Antifungal Activity of Ag(I) and Zn(II) Complexes of Sulfacetamide Derivatives

Reaction of sulfacetamide with arylsulfonyl isocyanates afforded a series of derivatives which were used as ligands (as conjugate bases) for the preparation of metal complexes containing Ag(I) and Zn(II). The newly synthesized complexes, unlike the free ligands, act as effective antifungal agents against Aspergillus and Candida spp., some of them showing activities comparable to ketoconazole, with minimum inhibitory concentrations in the range of 0.3 – 0.5 μg/mL. The mechanism of antifungal action of these complexes seems to be not connected with the inhibition of lanosterol-14-α-demethylase, since the levels of sterols assessed in the fungi cultures were equal in the absence or in the presence of the tested compounds. Probably the new complexes act as inhibitors of phosphomannose isomerase, a key enzyme in the biosynthesis of yeast cell walls.


Introduction
Several important classes of antifungal compounds are presently available, 5 such as the azoles inhibiting lanosterol-14-c-demethylase, 14 sterol-A4-reductase, or AV-A isomerase as well as the inhibitors of the zinc enzymes phosphomannose isomerase, 6'b or topoisomerase. 6 All these enzymes are involved in the biosynthesis of fungi/yeast cell walls, and their inhibition leads to impaired function of the membrane and as a consequence death of the pathogenic species. Recently, some metal complexes such as silver sulfadiazine were shown to possess effective antifungal properties against the pathogenic yeast Candida albicans. '7s The mechanism of action of this complex seems to be connected with the inhibition of phosphomannose isomerase, a key enzyme in the biosynthesis of yeast cell walls. 6.v.s Since resistance to the different antifungal agents constantly emerges, 2491 it is important to investigate new types of compounds, able to prevent this serious medicalsproblem. Metal complexes, containing mainly Ag(I) and Zn(II) seem to be a valuable such alternative. 7"s'-' Indeed, silver sulfadiazine is extensively used clinically for the prophylaxis and treatment of bacterial and fungal burn infections, alone or in combination with mafenide acetate 2, as well as cerium(IV) nitrate, s'-5 Considering compound as lead molecule, we prepared some new derivatives of sulfacetamide 3, another widely used clinical agent, 6 by its reaction with arylsulfonyl isocyanates of type 4. v The conjugate bases of the new derivatives 5-9 were then used as ligands for the preparation of the Ag(I) and Zn(II) complexes [10][11][12][13][14][15][16][17][18][19]. The new compounds and their complexes were assayed for their antifungal properties against two Aspergillus and one Candida albicans strains.

Materials and Methods
Elemental analyses were done by combustion with a Carlo Erba Instrument or gravimetrically for the metal ions. NMR spectra have been recorded at 200 MHz, with a Varian Gemmini 200 spectrophotometer; chemical shifts are reported as 5 values, relative to Me4Si as external standard, in solvents specified in each case. IR spectra were recorded with a Perkin-Elmer spectrophotometer using KBr pellets as reference. Conductimetric measurements were done at room temperature on a Radelkis KFT conductimeter.

