Antifungal activity of organotin compounds with functionalized

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Medical Mycology March 2010, 48, 373–383. Antifungal ... mentous fungi towards new organotin compounds: ( 1) [Sn(C. 4. H. 9. ) ... 2010 ISHAM, Medical Mycology, 48, 373–383 ..... growth in comparison with the growth control, drug-free.
Medical Mycology March 2010, 48, 373–383

Antifungal activity of organotin compounds with functionalized carboxylates evaluated by the microdilution bioassay in vitro MARIUSZ DYLA˛G*, HANNA PRUCHNIK†, FLORIAN PRUCHNIK‡, GRAZ˙YNA MAJKOWSKA-SKROBEK* & STANISŁAW UŁASZEWSKI* *Institute of Genetics and Microbiology, University of Wroclaw, Wroclaw, †Department of Physics and Biophysics, Institute of Natural Sciences,Wroclaw University of Environmental and Life Sciences, Wroclaw, and ‡Faculty of Chemistry, University of Wroclaw,Wroclaw, Poland

We investigated the susceptibility of 96 well-characterized strains of yeast-like and filamentous fungi towards new organotin compounds: (1) [Sn(C4H9)3(OOCC6H4SO3H-2)], (2) Sn(C4H9)3{OOC(CH2)3P(C6H5)3}]Br, and (3) [Sn(C6H5)3 [OOC(CH2)3N(CH3)3}] Cl. In the case of yeast-like fungi, the in vitro susceptibility tests were carried out according to the Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) reference method M27-A2, while for filamentous fungi the investigations were conducted according to the M38-A and M38-P methods. The organotin complexes 1, 2 and 3 are active antifungal agents. Minimal inhibitory concentrations (MIC) were in the range of 0.25–4.68 μg/ml for all tested fungal strains. Considerably larger differences were found for minimal fungicidal concentrations (MFC). In the case of yeast-like fungi, the fungicidal effect was generally observed at organotin compounds concentrations of 2.34–9.37 μg/ml. The MFC values for filamentous fungi were considerably higher and were in the range of 18.74–50 μg/ml. In conclusion, organotin compounds 1, 2 and 3 showed high fungistatic and fungicidal activities against different species of pathogenic and nonpathogenic fungi. However, they were also highly cytotoxic towards two mammalian cell lines. Keywords Organotin compounds, antifungal activity, broth microdilution, yeast, filamentous fungi

Introduction There are several classes of compounds showing antifungal activity, of which the azoles are one of the most important. They show mainly fungistatic activity [1–3], and as a result some fungi may become resistant to them during the therapy. Primary and induced resistance, especially with respect to the pathogenic yeast Candida krusei and Candida glabrata have also been found [4–7]. The additionally unfavorable phenomenon is interaction of these compounds with other

Received 19 February 2009; Received in final revised form 6 July 2009; Accepted 16 July 2009 Correspondence: Mariusz Dylag, Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/67, 51-148 Wroclaw, Poland. E-mail: [email protected]

© 2010 ISHAM

drugs [2,8]. Amphotericin B, which belongs to the polyenes antibiotic group, is well known to interact with ergosterol in cell membranes and is still universally applied in the therapy of systemic mycoses. Moreover, there is a resistance to use this drug because of its considerable toxicity for mammalian cells and because of the limited absorption in the case of oral application [1,2]. Other antifungal agents, like echinocandins and ciclopirox have limited possibilities of application [1,2] or like griseofulvin, have relatively narrow antifungal spectrum restricted mainly to dermatophytes [2,8–10]. Additionally echinocandins have no activity against Cryptococcus neoformans and Trichosporon spp. [1,3]. Similarly, flucytosine has a relatively narrow antifungal spectrum because a high number of filamentous fungi lack one or all of the enzymes which are very important in its mechanism of action. These enzymes include cytosine perDOI: 10.3109/13693780903188680

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mease, cytosine deaminase and uracil phosphoribosyl transferase. Therefore, the spectrum of flucytosine is restricted to the pathogenic yeasts, mainly to members of the genus Candida and Cryptococcus [1]. In the case of terbinafine weak activity was confirmed against species of Candida [1,11]. Another important group of antifungal agents are organotin compounds as it is well known that organometallic tin (IV) compounds display strong biological activity [12–14]. Their activity is essentially determined by the number of Sn-C bonds and nature of the organic groups bound to the central Sn atom. For the organotin series [SnRnX4-n], the most active are [SnR3X] compounds and then [SnR2X2]. In addition, the activity depends on the nature of the organic groups (R), e.g., tributyltins are the most active against Gram-positive bacteria and fungi [13]. Many organotin compounds are considerably more active anti-tumor agents than cis-[PtCl2(NH3)2 (cisplatin) [14–19]. A fundamental overview of the world literature which discusses the issue of anti-proliferative and anti-tumor activity of organotin compounds, was completed by Hadjikakou and Hadjiliadis [19]. These compounds also show antibacterial [12,13,18,22,23] and antifungal activity [12,13,18,20–23]. The activity of [SnRnX4-n] also depends to some extent on electronic and structural properties of ligand X. Many carboxylato triorganotin and diorganotin complexes have been synthesized and tested as antitumor [15–17], antibacterial and antifungal agents, e.g., [SnBu3 (OOCR)], [Sn4O2Bu8(OOCR)4] [18,20,24]. The triorganotin and diorganotin complexes are rather sparingly soluble in water. Introduction of polar substituent into RCOO ligand allows modification of their coordination properties and solubility of the carboxylato complex. In this paper, we report the antifungal activity of triorganotin complexes [Sn(C4H9)3(OOCC6H4SO3H-2)] (1), [Sn(C4H9)3{OOC (CH2)3 P(C6H5)3}]Br (2) and [Sn(C6H5)3 [OOC(CH2)3N (CH3)3}]Cl(3) with carboxylato ligands containing sulfo-, phosphonium- and ammonium- groups.

