The crystal structure of the most active antifungal compound 3 is also reported. We have previously reported the synthesis and NMR elucidation of these compounds.15 and 16 Sabouraud dextrose broth was inoculated with C. albicans and grown in an incubator (37 °C; optical density of 0.5 at 600 nm). C. albicans (ATCC strain 10231) culture was obtained from American Type Culture Collection (Manassas, VA, USA). The broth was prepared according to the manufacturer’s protocol.
The fungal susceptibility assay was based on a microplate method but with modifications. 17 Compounds (1–7) were prepared in pure DMSO at stock concentrations of 1.5, 2.5, 3.5, 5, 7.5, 10, 12.5 and 15 mM. Firstly, 100 μl/well of sterile broth was added into a clear, sterile 96-well microlitre plate (Corning Life Sciences, Acton, MA, USA). Fulvestrant purchase Secondly, 6 μl/well of the compound at the appropriate concentration above was added
and the plate tapped to mix the contents. Thirdly, 94 μl/well of sterile GSK1349572 water was added and the plate tapped. Finally, 100 μl/well of the culture was added and the plate tapped and incubated (37 °C; 18 h). Therefore, with a final volume/well of 300 μl and a dilution factor of 50×, the final concentration of DMSO/well was 2% v/v and the final concentrations of each compound/well were 30, 50, 70, 100, 150, 200, 250 and 300 μM. Fungal growth was not significantly inhibited by the 2% v/v DMSO (data not shown). The positive control used was the known antifungal drug clotrimazole. Fungal growth was quantified by optical density (600 nm) in a microplate reader (BioTek ELx800, Winooski, VT, USA). In vitro cytotoxicity of the synthesized homoisoflavanones was tested against a Chinese Hamster Ovarian (CHO) cell line using the 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazo-liumbromide (MTT) assay. The MTT assay is a colourimetric assay to determine cellular Oxygenase growth and survival, and compares well with other available assays. 18 and 19 The tetrazolium
salt MTT was used to measure cell viability. The homoisoflavanones were prepared in a 2 mg/ml stock solution containing 10% v/v DMSO. Emetine was used as the reference drug at an initial concentration of 100 μg/ml serially diluted in 10-fold to obtain 6 concentrations, the lowest being 0.001 μg/ml. Homoisoflavanones were diluted similarly. The DMSO solvent system had no measurable effect on cell viability (data not shown). Data are reported as the mean ± standard error of the mean of four independent experiments with duplicate measurements. Fungal growth was quantified as a percentage of the control without the test compound. GraphPad Prism (version 5.02; GraphPad Software, San Diego, CA, USA) was used to present and analyze the data. MIC50 values were deduced from the graphs. Statistical comparisons between 0 and each concentration for each compound were made by one-way ANOVA followed by Bonferroni’s post-test to determine P values. A value of P < 0.05 was considered significant.