Complexes With Biologically Active Ligands. Part 2. Preparation of Copper(II) Complexes of Positively-Charged Derivatives of Aminoglutethimide

Cu(II) complexes of 1-[4-(3-ethyl-piperidine-2,6-dione)-3-yl]-phenyl-2,4,6-trisubstituted pyridinium perchlorates, containing alkyl, aryl and combinations of these two types of moieties in their molecule were synthesized and characterized by elemental analysis, spectroscopy, magnetic, thermogravimetric and conductimetric measurements. In these complexes, Cu(II) ions are in octahedral geometry with four water molecules occupying the equatorial coordination sites and the two organic ligands in deprotonated state the remaining axial ones. The donor atom of these ligands is constituted by the ionized nitrogen of the glutarimide moiety. The new derivatives possess weak inhibitory activity towards the zinc enzyme carbonic anhydrase.


Introduction
A large series of biologically active coordination compounds containing heterocyclic sulfonamides as ligands were reported recently 2-4. Such derivatives behave as potent inhibitors of the ubiquitously spread zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) 2a,5 and since ligands such as acetazolamide 1, methazolamide 2 or ethoxzolamide 3 are widely used pharmacological agents in the treatment of a varie of disorders, 5,6 presumably, their complexes might lead to the development of novel types of such drugs. 2a In addition to these classical CA inhibitors, complexes were also prepared from positively-charged sulfonamides of type 4 (developed as isozyme-specific inhibitors for membrane-bound CA isozymes 7) as A Me well as saccharin 5 (for which CA inhibitory properties were recently reported8) or diazoxide 6, a compound possessing a completely different type of biological activity, but a coordination chemistry similar to that of compounds 1-4. 1 All these ligands coordinate transitionor main-group metal ions by means of the sulfonamido nitrogen atoms, in a monodentate or bidentate fashion (in which case endocyclic atoms such as the thiadiazolic N-3 or N-4 of acetazolamide 1, or the nitrogen from the benzothiazole ring of ethoxzolamide 3, participate by interaction with the cations too). 2-4 One of their specific features is the presence of a rather acidic NH proton in their molecule, with PKa's in the range 7.0 9.5 in derivatives 1-4 and 6 (saccharin 5 being a much stronger acid, with pKn of 1.3). 9 As a consequence, ligands of type 1-6 generally coordinate metal ions in their deprotonated state. [2][3][4] In addition to studies previously mentioned by us, 2"4 few other metal complexes were reported containing this type of ligands. A notable exception is constituted by the Cu(II) complexes of arylsulfonylated amino acids, such as tosylglycine 10 or the 4-aminophenylsulfonyl derivatives of glycine, alanine, valine and glutamic acid. 11 The interesting biological activity of complexes containing such ligands, 2-4,10,11 as well as the scarcity of literature data in this field, prompted us to extend the studies to related systems, containing a rather acidic NH group, because of the presence of SO 2 or CO moieties in its vicinity. Aminoglutethimide (3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione, 7) derivatives, such as the positively-charged compounds of type $, recently reported, 12 appeared as interesting candidates for several reasons. Thus, aminoglutethimide is a clinically used dru in the treatment of breast and prostate cancer, 13 adrenocortical carcinoma 14 and Cushing syndrome 15 among others. It inhibits the conversion of cholesterol to 20--hydroxycholesterol, inhibiting in fact the cytochrome P-450 hydroxylation of this substrate. 16 In this way the first step of steroidogenesis is inhibited, a fact that explains the efficiency of the drug in the treatment of tumors sensitive to such hormones. 13"16 On the other hand, the NH proton in derivatives of type $ is sufticiently acidic (PKa-s in the range 9.0-9.6 12) to allow easy deprotonation and participation in interactions with metal ions, similarly with derivatives of types 5 and 6 previously investigated by us. 1,2-4 It is also to expect that such complexes would be biologically active, since the ligands possess prominent such properties. 12 In this paper I report the preparation and characterization of Cu(II) complexes of positivelycharged derivatives of aminoglutethimide of type $. The new complexes were characterized by elemental analysis, IR and electronic spectroscopy, as well as magnetic moment measurements, conductimetric and thermogravimetric data. They were also tested for their ability to inhibit CA, showing weak such properties.