Materials and methods Reagents and solvents (pro analysis) were purchased from the Polish company POCH and Sigma-Aldrich and were used as received. The triorganotin carboxylates were prepared with the modified method described earlier [25]. Infrared spectra (KBr pellets and nujol) were recorded with a Bruker IFS 113v and Bruker 66/s, UV-Vis spectra on a Cary 500 and 1H, 13C and 119Sn NMR spectra on a Bruker AMX 300 and Bruker Avance 500. Proton chemical shifts () were reported with reference to the residual CD3OD ( 3.30 ppm) in CD3OD or DMSO ( 2.49 ppm) in DMSOd6; 13C chemical shifts were given with respect to the natural contents of 13CD3OD ( 49.00 ppm) in CD3OD or DMSO ( 39.52 ppm) in DMSO-d6 and 119Sn chemical shifts were reported with reference to the external Sn(Bun)Cl3 in

CDCl3 ( 6.1 ppm). All elemental analyses were performed with a Vario EL3 CHN analyzer. The minimal inhibitory concentration (MIC) [26] and minimal fungicidal concentration (MFC) values [26] for all complexes were determined according to standard procedures. The biological in vitro activity was determined by the microdilution bioassay according to the M27-A2 method, which is recommended for yeast by National Committee for Clinical Laboratory Standards (NCCLS) [27,28]. In the case of filamentous fungi, the evaluation of the in vitro activity was assessed using the methods NCCLS M38-A and M38-P [29,30]. The evaluation of cytotoxicity of all the tested compounds was performed on human lung adenocarcinoma cell line A 549 and mouse fibroblast cell line L 929. Both lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and maintained in the Cell Culture Collection of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland. A total of 549 cells were cultured in Alpha-MEM and L 929 cells in RPMI-1640 (Gibco, Warsaw, Poland). The basic culture media were supplemented with 2mM L-glutamine (Gibco, Warsaw, Poland), 1% antibiotic-antimycotic solution (PAA) and 10% heat-inactivated fetal bovine serum (Gibco, Warsaw, Poland). The cell cultures were maintained at 37°C in a humidified atmosphere consisting of 5% CO2/ 95% air. Organotin compounds Compound 1 Tributyl(2-sulfobenzoato)tin(IV) [Sn(C4H9)3 {OOCC6H4(SO3H-2}] The mixture of {Sn(C4H9)3}2O (2.54 cm3, 5  10−3 mol) and 2-sulfobenzoic acid monohydrate (2.202 g, 10−2 mol) in ethanol (10 cm3) was heated at 333 K for 1 h. The solvent was evaporated and a residue dissolved in diethyl ether and stirred at reflux for 30 minutes and evaporated to about 5 ml. The white product was filtered off and dried in vacuo. Yield: 3.97 g, 79%. Anal. Calcd. for [Sn(C4H9)3{OOCC6H4(SO3H-2)}]: C 46.46, H 6.57, S 6.53. Found: C 46.41, H 6.38, S 6.29. 1H NMR (CD OD) 0.92 ppm (t, 9H, CH , 3J(HH)  7.2 3 3 Hz), 1.27–1.33 ppm (m, 6H, αCH2), 1.34–1.41 ppm (m, 6H, γCH2), 1.61–1.69 ppm (m, 6H, βCH2), 7.51–7.54 ppm (m, 2H, H3, H4), 7.75–7.61 ppm (m, 1H, H6), 7.94–7.973 ppm (m, 1H, H3). 1H NMR {(CD3)2SO} 0.87 ppm (t, 9H, CH3, 3J(HH)  7.2 Hz), 1.13 ppm (m, 6H, αCH2), 1.31 ppm (m, 6H, γCH2), 1.57 ppm (m, 6H, βCH2), 7.46 ppm (broad) (3H, C6H4), 7.78 ppm (broad) (1H, H6), 14.16 ppm (s, 1H, SO3H). 13C NMR (CD3OD) 13.97 ppm (CH3), 19.02 ppm (αCH2, 1J(119/117Sn13C)  441.8/422.9 Hz), 28.02 ppm (γCH2, 3J(119/117Sn13C)  79.8 Hz), 28.94 ppm (βCH2, 2J(119/117Sn13C)  27.9 Hz), 128.66 ppm (Caryl), 130.27 ppm (Caryl), 131.04 ppm (Caryl), 131.61 ppm (Caryl), © 2010 ISHAM, Medical Mycology, 48, 373–383

