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New copper(I), copper(II) and zinc(II) azasterol derivatives were prepared using 20-hydrazone-imidazolin-2-yl-5α-pregnan-3β-ol (H1) as a ligand. The reactions were carried out under mild experimental conditions with different zinc(II) and copper(II) salts to produce [Zn(H1)2(OH)2] (1), [Zn(H1)2Cl2] (2), [Zn(H1)3(NO3)]NO3 (3), [Cu(H1)2(OH)2] (4), [Cu(H1)2]Cl (5) and [Cu(H1)2]NO3 (6). They were fully characterized by analytical and spectroscopic techniques. The molecular structure of these six metal complexes was also investigated by computational methods (DFT) to get information on the most stable geometry configuration around the metal atoms Zn and Cu. Antifungal activities of H1, compounds 1–6, and itraconazole (ITZ) as positive controls were tested in vitro against S. brasiliensis. The results showed that the fungus was more susceptible to all metal–azasterol complexes than H1. The most efficient metal–azasterol complex was [Zn(H1)2(OH)2] (1), with IC50 equal to that of itraconazole (0.25 μM). These biological results demonstrated that metal–azasterol complexes are promising molecules in the search and development of new antifungal compounds. [ABSTRACT FROM AUTHOR]

Objectives: Leishmaniasis, one of the most significant neglected diseases around the world, is caused by protozoan parasites of the Leishmania genus. Nowadays, the available aetiological treatments for leishmaniasis have variable effectiveness and several problems such as serious side effects, toxicity, high cost and an increasing number of resistance cases. Thus, there is an urgent need for safe, oral and cost-effective drugs for leishmaniases. Previously, our group has shown the effect of the ergosterol biosynthesis inhibitors on Leishmania amazonensis. Herein, we showed the effect of ravuconazole against L. amazonensis; ravuconazole is a second-generation triazole antifungal drug that has good bioavailability after oral administration and a long terminal half-life in humans, a broad activity spectrum, high effectiveness in treatment of mycosis and negligible side effects.Methods: Several methodologies were used: cell culture, fluorescence and electron microscopy, high-resolution capillary GC coupled with MS, fluorimetry and flow cytometry.Results: Our results showed that ravuconazole was able to inhibit the proliferation of L. amazonensis promastigotes and intracellular amastigotes in vitro, with single-digit to sub-micromolar IC50 values, causing several alterations in the morphology, ultrastructure, cell viability and physiology of the parasites. The mitochondrion was significantly affected by the treatment, resulting in a collapse of the mitochondrial transmembrane potential that consequently led to inhibition of ATP production, combined with an increase in reactive oxygen species and mitochondrial superoxide production; by transmission electron microscopy, the organelle displayed a completely altered ultrastructure. The treatment changed the lipid profile, showing a profound depletion of the 14-desmethyl endogenous sterol pool.Conclusions: These results suggest that ravuconazole could be an alternative option for the treatment of leishmaniasis. [ABSTRACT FROM AUTHOR]

Inhibition of Δ24-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol- 5-ene-3β-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker® Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole. [ABSTRACT FROM AUTHOR]