Materials and Methods
IR spectra were obtained KBr pellets, with a Beckmann 4260 instrument, in the range 200 4000 cm1. Electronic spectra were obtained by the diffuse reflectance technique in MgO as reference, with a Perkin Elmer Lambda 17 apparatus. Conductimetric measurements were done in DMF solutions, at 25C (concentrations of 1 mM of complex) with a Fisher conductimeter. Magnetic susceptibility measurements were done at room temperature by Faraday's method, using CoHg(NCS)4 as standard. Elemental analyses were done by combustion for C,H,N with an automated Carlo Erba analyzer, and gravimetrically for the metal ion, and were + 0.5% of the theoretical values. Thermogravimetric measurements were done in air, at a heating rate of 10C/min., with a Perkin Elmer 3600 thermobalance.
Aminoglutethimide 7 (pure L stereoisomer) used in the syntheses was from Sigma. Pyrylium perchlorates used to prepare derivatives 8 (by reaction with aminoglutethimide) were synthesized by literature procedures, by bisacylation of olefins or their precursors, as originally described by Nenitzescu and Balaban 17, from commercially available raw materials. Compounds 8 were synthesized as reported previously, 12 from 7 and trisubstituted pyrylium salts in alcohol, by the general method involved in reaction of such reagents with N-nucleophiles. 18 Copper(II) perchlorate was from Aldrich and was used without additional purification. Bovine CA II and human CA I were from Sigma Chemical Co. Inhibitors Maren's micromethod, in the conditions of the E-I (enzyme-inhibitor) technique, at 0C in were assayed bYl9 veronal buffer. ICso values represent the molarity of inhibitor producing a 50% decrease of CA specific activity for the CO2 hydration reaction.

Synthesis of coordination compounds 9a-f
An amount of 20 mMoles of pyridinium perchlorate 8a-f was suspended in 50 mL of ethanol and 20 mMoles of NaOH dissolved in 10 mL water were added. This solution was treated with an aqueous solution obtained by dissolving 10 mMoles of Cu(II) perchlorate in 10 mL of water, under intense stirring. The reaction mixture was stirred at room temperature for hour, complexes 9a-f which precipitated were filtered and air-dried. Yields were of 75-80%.

Results and Discussion
Compounds 8a-f used in this study were previously re!orted, 12 and were obtained by reaction of 2,4,6-trisubstituted pyrylium salts with aminoglutethimide 7.18 Compounds used in the present study, shown in Table I together with some selected IR bands (relevant for their complexation behavior), possessed aliphatic, aromatic and the combination of these two moieties in their molecules, which were chosen in order to investigate the role of substitution pattern upon properties and biological activity of the obtained complexes. Complexes 9 were prepared from Cu(II) perchlorate and the sodium salts of 8 (obtained in situ from 8 and the stoichiometric amount of NaOH), in molar ratios of 1:2. The blue-green complexes obtained in this way are shown in Table II, together with their elemental analysis and thermogravimetric data (:t0.5 % of the theoretical values for Cu, C, H, N and water). Spectroscopic (IR and electronic), magnetic and conductimetric data of complexes 9a-f are presented in Table III.
In the IR spectra of the prepared complexes 9, the following modifications were detected, as compared to the spectra of the corresponding derivatives 8: (i) the absence of v(NH) vibrations, which for Sa-f appeared at 3050-3070 cm-1; (ii) the shift of the very intense "amide I" band (v(C=O)) with 20-30 cm -1 towards lower wavenumbers for complexes 9 (Table III) as compared to the corresponding vibrations of derivatives 8 (Table I); (iii) the other amide bands ("amide II" around 1500-1520 cm"1, and "amide III" around 1270-1300 cm "1 in the ligands) appear in complexes 9 at almost the same wavenumbers (data not shown), but they are not so well resolved and have a reduced intensity; (iv) the very intense perchlorate bands, at 625 and 1100 cm"1, are present in both compounds $ and 9; (v) at wavenumbers under 400 cm-1, all complexes 9 show low intensity bands at 380 and 290 cm-1, tentatively assigned as due to Cu-O and Cu-N stretching vibrations.20, 21  Table IV it can be seen that, as expected, derivatives 8,9 behave as weak inhibitors of both CA isozymes. Still, it is obvious that the substitution pattern of the pyridinium ring strongly influences biological activity,, as for positively-charged sulfonamides of type 4 possessing the same type of groups in their molecules. 7 Thus, derivatives 8 are extremely weak inhibitors for both isozymes, the strongest one being the 2,6-dimethyl-4-phenyl-pyridinium substituted compound 8d (significant inhibition in the millimolar range). The Cu(II) complexes 9 behave as much stronger inhibitors, presumably due to a dual inhibition, as observed for the metal complexes of heterocyclic sulfonamides. 1-4 As expected, complexes 9 inhibit these two isozymes 2 to 6 times stronger as compared to the corresponding compounds $ from which they are derived. The strongest inhibitor was always the one containing the 4-phenyl-2,6-dimethyl moiety, followed by the complexes possessing only aliphatic groups at the pyridinium ring. In contrast, the weakest inhibitors were those possessing more than one aromatic group in that position ($e,f and 9e,f, respectively). This is probably due to the very bulky nature of the last derivatives and an impaired access within the CA active site.