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142.79 ppm (Caryl), 173.33 (COO). 119Sn NMR (CD3OD): 45.6ppm. IR (KBr, cm−1) 445.48 w, 554.43 w, 570.83 m, 619.04 m, 622.8 s, 660.5 m, 713.5 m, 731.8 m, 757.8 m, 800.3 w, 879.3 m, 961.3 w, 994.1 m, 1021.1 s, 1037.5 m, 1079.9 s, 1130.0 s, 1141.6 s, 1187.9 vs, 1210.1 s, 1238.0 s, 1271.8 s, 1341.2 w, 1377.8 m, 1405.8 s, 1464.6 m, 1538.9 m, 1577.4 m, 1592.9 m, 1722.1 s, 2855.0 s, 2871.4 s, 2923.5 vs, 2957.3 vs, 3064.33 m, 3100.01 m, 3140.51 m, 3439.42 s. Compound 2 (4-oxo-4-(tributylstannyloxy)butyl)(triphenyl)phosphonium bromide-ethanol [Sn(C4H9)3{OOCCH2 CH2CH2P(C6H5)3}]Br·C2H5OH. Solution of {Sn(C4H9)3}2 O (2.27 cm3, 2.5 mmol) and (3-carboxypropyl)triphenylphosphonium bromide (2.147 g, 5 mmol) in ethyl alcohol (20 cm3) was heated at 60°C for 3 h. Solvent evaporated in vacuo. The yield of a viscous colorless product was 2.49 g, 72%. Anal. Calcd for C36H54O3PSnBr: C 56.57, H 7.12, P 4.05 Br 10.45. Found: C 56.11, H 7.30, P 3.55 Br 11.05. 1H NMR (CD OD): 0.87 ppm (t, 9H, CH , 3J(HH)  7.3 3 3 Hz), 1.17 ppm (t, 3J(HH)  7.1 Hz), 1.19 ppm (t, 6H, αCH2, 3J(HH)  8.0 Hz), 1.32 ppm (sextet, 6H, αCH2, 3J(HH)  7.4 Hz), 1.61 ppm (kwintet, 6H, βCH , 3J(HH) 2  7.9 Hz), 1.86–1.95 ppm (m, 2H, CH2CH2COO), 2.47 ppm (t, 2H, CH2COO, 3J(HH)  6.8 Hz), 3.42–3.50 ppm (m, 2H, CH2P), 7.76 ppm (dt, 6H, meta-H, 3J(HH)  7.8 Hz, 4J(PH)  3.5 Hz), 7.83 ppm (ddd, 6H, orto-H), 3J(HH)  7.8 Hz, 3J(PH)  12.6 Hz, 3J(HH)  1.5 Hz), 7.89 ppm (dt, 3H, para-H, 3J(HH)  7.5 Hz, 3J(HH)  1.7 Hz),. 13C NMR (CD3OD): 14.07 ppm (s, 3C, CH3), 18.37 ppm (s, 1C, CH3CH2OH), 19.03 ppm (s, 3C, αCH2, 1J(119/117Sn13C)  440.6/421.7 Hz), 20.40 ppm (s, 1C, CH2CH2COO), 23.15 ppm (s. 1C, CH2COO), 27.97 ppm (s. 3C, γCH2, J(119/117Sn13C)  73.8 Hz), 29.23 ppm (s, 3C, βCH2, 2J(119/117Sn13C)  27.5 Hz), 37.16 ppm (s, 1C, CH P), 2 58.28 ppm (CH2OH), 115.31 ppm (d, 1C, ipso-C, 1J(PC)  86.4 Hz), 131.55 ppm (s, 2C, o-C), 134.81 ppm (s, 2C, m-C), 136.30 ppm (s, 1C, p-C), 178.96 (s, 1C, COO). 119Sn NMR (CD3OD): 22.95 ppm. IR (KBr, cm-1) 434w, 451w, 490s, 508s, 519m, 531m, 608m, 690s, 723s, 741s, 788w, 848w, 877m, 930vw, 963w, 997m, 1025m, 1050m, 1076m, 1113s, 1138m, 1164m, 1182m, 1269s, 1303s, 1315m, 1341s, 1382s, 1415m, 1439vs, 1455s, 1464s, 1485m, 1537w, 1575s, 1588s, 1616vs, 2854s, 2869s, 2922vs, 2953vs, 3010m, 3022m, 3058m, 3081m, 3151w, 3175w. Compound 3 trimethyl{4-oxo-4-(triphenylstannyloxy) butyl}ammonium chloride [Sn(C6H5)3 {OOCCH2CH2CH2 N(CH3)3}]Cl. Mixture of Sn(C6H5)3OH (1.835 g, 5 mmol) and (3-carboxypropyl) ammonium chloride (0.908 g, 5 mmol) in ethyl alcohol (60 cm3) was heated at 60°C for 3 h. White product was filtered off, washed with ethanol and dried in vacuo. Yield: 2.043 g, 77%. Anal. Calcd for C25H30O2NSnCl: C 56.58, H 5.70, N 2.64, Cl 6.68. Found: C 56.81, H 4.70, N 2.10, Cl 7.66. 1H NMR (CD3OD) 1.91 © 2010 ISHAM, Medical Mycology, 48, 373–383

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ppm (m, 2H, CH2CH2COO), 2,27 ppm (t, 2H, CH2COO,  6.8 Hz), 2.99 ppm (s, 9H, CH3), 3.14 ppm (m, 2H, CH2N), 7.39–7.49 ppm (m, 9H, SnC6H5, m,p-H), 7.82 ppm (dd, 6H, SnC6H5, o-H, 3J(HH)  7.8 Hz, 4J(HH)  1.5 Hz, 3J(119/117SnH)  63.0 Hz). IR (KBr, cm-1) 445w, 456s, 486w, 542w, 593w, 600w, 619w, 658w, 678w, 700vs, 735s, 791vw, 856vw, 878vw, 935w, 951w, 969w, 997m, 1023w, 1072m, 1125w, 1157w, 1188w, 1209w, 1262w, 1317m, 1334w, 1410s, 1428s, 1443m, 1480s, 1553vs, 1572s, 1581s, 1630m, 1773w, 1830w, 1887w, 1904w, 1964w, 2911m, 2951m, 2988m, 3031m, 3046m, 3054m, 3066m. 3J(HH)

Fungal strains Yeast-like fungi. A total of 41 strains of 8 yeast-like species were included in the studies. The following are the members of the test population: Cryptococcus neoformans (Sanfelice) Vuillemin, six clinical strains isolated from cerebrospinal fluid (CSF) and blood; Candida krusei (Castellani) Berkhout, six clinical strains derived from nail, oral and cutaneous specimens in cases of candidiasis; Candida albicans (Robin) Berkhout, eight clinical strains obtained from patients with nail, cutaneous and vulvovaginal candidiasis and one reference strain ATCC90028 from American Type Culture Collection; Candida glabrata (Anderson) Meyer et Yarrow, four clinical strains isolated from patients with cutaneous and vulvovaginal candidiasis; Candida famata (Harrison) Meyer et Yarrow, three clinical strains obtained from patients with nail and cutaneous candidiasis; Rhodotorula rubra (Demme) Lodder, four clinical strains isolated from nail samples; Geotrichium candidum Link, four clinical strains, all from patients with oral infections, and Trichosporon cutaneum (de Beurmann et all) Ota, five clinical strains isolated from skin and nail infections. Filamentous fungi. A total of 55 strains belonging to 11 species were tested. The test isolates comprised: Trichophyton rubrum (Castellani) Sabouraud, eight clinical strains, all isolated from patients with onychomycosis; Trichophyton mentagrophytes var. mentagrophytes (Robin) Blanchard, four clinical strains recovered from patients with tinea capitis trichophytica and tinea barbae; Trichophyton tonsurans Malmsten, three clinical strains, from patients with tinea capitis trichophytica; Microsporum canis Bodin, six clinical strains isolated from children with tinea capitis microsporica; Microsporum gypseum (Bodin) Guiart et Grigorakis, four clinical strains isolated from skin infections; Epidermophyton floccosum (Harz) Langer. Et Milochevitch, five clinical strains obtained from patients with tinea inguinalis and tinea cutis glabrae; Aspergillus flavus Link, five environmental

