinically used such drugs, with acetazolamide 1, methazolamide 2 and ethoxzolamide 3 being first generation inhibitors (without specificity for different CA isozymes or selectivity for diverse organs where these enzymes are present), benzolamide 5 and chlorzolamide 6 having the status of orphan or experimental drugs 4 (and also showing specificity for some CA isozymes in certain concentrations3'4, whereas sezolamide 4 is the representative of a novel class of such inhibitors (third eneration) recently introduced in clinical medicine as topically effective drugs in the treatment of glaucoma. 5-Amino-1,3,4-thiadiazole-2-sulfonamide 7 is on the other hand the key compound for synthesis of diverse inhibitors such as 1, 2, 5 or 8. This last compound is a member of a novel class of recently developed membrane-impermeant derivatives which selectively inhibit membrane-bound isozyme CA IV.
Recently, our 7roups proved that metal complexes of heterocyclic sulfonamides behave as even stronger CA inhibitors, and their mechanism of action was also explained, as being due to a dual inhibition by sulfonamido anions and metal ions, formed by dissociation of the complex inhibitor, which subsequently 9 10II bnd to dfferent stes of the enzyme. Metal ons probably bnd to catalytically critical hstdne residues whereas the sulfonamido anions coordinate to the Zn(II) ion within CA active site. 12 A large series of such complexes was reported in the last years, containing mainly acetazolamide 1, methazolamide 2 and ethoxzolamide 3 as ligands, and structures were determined for some of them by means 81314 of X-ray crystallography and spectroscopic methods," although some other ligands were investigated too. '7'4 As the coordination chemistry of this type of ligands is sometimes complicated due to their versatility and large number of donor atoms present in their molecules, it is of great interest to investigate some other ligands, as well as ternary complexes containing heterocyclic sulfonamides and amine-type 27 hgands, also due to their possible pharmacological apphcatons.
In this paper we report the preparation and characterization by spectroscopic, magnetic, conductimetric and thermogravimetric methods of Ni(II) complexes of sulfonamides 1-8 and hydrazine as ligands. The prepared complexes behave as very efficient inhibitors of human isozymes CA I and CA II. #  To this solution was added l0 mL of aqueous solution containing 5 mMoles Ni(II) salt (nitrate, or chloride) and the obtained solution was treated with an excess (3 mL) of hydrazine hydrate 80%. The mixture was stirred magnetically at room temperature for 2 hours, then the obtained precipitates were filtered and air-dried. Yields were in the range of 56-95%.

Results and Discussion
The prepared complexes and their proposed formulas (based on elemental analysis, 0.5% of the theoretical values) are shown in Table I. aBy gravimetry; bBy combustion.
The new compounds 9-16 were also characterized by means of spectroscopic (IR and electronic spectra), thermogravimetric, conductimetric and magnetic measurements. Some of these data are shown in Tables II and IV. In the IR spectra of the complex derivatives, the main change as compared to the uncomplexed sulfonamides was the shift of the SO 2 vibrations (in the range 1100-1180 cm for the antisymmetric vibrations, and 1300 1376 cmfor the symmetric vibrations, respectively, in derivatives 1-8) 2"5.7 to lower wavenumbers (with 5 20 cm-), as for previously reported such complexes. 7.8.t3-5 The thiadiazole bands around 1500 1600 cmare also slightly shifted to lower wavenumbers in the spectra of the complexes as compared to the corresponding sulfonamide (data not shown), suggesting a possible interaction of Ni(II) with some of the endocyclic nitrogens of compounds 1-8 too. Moreover, the hydrazine vibration, TM around 970 -1 cm ,was also detected in the IR spectra of complexes 9-16. Vol. 3, No. 3, 1996 Complexes with Biologically Active Ligands. Part 6. As seen from data of Tables II, the effective magnetic moments of complexes 11, 12, 14 and 15 suggest an octahedral environment of Ni(II), whereas for 16, a tetrahedral geometry of the metal ion is assumed. The other three complexes (9, 10 and 13) have magnetic moments lower than the only spin component (2.83 BM), suggesting that any magnetic coupling between the two Ni(II) ions appears for the dinuclear derivatives, 9 and 10.19 Reflectance diffuse (RD) spectra of the newly synthesized complexes are shown in Table III, together with some calculated parameters.
As seen from data of Table III, in the reflectance spectra of complexes 9-15, a band appears in the visible range (around 18,000 cm-1) and another one in the near infrared region, around 11,000 cm-, suggesting an octahedral environment of the Ni(II) ion(s). 19 The spectrum of compound 16 on the other hand shows a broad band centered at approximately 19,870 cm -1 and another one at 11,480 cm -I which indicate a distorted tetrahedral geometry of Ni(II) in this complex. 9 Table IV also shows the assignment of the bands using the 2o Lever tables. The results of RD spectra are thus in agreement with those of magnetic measurements, suggesting octahedral Ni(II) ions in all but the last complex (16), which presumably contains tetrahedral Ni(II).
The donor system of the conjugated base of sulfonamides 1-7 is probably constituted by the anionic nitrogen of the sulfonamido moiety and the endocyclic nitrogens, excepting for sezolamide 4 where probably the endocyclic sulfur participates in coordination. The positively-charged sulfonamide 8 on the other hand seems to act as monodentate ligand when deprotonated at the sulfonamido moiety, by means of the sulfonamido nitrogen. Hydrazine probably acts as both mono., bidentate and bridging bidentate ligand in the prepared complexes. TG and conductimetric data (Table II) are in agreement with the above proposals. As no appropriate crystals for X-ray diffraction experiments could be obtained, the precise structure of the prepared complexes is not assigned for the moment. J. Borras, J. Casanova, T. Cristea, A. Gheorghe, A. Seozzafava, C.T. Supuran and K Tudor

Metal-Based Drugs
Biological activity data with the new complexes (compared to that of the sulfonamide from which they derive) are presented in Tables IV and V. As seen from data of Table IV, the new complexes prepared by us behave as strong inhibitors for both investigated CA isozymes, with potencies larger than those of the corresponding sulfonamides. Generally, complexes were 1.1 5.5 times more active than the free sulfonamides from which they were prepared. The notable exception is 12, which is more than 6000 times more inhibitory towards CA I, as compared to sezolamide 4. No explanation for this large difference of activity is available for the moment, but in Table V some comparative data are shown regarding CA I inhibition with sezolamide 4, Cu(II) ions as well as complex 12. From such data, one can conclude that at least for isozyme CA I, the enzyme inhibition observed with this class of derivatives is due to the metal complex per se, and not to its dissociation products formed in dilute solution (metal ions and sulfonamido anions), as hypothesized eralier by us. 7'8  aCA activity in the same conditions and in the absence of inhibitors is taken as 100%.
The increased activity as enzyme inhibitors of metal complexes of sulfonamides might be thus accounted on their direct binding to the enzyme, in undissociated formed, in addition to the previous explanation that the effect is due to a dual inhibition by metal ions and sulfonamido anions formed in dilute solution by dissociation of the complex. 7'8