1,3,4-Thiadiazole Derivatives. Part 91. Synthesis and Biological Activity of Metal Complexes of 5-(2-Aminoethyl)-2-Amino-1,3,4-Thiadiazole

Metal complexes of the title ligand (L) containing Co(II), Ni(II) and Cu(II) were prepared and characterized by elemental analysis, IR, electronic spectroscopy and conductimetry. The new derivatives, possessing the following formulae, CuL2(OH)2, NiL2Cl2, and [Co2LCl4]n showed in vitro antifungal activity against Aspergillus and Candida spp.

N---N 1" R=H, Z=SH 2: R=Ac, Z=SO2NH2 3: R=H, Z=SO2NH2 These compounds, have also been investigated as complexing agents, 316 in order to obtain substances with a diversified biological activity, conferred to them among others by the presence of the metal ions, except for that of the thiadiazole ligand. Thus, some metal complexes of ligands of type 1-3 have been recently reported as in vitro inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), 1'14'16 whereas m vivo studies showed good antiepileptic action for some Cu(II) and Zn(II) complexes of the sulfonamide type ligands. TM Finally, some 2,5-disubstituted-l,3,4-thiadiazoles as well as their Cu(II) complexes were 16h reported to act as fungitoxic agents.
Recently, another group of biologically active 1,3,4-thiadiazoles was reported by us. 17 5-(2-Aminoethyl)-2-amino-l,3,4-thiadiazole 4 and some of its positively charged pyridinium derivatives showed very good activatory effects on carbonic anhydrase, 17 making such compounds attractive candidates for the development of pharmacological agents for the treatment of CA defficiency syndrome, a rare but dramatic genetic disease which is caused by the lack ofisozyme CA II in the human organism. 8'9 As 4 possesses an interesting donor atoms system besides its prominent biological activity, 17'9 a study for preparation and biological assay of some of its metal complexes was initiated in our laboratory. Fungitoxicity has not been investigated previously for amines of type 4, and since many imidazoles and triazoles such as ketoconazole, fluconazole, clortrimazole, etc possess outstanding fungistatic properties, being widely used clinically, 2 it appeared of interest to test thiadiazole 4 and some of its metal complexes for this type of biological activity. In this paper we report the synthesis of Co(II), Ni(II) and Cu(II) complexes of ligand 4, and their activity as fungistatic agents against Aspergillus and Candida spp.

Materials and Methods
Infrared spectra were recorded in CsBr pellets with a Nicolet 2DXFT-IR apparatus. Electronic spectra were recorded by the diffuse reflectance technique using MgO as a reference material. Conductometry was done in DMF solution with a Radelkis 1200K/1 apparatus at 25C. Elmental analyses were done by combustion (C,H,N,S) with an automated Carlo Erba analyser, or by atomic absorption spectroscopy for the metal ions. Ligand 4 was prepared as described in the literature. 7 Metal salts and solvents were analytical grade (from Merck or Aldrich) and were used without further purification.
Synthesis of complexes [5][6][7] All complexes reported in this paper have been synthesised according to the following general procedure: methanolic solutions containing the metal chloride (CuC12 2H20, CoC12 6H20, NiCI 6HO) and the heterocyclic diamine, 4, (L.2HC1), 17 were mixed with stirring in molar ratios of 1:1 and 1:2, respectively. The dihydrochloride of amine 4 was used in synthesis, due to its good methanol solubility. 17 The resulting mixture was treated with a stoichiometric amount of triethylamine (in order to neutralize the HC1 of the dihydrochloride) and the obtained mixture was heated for 60 min. on a water bath. The complexes precipitated from these solutions, were filtered, washed with diethylether and dried in vacuum. The following coordination compounds were synthesised and characterised: CuL2(OH)2 (green blue) 5, NiL2C12 (blue) 6, and Co2LC14 (red brown) 7. In the above formulas L stands for the corresponding diamino ligand. It is to note that, even when working in molar ratio MClx: L of 1:1 or 1:2, only the above mentioned compounds were obtained. Assay ofmgistatic activity of compounds 4-7

Metal-BasedDrugs
The fungistatic activities of derivatives 4-7 were determined by a modification of the growth method 21 recently reported by us, 22 utilizing two Aspergillus and one Candida spp. The fungi were cultivated in agar plates at 25C, in the absence and in the presence of compounds 4-7, in concentrations ranges of 10 -3 10 -7 M (solutions in DMSO). No significant fungistatic effects were observed at concentrations of 10 -6 and 10 .7 M of the new compounds, except for the Cu(II) derivative 5 (the most active in the series), which showed interesting inhibitory activity in micromolar concentrations too. Percentual inhibition of growth was calculated with the following formula: 21'22 % inhibition 100 x (Dc-Di)/Dc where Dc represents the average diameter of the fungi colony in the control plate after 48 hours, whereas Di the same parameter in the presence of tested compound.