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strains isolated from buildings and a library with humidity problems; Aspergillus fumigatus Fresenius, two clinical strains isolated from a patient with pulmonary aspergillosis and a patient with otomycosis and two environmental strains isolated from the library with humidity problems; Scopulariopsis brevicaulis (Sacc.) Bainier, six clinical strains, all obtained from patients with acauliosis; Fusarium avenaceum (Corda ex Fries) Saccardo, four environmental strains isolated from agricultural plants, and Mucor plumbeus Bonorden, four environmental strains obtained from buildings with humidity problems, wood and soil. We should emphasize that the great majority of strains used in our investigation were clinical cases involving patients who had not previously been given any antifungal therapy. Preparation of solutions Complexes 1–3 are soluble in methanol, ethanol, dimethyl sulfoxide (DMSO) and in other polar solvents. Solutions of these compounds are stable for at least two weeks. They are also stable in a mixture of water/methanol (50/50). The 1H NMR spectra of complexes in CD OD and D O/CD OD 3 2 3 did not change after this period. Compounds in solid state have most probably pseudo-tetrahedral structure with carboxylato ligand coordinated strongly via one oxygen atom, and distance between Sn atom and O atom of C  O group is considerably longer [16,18,24]. Signal at 14.16 ppm in the 1H NMR spectrum of complex 1 in (CD3)2SO evidences the presence of SO3H proton. The 1H, 13C and 119Sn NMR spectra indicate that compounds 1 and 2 in methanol have trigonal bipiramidal structure with methanol molecule coordinated with central atom. The 13C (αCH2) chemical shifts are at 16.0–16.5 ppm for four-coordinated [Sn (C4H9)3(OOCR)] compounds, in solutions in non coordinating solvents (e.g., chloroform) and they are shifted downfield for 1–2 ppm in more polar, more strongly coordinating solvents (DMSO, MeOH). The same dependence has been found in the case of 119Sn chemical shifts [31,32]. The 13C (αCH2) and 119Sn chemical shifts for complex 1 are equal to 19.02 ppm (1J(13C119/117Sn  441.8/422.9 Hz) and 45.59 ppm, and for complex 2: 19.03 ppm (1J(13C119/117Sn  440.6/421.7 Hz) and 22.95 ppm. Thus all these values confirm that complexes 1 and 2 in methanol and other coordinating solvents are five-coordinated and have trigonalbipiramidal structure with three butyl groups in equatorial positions and with carboxylato ligand and methanol molecule in axial coordination sites [16,31,32]. The 1H NMR spectrum of compound 3 is similar to the spectra of other triphenyltin (IV) compounds. Therefore, this complex in the methanol solution also probably has trigonal-bipiramidal structure [31,32]. The compounds 1 and 2 were dissolved in the 96% ethanol (Polmos). Complex 3 was

dissolved in DMSO (Fluka), because of its better solubility in this solvent than in ethanol and because of precipitation which was observed if the ethanolic compound solution mixed with RPMI 1640 medium. All three tested compounds were readily soluble in the above mentioned solvents. The compounds stability in DMSO and ethanol and the level of their antifungal activity was also confirmed. It was examined using fresh, directly prepared stock solutions and then stored for two weeks at 2–8oC. Using all C. albicans isolates we found no differences in the level of antifungal activity between fresh stock and stock solutions of compounds which had been stored for two weeks. The solubility of these compound solutions in standard culture media used in this study also assessed. Complexes 1 and 2 dissolved in ethanol and compound 3 dissolved in DMSO were soluble and stable in Sabouraud dextrose [26], YPD [33], PDA [34] and RPMI 1640 media. To examine stability and check how constant their antifungal activity level was, identical tests were performed for a few chosen strains of tested fungi with the use of the same microdilution plates containing liquid medium with proper compound concentrations in each well. These control microdilution plates without inoculum suspension of a tested strain were kept in a dark-room, at 2–8°C for one week. After this time the procedure was continued and the inoculum suspension was added to each well. Comparisons of MIC values for direct inoculation were made and after one week the high stability of compounds was confirmed in the medium as well as the lack of differences between MIC values. Additionally, the agar dilution technique [35] was specially used for filamentous fungi to confirm lack of any differences in the colony diameter. In this method, culture media (PDA and Sabouraud dextrose agar) containing compounds in different concentrations were inoculated immediately after preparation and after expiration of a two-week storage period at 2–8°C. As described in the NCCLS M38-P [30] and M27-A2 [27,28] documents, solutions of the test compounds were prepared at concentrations 100 times higher and then were diluted 50 times with the NCCLS standard liquid RPMI 1640 medium containing L-glutamine, 2% glucose (without sodium bicarbonate), and buffered using 0.165M morpholinepropanesulfonic acid (MOPS) to obtain the final pH 7.0 (Sigma-Aldrich Inc., St.Louis, USA). The concentration of the solution prepared in this manner was two times higher than required for the test [27,28,30]. The final concentrations of compounds used in broth microdilution bioassay were 0.068–50 μg/ml. Inoculum preparation All yeast-like strains were subcultured onto solid YPD medium and then after 48 h of incubation at 28°C (37°C in the case of C. neoformans) one colony of each strain © 2010 ISHAM, Medical Mycology, 48, 373–383