Derivatives
Sulfacetamide, sulfadiazine, mafenide hydrochloride, arylsulfonyl isocyanates, metal salts and solvents were commercially available (from Sigma, Aldrich or Janssen) and were used without further purification.
General procedure for preparation of arylsulfonylureido sulfacetamides [5][6][7][8][9] An amount of 2.15 g (10 raM) of sulfacetamide was suspended in 100 mL of highly anhydrous (kept on molecular sieves) acetonitrile and magnetically stirred at 4C for 10 rain. The stoichiometric amount of arylsulfonyl isocyanate 4 (eventually dissolved in the same solvent for the solid compounds, or in pure state for the liquid ones) was then added dropwise, maintaining the temperature under 10 C. The reaction mixture was stirred at room temperature for 2-4 h (tic control), then the'solvent was evaporated in vacuo and the residue crystallized from ethanol-water. Yields were practically quantitative. Assay of sterols present in the fungi cultures A reverse-phase HPLC method adapted from the literature,has been used to determine the amount of sterols (ergosterol and [anosterol) present in the fungi cultures. The fungi have been cultivated as mentioned above for 5 days, with or without inhibitors added in the nutrient broth. Culture media were suspended in a small volume of MOPS buffer (pH 7.4) and the cells centrifuged at 20000g for 30 rain. Cells were weighed (wet paste) and broken by sonication. Sterols present in the homogenate were then extracted in chloroform, the solvent has been evaporated to a small volume and the extracts applied on a la-Bondapak-Cl8 column, with acetonitrile as eluting solvent. Authentic ergosterol and lanosterol (from Sigma) were used as standards. The flow rate was of 3 mL/min. The retention times were: 8.87 rain for ergosterol; and 7.62 rain for lanosterol, respectively. Blank assay were done for cultures which were not treated with inhibitors in order to assess the normal levels of sterols present. The amount of ergosterol tresent in the same amount of wet cells from the culture grown in the absence of inhibitor was taken as 100%. 21"24 Results and Discussion Reaction of sulfacetamide 3 with arylsulfonyl isocyanates 4, afforded the ureido derivatives 5-9, by the procedure already reported previously by this group. 7 The new compounds obtained as outlined in Scheme 1, were characterized by spectroscopic and analytic data that confirmed their structures.
Metal complexes 6-11 containing the conjugate base of sulfonamides 5-9 (LH) and Ag(1) or Zn(lI) ions, of types 10-19, were also obtained (Scheme 1), and their elemental analysis data are shown in Table I The new complexes have been characterized by spectroscopic, conductimetric and thermogravimetric measurements (Table II). By comparing the IR spectra of the complexes and the free ligand, the following differences were evidenced: (i) the shift of the two sulfonamido vibrations (both the symmetric as well as the asymmetric one) belonging to the SO2NHAc moiety (at 1173-1176 cm , and 1378-1382 cm") towards lower wavenumbers in the spectra of the complexes, as compared to the spectra of the corresponding ligand (Table   5.9 II), as already documented previously for similar complexes.-One should note that only one pair of such vibrations underwent the above-mentioned shift, presumably those of the SO2NHAc moiety, whereas the second sulfonamide (X-C6H4SO_NHCONH) moiety appeared at the same wavenumbers both in ligands as well as in their metal complexes (data not shown). This is a direct indication that the deprotonated SO_NHAc moiety of the ligand interacts with the metal ions in the newly prepared coordination compounds; (ii) the acetamide stretching vibration in the spectra of the prepared complexes are shifted with 15-20 cm towards lower wavenumbers, as compared to the same vibration in the spectrum of sulfonamides 5-9 indicating again that this moiety is probably in the vicinity of the metal ions (data not shown); (iii) the ureido vibration in the spectra of complexes 10-19, assigned as the intense band at 1680 cm appear at the same wavelength as that of the corresponding free ligands (Table II), suggesting that these moieties do not participate in the coordination of the metal ions. One may reach the same conclusion by studying the H-NMR spectra of the Ag(I) and Zn(II) complexes, as compared to the corresponding spectra of the ligands. Thus, the only difference between the two types of spectra concerns the signal of the methyl group of the AcNHSO2 moiety, which in the complexes appears at 2.55 2.68 ppm, whereas in the ligands at 2.75-2.77 ppm (Table II). Similar behaviors were also evidenced previously for other sulfonamide metal complexes, by our group, ,_5-29 and probably indicate that this methyl moiety is in the vicinity of the zinc/silver ions in the prepared complexes. Conductometric data (Table II) also indicate that the new complexes 10-19 are non-electrolytes, being undissociated in DMF (or DMSO) as solvent.

Derivatives
Biological activity data with the new derivatives 1-19 and the standard azole antifungal agent ketoconazole 20 are shown in Table III.  [10][11][12][13][14][15][16][17][18][19] reported here represent a new class of antifungals with MIC-s (minor inhibitory concentration) in the micromolar range, which might induce strong in vivo antifungal effects. Furthermore, the ligands from which the complexes were prepared, as well as the related sulfonamide 2, are devoid of such antifungal properties, against the three strains investigated here. The most active derivative were the Ag(I) complexes 10-14, followed by the Zn(II) ones containing the same type of ligand. From this point of view, the halogeno-substituted ligands led to more active antifungal complexes as compared to the phenyl-and tosyl-ureido derivatives. Candida was most susceptible to inhibition, followed by A. flavus, whereas A. niger was the most resistant to this type of antifungals. In this respect, the complex derivative parallel the biological activity of ketoconazole, although they are less active. One should anyhow note that some of our new complexes are more active antifungals than silver sulfadiazine 1, a clinically widely used derivative (Table III).
Ketoconazole 20 is known to act as an inhibitor of lanosterol 14-c-demethylase (CYP51A1), a microsomal cytochrome P-450 dependent enzyme system belonging to a gene superfamily involved in sterol biosynthesis in fungi, plants and animals. 24 CYP51A1 has been shown to catalyze the conversion of lanosterol to the 14-desmethylated derivative, ergosterol, through a complicated oxidative sequence. Its inhibition in fungi causes the depletion of ergosterol and accumulation of 14-methylsterols in the membrane of the cells, disturbing thus membrane function and causing the death of these organisms. 24 Thus, in order to investigate whether the complexes reported here act as ergosterol biosynthesis inhibitors, similarly to the azole antifungals, the amounts of ergosterol present in C. albicans cultures after treatment with different concentrations of the new the inhibitor 11 and ketoconazole 20, a potent CYP51A1 inhibitor, 2 have been determined by means of a HPLC method (Table IV) *Mean + standard deviation (n 3); The amount of ergosterol present in the same amount of wet cells from the culture grown in the absence of inhibitor is taken as 100 %.
The data of Table IV show that, in contrast to ketoconazole, the silver complex investigated here, 1, does not act as inhibitor of CYP51A1, possessing thus a different mechanism of antifungal action. Probably, as many other Ag(1) derivatives, the new antifungals might exert their effects through poisoning some components of the respiratory enzymes in the fung, al/microbial electron transport system, or even through interaction with the DNA of the pathogenic species. "3 In conclusion we report here the preparation and antifungal activity of some Ag(I) and Zn(II) complexes of sulfacetamide derivatives, possessing interesting biological activity against several Aspergilhts and Candida strains.