Six new pyranonaphthoquinone derivatives, gunacin A-E (2-7), along with the known compounds gunacin (1) and the isocoumarin derivative (+) orthosporin (8), were isolated from the fungus Exobasidium sp. Their chemical structures were elucidated by X-ray crystallography, extensive spectroscopic analysis supported by ROESY experiments, and mass spectrometry. Two tested compounds (1, 5) demonstrated high activity against Leishmania mexicana and four salivarian Trypanosoma species, with the lowest detected EC50 value of 0.02-0.24 μM, a value that is comparable to those of currently used drugs. In addition, compounds 1, 3, 5, 6, and 7 demonstrated antibacterial properties at micromolar concentrations, while 1, 5, 6, and 7 exhibited moderate antifungal activity (MIC 33.3-66.7 μM). In cytotoxicity assays, the compounds exhibited a range of toxicity against mammalian Jurkat, RAT2, MDCK cell lines, HeLa cells, and fibroblasts, with inhibition levels varying from strong to minimal inhibition (EC50 = 0.03-125 μM). This study is among the first to explore Exobasidium, a genus of phytopathogenic fungi and highlights the untapped potential of smut fungi (Basidiomycota: Ustilaginomycetes). The discovery of gunacins, which exhibit potent antiprotozoal activity at submicromolar concentrations, suggests a promising avenue for the development of antiprotozoal agents.

Department of Chemistry Faculty of Sciences Egerton University P O Box 536 Egerton 20115 Kenya

Department of Inorganic Chemistry Faculty of Science Charles University Hlavova 2030 8 Prague 2 12843 Czechia

Department of Microbial Drugs Helmholtz Centre for Infection Research GmbH Inhoffenstraße 7 Braunschweig 38124 Germany

Department of Organic Chemistry Faculty of Science Charles University Hlavova 2030 8 Prague 2 12843 Czechia

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czechia

Institute of Parasitology Biology Centre Branišovská 31 České Budějovice 370 05 Czech Republic

Laboratory of Cell Immunology Department of Cell Biology Faculty of Science Charles University Viničná 1594 7 Praha 2 12843 Czechia

Laboratory of Cell Signaling Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czechia

Laboratory of Environmental Biotechnology Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czechia

Laboratory of Fungal Genetics and Metabolism Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czechia

Laboratory of Molecular Structure Characterization Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czechia

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