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was transferred to initiate cultures in YPD broth, which were incubated at 28°C (37°C for C. neoformans). Each yeast-like strain used to prepare inoculum suspension for microdilution bioassay was in the exponential (log) phase of growth, which was identified on the basis of a growth curve of each [36]. This phase in our investigation was usually evident after 18 to 24 h of incubation. Inoculum suspensions were made in liquid RPMI 1640 medium using small volumes of YPD broth medium and adjusted to a final optical density (OD) of 0.5 in the McFarland scale (Densimat, Biomerieux). At least a 1:1000 dilution was performed to yield a suitable inoculum of 0.5  103 to 4.5  103 CFU/ml according to method M27-A2 [27,28]. The inoculum suspensions of filamentous fungi were prepared in sterile RPMI 1640 medium in accord with the NCCLS method [29,30,34,37]. All tested strains were subcultured onto potato dextrose agar (PDA; Difco) plates at 28°C (for dermatophytes) [34,38] and at 35°C (for other filamentous fungi) [28]. Inocula of each strain were prepared from 7- to 14-day-old cultures grown on PDA. Colonies of the test strains were covered with 5–10 ml of sterile 0.85% NaCl with Tween 80 (to prevent conidia aggregation) at final concentration of 0.025%, and the suspension was prepared by gently scrapping the surface with the sterile swab wetted in saline. Finally the mixture of conidia and hyphal fragments was transferred to a new sterile tube. This mixture was shaken for 20 sec and then kept for 15 to 20 min for sedimentation of heavy hyphal fragments and the next upper homogeneous suspensions were collected [37,38]. Inoculum suspensions were adjusted spectrophotometrically to optical densities [OD] that were approximately 0.125 at 550 nm. The inoculum suspensions were diluted 1:50 in RPMI1640 medium [29,30,37]. Homogenous suspensions contained mostly conidia and the final sizes of the stock inoculum suspensions ranged from 0.5  104 to 4.5  104 CFU/ml (colony forming units per mililiter), as confirmed by quantitative colony counts after plating serial dilutions of the inoculum suspensions on Sabouraud dextrose medium [26,37]. Broth microdilution method Investigations of biological activity for all tested compounds were carried out through the use of special, sterile 96-well round-bottom microtiter plates (Nalge Nunc International, Dk-400 Roskilde, Denmark). Serial dilution of compounds were made in liquid RPMI 1640 medium (pH 7.0), as used to prepare inocula for each strain. Each well contained 100 μl of a diluted compound solution in liquid medium and was inoculated with 100 μl of inoculum suspension. Controls of growth, sterility and solvents influence were included for each tested strain. To evaluate the quality of the test, the reference strain C. albicans (ATCC © 2010 ISHAM, Medical Mycology, 48, 373–383

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90028) was included. In this case fluconazole (Pfizer, UK) dissolved in sterile distilled water was used instead of the organotin compounds. The MIC values of fluconazole for the reference strain ranged between 0.275 μg/ml to 0.39 μg/ml. For each tested strain, microdilution MIC values of the organotin carboxylates were evaluated in the study, and MIC ranges for quality control strain were well established [39]. We must point out that for all three tested organotin carboxylates MIC100% values were fixed as distinct from MIC50% values for fluconazole, according to recommendations for azoles [29,38]. The microtiter plates were incubated at 28°C for all tested strains and at 37oC in the case of C. neoformans. The MICs were read after the 24 h of incubation for Candida and Rhodotorula species and after 48 h for slower growing C. neoformans, G. candidum and T. cutaneum. For strains of the filamentous fungi the results were read after 24 h in the case of M. plumbeus, after 48 h in the case of Aspergillus and Fusarium species and after 72 h for more slowly growing S. brevicaulis [27–29]. In the case of remaining filamentous fungi, the plates were read after 4–5 days of incubation (for T. rubrum, T. mentagrophytes, T. tonsurans, M. canis and M. gypseum) and after 7 days for E. floccosum [37]. The concentration at which no growth was observed was accepted as MIC100% values [34] (100% inhibition of growth in comparison with the growth control, drug-free well) (Fig. 1 & 2). For all tested strains of each species, the MIC value ranges were defined. Apart from marking the MICs endpoints for different strains of fungi, the aim of our study was also to determine the value of MFC and to do so after a long incubation time, 20 μl of sample taken from several microwells were cultured in triplicate [40] on Petri dishes with solid YPD medium for yeast-like fungi and with PDA medium for filamentous fungi. The samples were taken from control microwells, from a microwell with minimal inhibition concentration (MIC) and from all microwells with organotin compound concetration higher than the MIC. Reading was performed only when growth was seen on plates where samples from the growth control microwells were inoculated. MFCs were defined as the lowest compound concentration for which approximately 99–99.5% killing activity was observed [40]. The latter was determined by counting colonies on plates designed for control growth and plates designed for cultivating the fungi from microwells including different concentrations of tested compounds. For all tested strains from each species MFC value ranges were determined. These studies were performed with triplicate rows. Cytotoxicity tests The sulphorhodamine B (SRB) bioassay was performed according to the method of Skehan et al. [41]. Twenty-four

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Fig. 1 MIC100% value determination for compound 3 [Sn(C6H5)3[OOC(CH2)3N(CH3)3}]Cl against one of the clinical strains of Candida albicans isolated from a nail infection (microwell A10- MIC100% value, concentration 1.17 μg/ml, microwell A11-concentration 0.78 μg/ml, microwell B1sterility control of medium, microwells B2, B3, B4-controls of growth) and Candida krusei isolated from a nail infection (microwell C7-MIC100% value, concentration 3.125 μg/ml, microwell C8-concentration 2.34 μg/ml, microwell D1-sterility control of medium, microwells D2, D3, D4-controls of growth).

hours before addition of the tested compounds, the exponentially growing cells were seeded in 96-well tissue culture plates (Nunc) at a density of 2  104 cells per well in 100 μl of culture medium for A 549 and a density of 4  104 cells per well for L 929. An assay was performed after 24 to 72 h of exposure of the cultured cells to varying concentrations (0.1–100 μg/ml) of the tested agents. A negative control containing culture medium alone was also evaluated. The cells attached to the plastic were fixed by gently layering cold 50% trichloroacetic acid (SigmaAldrich, Germany) on the top of the culture medium in each well. The plates were incubated at 4°C for 1 h and then washed five times with tap water. The cellular material fixed was stained with 0.4% sulforhodamine B (Sigma-

Aldrich, Germany) dissolved in 1% acetic acid (POCh, Gliwice, Poland) for 30 min. Unbound dye was removed by rinsing (4  ) with 1% acetic acid. The plates were air-dried and the protein-bound dye was extracted with 10 mM non-buffered Tris base (POCh, Gliwice, Poland). The optical density at 540 nm was determined using an ELISA plate reader (Asys UVM340). The background optical density was measured in the wells filled with culture medium, without the cells. The results of cytotoxic activity were expressed as an ID50 – the dose of compound (μg/ml), which inhibits the proliferation of 50% tumor cells as compared to the control untreated cells. Each compound in given concentration was tested in triplicates in each experiment, which was repeated 3–5 times.

