Synthesis, Characterization and in Vitro Antifungal Effect of Some Butyltin(IV) N-substituted 2-aminoethanethiolates

Six new N-substituted di- and tributyltin 2-aminoethanethiolates (cysteaminates) have been prepared and characterised by 1H, 13C and 119Sn NMR spectroscopy. All these compounds exhibit a good in vitro antifungal effect against selected types of human pathogenic fungi (Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, Trichosporon beigelii, Aspergillus fumigatus, Absidia corymbifera, Trichophyton mentagrophytes) and their activity is comparable with that of some antifungal drugs commonly used in the clinical use like ketoconazole. The structure-activity relationships in these compounds are discussed.


Introduction
The organotin 2-aminoethanethiolates (cysteaminates) have been studied previously for their potential polydonor abilities. However, biological properties, especially antifungal effects, of these compounds containing biologically interesting group SCH_,CHzN are almost unknown. It has been discovered recently that compounds of this type exhibit a relatively high in vitro antitumour activity-. Unfortunately, low solubility of these derivatives in water blocks further research in this context3. On the other hand, the lipophilic character of these derivatives seems to be no hamper for their antifungal effect. Thus, the set of new butyltin derivatives containing N-substituted group SCH2CH2N has been prepared and their in vitro activity against some medically important yeasts (Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, Trichosporon beigelii) and molds (Aspergilhts fitmigatus, A bsidia cotymbifera, Trichophyton mentagrophytes) has been investigated. Results

Scheme
The studied compounds have been prepared by the reaction of tri-or dibutyltin chloride with corresponding N-substituted 2-aminoethanethiole in benzene solution in presence of natrium methoxide as a base. The preparations were carried out under anaerobic conditions. All prepared organotin compounds are colourless oils stable on air.
In the case of tributyltin derivatives (1-3) the values of (9Sn) and J(9Sn,3C) are about 80 ppm and 330 Hz, respectively(s. Tab.IV.); the C-Sn-C angles, estimated 4'5 from these values of coupling constants are 108. These values indicate the presence of tetrahedrally bonded tin atom in the molecule, without any significant interaction between tin and nitrogen atom4". The values of (9Sn) do not show any concentration dependence and thus intermolecular association seems to be excluded. This conclusion is in agreement with A. Smicka, V. Buchta and K. Handlir Synthesis, Characterization and in vitro Antifungal Effect of Some Butyltin(IV) N-substituted 2-Aminoethanthiolates the results of previous study of Bu3SnSCHzCHzNEt2 .F or dibutyltin compounds (4-6) the values of (l9Sn) and IJ(II9Sn,13C) are about 130 ppm and 378 Hz, respectively (s.Tab.IV.), similar to these data for BuzSn(SCH,CHzNEtz)z , therefore, we suppose analogous structural features in these compounds (fourcoordinated tin atoms with C-Sn-C angle being 113).
In vitro antifungal screening.
The results of in vitro antifungal susceptibility testing against eight strains of potentially pathogenic fungi for humans are given in Table as a minimal inhibitory concentration (MIC, in lamol.l). For comparison, the MIC values of tri-, dibutyltinchloride, Bu3SnCl (7), Bu2SnClz (8), acyclic tributyltin-2-(N,Ndimethyl)aminoethanethiolate (9) and ketoconazole (KTZ) are given there, too. The parent N-substituted thioles without organotin moiety did not show any activity against the fungal strains tested. On the other hand, the studied organotin derivatives showed good antifungal effects which were comparable with the in vitro activity of antifungal drugs like ketoconazole6. Tributyltin compounds studied exhibit interesting antifungal effect towards some strains, against which ketoconazole is markedly poorly active (e.g. AF, AC).
Tab. I. The MICs (gmol.l) of studied compounds determined by microdilution broth test Fungal strain (incubation time) The data given in Table reveal higher antifungal effects of the tributyltin compounds 1-3 than that of the dibutyltin analogues 4-6. These results correspond with the dependence of antifungal and antibacterial activity of organotin compounds on the degree of tin atom alkylation in sequence RSnX3 < R2SnX2 < R4Sn << R3SnX. The organotin(IV) compounds with Sn-S bonding usually exhibit a considerable antifungal activity8'9. The studied N-substituted butyltin 2-aminoethanthiolates exhibit different act!vity in comparison with butyltin chlorides 7 and 8. Thus, dibutylstannyl derivatives 4 and 5 are more active than dibutylstannyldichloride against seven of the eight fungal strains applied (except the mold TM). On the contrary, the tributylstannyl derivatives 1-3 are less active than tributylstanny|chloride against six of the eight fungal strains applied. The derivatives 1-3 are more active than tributylstannylchloride only against the mold TM and the yeast CA (for one day incubation). These effects could be explained by different affinities of the studied compounds to water. An important role in the antifungal activity plays here the equilibrium between the lipophilic and hydrophilic character of the compounds during the transport through cell membranes.
The antifungal effect probably does not depend significantly on the structural changes of thiolate substituent. From the comparison with Bu3SnSCH2CHzN(CH3)_ (9) it seems to be obvious, that the presence of heterocyclic arrangement itself is not important for the antifungal effect of this type of compounds. The S-Metal Based Drugs Vol. 8, Nr. 3,2001 bonded substituent probably facilitates the transport through the cell membrane only, as it was already shown by van der Kerk and Luijten l. In the case of butyltin compounds, in which the substituent exhibits a biological effect as well, the synergic effect of substituent and butyltin group was observed lz.