Results and Discussion
Due to the complicated donor system of the ligand 4, at least four different more common complexation modes are probable, depicted as I-IV in the formulas bellow. The most favourable one for mononuclear complexes would be II or IV, due to the stability of six membered cycles formed in these cases, while I and III should lead to less stable four membered rings. From IR data discussed shortly, we ruled out coordination of types III or IV, as among others, no M-S bands were observed in the IR spectra. From the stereochemical point of view it is also hardly possible a coordination of type IV. Thus, as for histamine and histidine complexes, the most probable coordination mode is probably II, involving the aminoethyl NH2 group and the endocyclic nitrogen N-4 ofthe heterocyclic ring 23 H2 H2

NH2
In the IR spectra of complexes 5-7 the large stretching vibrations of the ammonium bands from the IR spectrum of 4 (as dihydrochloride salt), 17 around 3000 cm , are absent, due to the involvement of the free NH2 groups in coordinating the metal ions. Characteristic vibrations of the ligand (SNH: at 1630 cm4, Vc=N at 1564 cm 4 in the spectrum of 4) are also present in the spectra of complexes 5-7, but shifted towards lower wavenumbers as compared to the spectrum of 4. Thus the thiadiazolic band appears at 1510 1532 cm 1 (Vc=N) in the spectrum of these complexes. The shiR with 25-35 cm 1 towards lower wavenumbers of the NH: and C=N vibrations in the IR spectra of the complexes is indicative that the coordination of type II or I proposed above might describe the behaviour of 4 in its metal complexes. The coordination by two types of nitrogen atoms is also supported by the existence of large bands in the range 200-600 cm 4 in the spectra of complexes 5-7, presumably due to M-N vibrations. A band centred at 470 cm 4 was observed for the Cu(II) complex 5, at 440 cm "1 for the Ni(II) complex 6 and several peaks for the Co(II) complex $, at 316, 450 and 526 cml. 4 Supplementary stereochemical information for the new complexes 5-7 was obtained from the absorption electronic spectra of the three compounds.
In the electronic spectrum of the Cu(II) derivative 5, two large bands were observed: i) the first one is located in the range 5-18 xl03 cm 4 and offers information about d-dtransitions. By deconvolution using the Gauss method, 25 the following transition bands were identified, at 7 xl03, 11 xl03, and 15.7 x103 cm1, respectively. The lowest energy, according to the dx 2. y2 dz 2, transitions, is characteristic for the copper (II) ion in a (pseudo)-octahedral configuration. 25 The band appeared at a slightly higher energy due to the involvement of the 4s orbital in the coordination sphere, ii) The second transition observed in the range 18-25 xl 03 cm -1 can be attributed to charge transfer bands. 25 The diffuse reflectance spectrum of the nickel complex 6 is characteristic for a (pseudo)-octahedral arrangement with the following transition bands: v 10 xl0 cm (3T2g ::: 3A2g), v2:17.5 xl0 cm (3Tlg (F) <:= 3AEg), v3: at higher energies (charge transfer energy). After deconvoiution the first band (vl) was splitted in two components and the second band (v2) in three components, this supplementary division being probably due to the low simetry of the ligand field and to the non-equivalence of the donor atoms. 25'26 The electronic spectrum of the Co(II) complex 7 possibly contains two overlapped bands of two chromophores in different stereochemistries. The presence of an intense band at 15 xl0 cm "I confirms the tetrahedral sterochemistry, while other bands at 11.2 xl03, 16.3 xl03, and 21.5 xl0 cm"I, respectively, are characteristic for a (pseudo)-octahedral configuration of the Co(II) ion. 27 It is difficult to formulate this compound as octahedra! cation: tetrahedral anion complex, (due to its brown-red color characteristic of polynuclear complexes of Co(II)) 25  """ k The fungistatic activities of derivatives 4-7 against two Aspergillus species and Candida albicans are presented in Table I. Clotrimazole 8, a topically effective fungistatic agent with clinical applications was used 20,28 as a standard in these assays.
As seen from the data of Table I, the thiadiazole 4 is much less effective as fungitoxic agent as compared to clotrimazole, an imidazole derivative with a potent such activity, against all three fungi species investigated. The metal complexes 5-7 already show an increased fungitoxic activity (at the concentration of 10 laM, for which the data of Table I stand). At 0.1 laM concentrations no such activity was observed for the complexes, except for the Cu(II) derivative 5 (data not shown). At 10 laM, significant inhibitions of growth were detected for 5, which is almost as effective as clotrirnazole against A. niger, slightly more effective against A. flavum, and much less effective against Candida. The other two complexes showed a modest activity, which is anyhow greater against the Apergillus pp. as compared to Candida. The metal ion inducing best fungitoxic activity was Cu(II), followed by Co(II) and Ni(II), which possessed comparative efficiency.
As for the mechanism of action of these derivatives, probably it is similar to that of the imidazole, and triazole derivatives used clinically, i.e., the inhibition of sterol 14-c-demethylase, a microsomal cytochrome P-450 dependent enzyme system. 29 These compounds thus impair the biosynhesis of ergosterol for the cytoplasmic membrane and lead to the accumulation of 14-t-methyl sterols which may disrupt the close packing of acyl chains of phospholipids, impairing the function of membrane-bound enzymes and inhibiting growth. 29,3  aMean + standard error from 10 plates.