Fig. 2 MIC100% value determination for compound 1 [Sn(C4H9)3(OOCC6H4SO3H2)] against two of the clinical strains of Trichophytonmentagrophytes var. mentagrophytes from a patient with tinea capitis trichophytica (microwell A10-MIC100% value, concentration 1.17 μg/ml, microwell A11concentration 0.78 μg/ml, microwell B1-sterility control of medium, microwells B2, B3, B4- controls of growth) and from patient with tinea barbae (microwell C9-MIC100% value, concentration 1.56 μg/ml, microwell C10-concentration 1.17 μg/ml, microwell D1-sterility control of medium, microwells D2, D3, D4- controls of growth).

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© 2010 ISHAM, Medical Mycology, 48, 373–383

2.34–3.125 [b] 6.25–9.37 2.34–3.125 6.25–9.37 6.25–9.37 6.25–9.37 4.68–6.25 9.37–12.5

MFC

4.68–6.25 6.25–9.37 2.34–3.125 6.25–9.37 6.25–9.37 6.25–9.37 9.37–12.5 9.37–12.5

[b]

0.55–1.56 2.34–3.125 0.25–0.55 0.78–1,17 2.34–4.68 0.55–1.17 3.125–4.68 2.34–4.68

[a]

3 MIC MFC

C. neoformans (6) C. famata (3) T. cutaneum (5) G. candidum (4) R. rubra (4) C. albicans (9) C. glabrata (4) C. krusei (6)

0.55–1.56 1.56–2.34 0.25–0.55 1.17–2.34 1.56–3.125 0.55–1.17 2.34–3.125 2.34–4.68

[a]

*The MIC and MFC ranges of values are given for tests repeated at least three times. **The range of values of compound concentrations in microdilution bioassay: 0.068–50 μg/ml. [a]MIC value ranges. [b]MFC value ranges.

4.68–6.25 6.25–9.37 2.34–3.125 6.25–9.37 6.25–9.37 6.25–9.37 6.25–9.37 Ô12.5

[b]

0.78–3.125 2.34–3.125 0.25–0.55 1.17–2.34 2.34–4.68 0.55–1.17 3.125–4.68 2.34–3.125

[a]

2 MIC MFC 1 MIC Species (no. of tested strains)

Tested compounds (concentration values are given at μg/ml)**

A collection of different clinical and environmental strains of fungi was examined in terms of their susceptibility towards organotin compounds [Sn(C4H9)3 (OOCC6H4SO3H-2)] (1) [Sn(C4H9)3{OOC(CH2)3P(C6H5)3}]Br (2) and [Sn(C6H5)3[OOC(CH2)3N(CH3)3}]Cl (3). Table 1 shows the MICs and MFCs values of yeast-like fungi determined in vitro for all three tested compounds. Among all investigated clinical strains, the highest susceptibility was found with T. cutaneum. The total inhibition of growth (MIC) for these isolates was observed for concentration ranges of 0.25–0.55 μg/ml. The fungicidal effect for all three tested compounds was found to be at concentrations ranging from 2.34–3.125 μg/ml. All three compounds revealed slightly lower activity against C. albicans isolates with a MIC ranging from 0.55–1.17 μg/ml (Fig. 1). Fungicidal action of all three tested compounds was observed at concentrations ranging from 6.25–9.37 μg/ml. All C. neoformans strains also showed high susceptibility to the tested compounds. For these strains the MIC values were in the range of 0.55–1.56 μg/ml for compounds 1 and 3. Reduced effectiveness against all C. neoformans strains was observed for tributyltin compound 2 for which MIC values were slightly higher and ranged from 0.78–3.125 μg/ml. The analysis of the MFC values indicated that all strains of C. neoformans showed considerably higher susceptibility in comparison with the C. albicans strains and fungicidal effect for compounds 1 and 2 was observed at concentrations ranging from 4.68–6.25 μg/ml. The activity of compound 3 was considerably higher than that of complexes 1 and 2 and the MFC values were between 2.34– 3.125 μg/ml. In addition, the effectiveness of compound 3 was greater than that of the complexes 1 and 2 for G. candidum strains for which the MIC value ranged from 0.78– 1.17 μg/ml. For compounds 1 and 2 the MIC values ranged from 1.17–2.34 μg/ml. The value of minimal fungicidal concentrations (MFC) for all three compounds was in the range of 6.25–9.37 μg/ml. In the case of C. famata and R. rubra, compound 1 was considerably more effective than compounds 2 and 3. The MIC values for 1 against C. famata and R. rubra strains were from 1.56–2.34 μg/ml and 1.56 μg/ml–3.125 μg/ml, respectively. For compounds 2 and 3 the MICs were considerably higher. In the case of R. rubra strains MIC values were in the range of 2.34–4.68 μg/ml. Similar values were noted for C. famata strains for which MIC values ranged from 2.34–3.125 μg/ml for both mentioned compounds. It should be noted that fungicidal action against all R. rubra and C. famata strains was observed at concentrations ranging from 6.25–9.37 μg/ml with all three compounds. Smaller susceptibility to all three tested compounds was noted with C. krusei strains. For compounds 1 and 3 the minimal values of concentrations

Table 1 Antifungal activity of triorganotin complexes [Sn(C4H9)3(OOCC6H4SO3H-2)] (1) [Sn(C4H9)3{OOC(CH2)3P(C6H5)3}]Br (2) and [Sn(C6H5)3[OOC(CH2)3N(CH3)3}]Cl (3) against the selected strains of yeast-like fungi.*

Results and discussion

379

MFC MIC MFC

25–37.48 [b] 37.48 50 37.48–50 25–37.48 25–37.48 25– 37.48 25– 37.48 25–50 25–50 18.74–25

1.56–3.125 [a] 0.78–2.34 3.125–4.68 0.55–1.56 0.55–1.56 3.125–4.68 2.34–3.125 2.34–3.125 0.78–2.34 1.17–3.125 0.25–0.55

3

*The MIC and MFC ranges of values are given for tests repeated at least three times. **The range of values of compounds concentrations in microdilution bioassay: 0.068–50 μg/ml. [a]MIC value ranges. [b]MFC value ranges.