Syntheses
Starting amines were commercially available and were dried by distillation with CaH. Thiirane was prepared from ethylene carbonate and KSCN 13. Benzene was dried by refluxing with sodium under argon atmosphere and distilled before use. The preparations of sulfanylethylamines and butyltin derivatives were carried out under Ar atmosphere using standard Schlenk technique. N-(2-sulfanylethyl)-N'-methylpiperazine (X=-NCH3): N-methylpiperazine, (4 g; 40 mmol) was dissolved in benzene (25 ml), than thiirane (2.4g; 40 mmol) dissolved in benzene (5 ml) was added in three portions in the intervals of 30 minutes at 60 C. Then the mixture was stirred for 3 hours at 60C and refluxed for 2 hours. The benzene was evaporated off under reduced pressure. Vacuum distillation (113-114 C/1 kPa) of the residue afforded 4.4 g of colourless liquid (yield 68%). The syntheses of other two 2-sulfanylethylamines were carried out using the same procedure with the following results: was added and the reaction mixture was stirred at room temperature for 2 hours. The precipitate of NaCI was filtrated off and the solvent was evaporated under reduced pressure. Vacuum distillation (155-8 C/18 Pa) of the residue afforded 1.91 g of a colourless liquid (yield 68 %). The other butyltin compounds (1, 2, 4-6) were prepared using the analogous procedure. Boiling points and the results of the elemental analysis of the studied compounds are given in Table II In vitro antifungal testing. The in vitro antifungal testing was carried out by the modified microdilution broth format of the M27-A guidelines 6. Quality control strains (Candida albicans ATCC 90028, C. parapsilosis ATCC 22019, C. krusei ATCC 6258) and ketoconazole (Janssen-Cilag, Beerse) as a reference drug were involved. All fungal strains were passaged on Sabouraud dextrose agar at 35C prior to being tested. Minimal inhibitory concentration (MIC) was determined by the following method: Dimethyl sulfoxide (DMSO) served as a diluent for all compounds tested. DMSO did not exceed the final concentration of 2 %. As a test medium was used RPMI 1640 (Sevapharma, Prague), supplemented with L-glutamine and buffered with 0.165 M morpholinepropanesulfonic acid (Serva) to pH 7.0 using 10 M NaOH. Each well of the microdilution tray was filled with 200 ktl of the RPMI 1640 medium with a diluted compound tested and then inoculated with 10 lal of suspension of a given fungal strain in sterile water. Fungal inoculum was prepared 3 to give a final size of 5 10 4-0.2 CFU.mI. The trays were incubated at 35 C and the MICs were visually read after 24 h and 48 h for all fungal strains tested except T. mentagrophytes (72 h and 120 h). The MIC was defined as at least a 80 % inhibition of the growth of control.