MIC

1.56–3.125 [a] 1.17–3.125 3.125–4.68 0.78–1.56 0.78–1.56 3.125–4.68 3.125–4.68 3.125–4.68 1.17–2.34 1.17–3.125 0.25–0.55

MFC

25–37.48 [b] 37.48 50 25–37.48 25–37.48 25–37.48 25–37.48 25– 37.48 25–50 25–50 18.74–25

MIC

1.56–3.125 [a] 1.17–3.125 3.125–4.68 0.55–1.56 0.55–1.56 3.125–4.68 3.125–4.68 3.125–4.68 0.78–2.34 1.17–3.125 0.25–0.55 S. brevicaulis (8) T. rubrum (8) T. tonsurans (3) T. mentagrophytes var. mentagrophytes (4) E. floccosum (5) A. flavus (5) A. fumigatus (4) F. avenaceum (4) M. canis (6) M. gypseum (4) M. plumbeus (4)

Species (no. of tested strains)

Tested compounds (concentration values are given at μg/ml)**

2 1

necessary for complete inhibition of growth for all strains were in the range of 2.34–4.68 μg/ml. The most effective was compound 2, for which the MIC values ranged from 2.34–3.125 μg/ml. For all of C. krusei strains quite high MFC values were observed. They ranged from 9.37–12.5 μg/ml for compounds 2 and 3 for all tested C. krusei strains. Fungicidal effect in the case of compound 1 was observed only at concentration 12.5 μg/ml for all C. krusei strains. Similar susceptibilities to the tested organotin compounds as found with C. krusei strains were found with the C. glabrata strains for which total inhibition of growth was observed at concentrations in the range from 2.34 μg/ml to 3.125 μg/ml for compound 1, and from 3.125 μg/ml to 4.68 μg/ml for compound 2, and 3. Similar differences in MFCs were found for tested organotin compounds as they ranged from 4.68–6.25 μg/ ml for compound 3, 6.25–9.37 μg/ml for 1 and 9.37–12.5 μg/ml for 2. It is worth noting that the MFC values are relatively low, i.e., they are equal to 6.25 μg/ml or 9.37 μg/ ml for the majority of the tested strains of yeast-like fungi. The MICs for filamentous fungi were similar to those of yeast-like fungi in that the values ranged from 0.25–4.68 μg/ml (Table 2). The highest susceptibility to all three tested compounds was found with M. plumbeus strains for which MIC values generally were in the range of 0.25–0.55 μg/ml. A fungicidal effect for each of three compounds was observed only at high concentration and the MFC values were in the range of 18.74–25 μg/ml. High susceptibility was also found in the case of all T. mentagrophytes and E. floccosum strains. The MICs for compounds 1 and 3 were found in the range of 0.55–1.56 μg/ml. (Fig. 2). In the case of less active compound 2 the range of the MIC values was from 0.78–1.56 μg/ml for all T. mentagrophytes and E. floccosum strains. Similar differences between compounds were observed in the case of the MFC values which were also higher for compound 2 and were in the range of 37.48–50 μg/ml for T. mentagrophytes strains and 25–37.48 μg/ml for E. floccosum strains. More active were compounds 1 and 3 for which the MFC values were in the range of 25–37.48 μg/ml for both T. mentagrophytes and E. floccosum strains. Susceptibility of M. gypseum and M. canis strains was quite good as those found for M. plumbeus or E. floccosum. The MIC values in the case of M. canis strains were in the range of 0.78–2.34 μg/ml for both 1 and 3 compound. The MIC values were higher for compound 2 and were in the range of 1.17–2.34 μg/ml. For M. gypseum strains MIC values for all three compounds were in the range of 1.17–3.125 μg/ml and fungicidal activity for all three compounds was noted only for concentrations in the range of 25–50 μg/ml for both M. canis and M. gypseum strains. Satisfactory results were also obtained for T. rubrum strains. The MICs for all eight tested isolates

25–37.48 [b] 37.48 50 25–37.48 25–37.48 25–37.48 25– 37.48 25– 37.48 25–50 25–50 18.74–25

Dylag et al.

Table 2 Antifungal activity of triorganotin complexes [Sn(C4H9)3(OOCC6H4SO3H-2)] (1) [Sn(C4H9)3{OOC(CH2)3P(C6H5)3}]Br (2) and [Sn(C6H5)3[OOC(CH2)3N(CH3)3}]Cl (3) against the selected strains of filamentous fungi.*

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were in the range of 1.17–3.125 μg/ml in the case of compounds 1 and 2. Higher activity was observed with compound 3 for which MIC values were in the range of 0.78–2.34 μg/ml. Fungicidal effect for all T. rubrum strains was observed at concentrations 37.48 μg/ml. Satisfactory results were found in the case of other filamentous fungi. Particularly satisfying results were obtained for S. brevicaulis strains, for which the MIC values were in the range of 1.56–3.125 μg/ml and for most isolates concentrations of 2.34 μg/ml caused full inhibition of growth for all three tested organotin carboxylates. Minimal fungicidal concentrations for strains from other species were also high and were in the range of 25–37.48 μg/ml. Considerably lower susceptibility was shown by the F. avenaceum and A. fumigatus strains, for which the MIC values in the case of compound 1 and 2 were in the range of 3.125–4.68 μg/ml. More active was compound 3 for the F. avenaceum and A. fumigatus strains for which the MIC values were in the range 2.34–3.125 μg/ml. Fungicidal activity of these three compounds was similar in A. fumigatus and F. avenaceum strains and were in the range of 25–37.48 μg/ml. Insignificantly lower susceptibility was found in the case of A. flavus isolates for which MIC values were generally in the range of 3.125–4.68 μg/ml for all three compounds. A fungicidal effect was noted for concentration values in the range of 25–37.48 μg/ml. The lowest susceptibility was observed for all T. tonsurans strains, for which the MIC values of all three tested compounds were in the range of 3.125–4.68 μg/ml. It should be emphasized that the fungicidal effect for all three compounds was observed only at concentrations 50μg/ml. There are considerable differences between MFC and MIC values. The largest differences were found in the case of filamentous fungi, for which fungicidal effect for the largest number of the tested strains was observed as a rule at concentrations in the range of 37.48–50 μg/ml for all organotin compounds, while the MIC values were in the range of 0.25–4.68 μg/ml. Major differences were not found between MIC values of yeasts and filamentous fungi in this investigation. This could indicate that all three organotin carboxylates show very similar inhibitory activity against all fungi. The MFC values obtained with filamentous fungi also indicate that the fungicidal effect for these compounds were at similar concentrations. Compounds 1 to 3 were also evaluated for their in vitro cytotoxicity against two cell lines, i.e., A 549 (human lung adenocarcinoma) and L 929 (mouse fibroblast) using the SRB assay [41]. The ID50 value obtained for compound 1 was 2.46 μg/ml for A 549 cell lines and 1.73 μg/ml for L 929 cell lines with the standard deviation value (SD)  0.16 and 0.12, respectively. The ID50 values, obtained for compounds 2 and 3 were very similar and in the case of A 549 cell line were 2.51 μg/ml with SD  0.23 and 2.1 μg/ © 2010 ISHAM, Medical Mycology, 48, 373–383

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ml with SD  0.4, respectively. The value of cytotoxic concentrations in the case of mouse fibroblast cell line obtained for compounds 2 and 3 were equal to 1.94 μg/ml  0.54 and 2.35 μg/ml  0.09, respectively. In general all the compounds exhibited a comparable potent cytotoxic activity against human and murine cell lines with ID50 values in the range 1.73–2.51 μg/ml. Our results point to high antifungal effectiveness of the investigated organotin compounds. An important advantage of these new compounds is their wide, non-specific spectrum of antifungal activity for yeast-like and filamentous fungi as seen in the present work. Similarly, a wide spectrum of antifungal activity is found with azole derivatives and terbinafine, but the first one has mainly fungistatic activity [1,2,11]. In the case of terbinafine there is a considerably lower activity against species of Candida 1, [11]. Amorolfine and ciclopirox, which are fungicidal, have a very wide spectrum of antifungal activity as well, but both of them have use limitations and are designed for local application [1,2,11]. In contrast to our tested compounds, which show a clear fungicidal action at higher concentrations, 5-fluorocytosin, azoles and griseofulvin show only fungistatic action [1,2,411]. Additionally, in contrast to echinocandins the tested organotin compounds are very effective against C. neoformans and T. cutaneum strains [3]. In the case of filamentous fungi strains used in our study considerably higher differences were found between the MIC and MFC values for the same strain. The MICs in the case of all filamentous fungi strains used in our investigations were in the range of 0.25–4.68 μg/ml, and the fungicidal effect was observed at concentrations in the range of 18.74–50 μg/ml. Among all tested filamentous fungi strains the highest susceptibility was shown by M. plumbeus strains and the lowest by all T. tonsurans strains. It is worthwhile to note the high effectiveness of all three tested compounds against all S. brevicaulis clinical and environmental strains. MIC100% for these strains was observed for compound concentrations in the range of 1.56–3.125 μg/ml and for most isolates at a concentration equal to 2.34 μg/ml for full inhibition of growth for all three tested organotin carboxylates. Thus the complexes 1, 2 and 3 are promising antifungal agents considering that S. brevicaulis is quite a resistant organism [38,42,43]. At present, the cell targets of organotin compounds have not been definitely determined. There are only single reports in the literature on the subject. Documented reports exist which indicate that organotin (IV) compounds can influence mitochondrial ATP-ase and lead to oxidative stress [44]. Additionally, Bragadin and Marton reported that the uptake of trialkyltin compounds stimulate opening of a selective anionic channel which leads to the swelling of mitochondria [45]. According to Nopp et al. the tributyltin (TBT) is a strong apoptotic stimulus by induction of

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caspase-3 activity [46]. Tiano et al. described a genotoxic effect for tributyltin-chloride (TBTC) [47]. Recently reported results by Ballmoos et al. indicate ATP-ase as a target of tributyltin chloride [48]. The authors of the latest paper on cell targets for organotin compounds suggest the proteasome as one of the molecular targets for these compounds [49]. Our new triorganotin carboxylates differ from these reports and we have recently begun investigation about their mechanism of action.

Conclusion Antifungal activity of organotin complexes [Sn(C4H9)3 (OOCC6H4SO3H-2)] (1) [Sn(C4H9)3{OOC(CH2)3P(C6H5)3}] Br (2) and [Sn(C6H5)3[OOC(CH2)3N(CH3)3}]Cl (3) against yeast-like, and filamentous fungi has been investigated. All complexes appear to be very effective antifungal agents with MIC values in the range of 0.25–4.68 μg/ml. The MFC values for yeast-like fungi were in the range of 2.34– 12.5 μg/ml for all three tested compounds, but for filamentous fungi they were in the range of 18.74–50 μg/ml. Differences between fungistatic activity of compounds 1, 2 and 3 against all species of fungi are relatively small. Generally, activity of organotin compounds against yeastlike fungi changed in the order 1  32 and for filamentous fungi 3  12. Antifungal activity of [SnR3X] compounds depends mainly on [R3Sn] moiety. However, their effectiveness is also influenced by X ligands. Thus compound 1 with OOCC6H4SO3H ligand is considerably more active than compound 2 and 3 against strains of the yeast-like fungi and also has higher antifungal activity than compound 2 in the case of the filamentous fungi. In the case of a great number of tested strains, the MIC values for compound 2 containing phosphoniumcarboxylate OOC(CH2)3P(C6H5)3 were one and a half to two times higher than those for complex 1. Our results indicate a very strong antifungal activity of all three tested organotin carboxylates, however, they also showed quite high cytotoxic properties towards two mammalian cell lines, specifically at concentrations of 1.73–2.51 μg/ml. The studies of these compounds and other [SnR3X] and [SnR2X2] complexes (X  carboxylate, amidates, amidinates and their derivatives) should provided the basis for finding complexes showing lower toxicity.

Acknowledgements The authors are grateful to the Ministry of Science and Higher Education for the support to this research (Grants: PBZ –Min- 015/P05/2004; 1016/S/IGiM/09 and No 3 T09A 01029).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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