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Abstract Three new amide derivatives (alteralkaloids A–C, 1–3) and three known alkaloids (4–6) were afforded after phytochemical investigation of fungus Alternaria brassicicola. The structures of these compounds were confirmed by NMR spectroscopic and HRESIMS data. Furthermore, the absolute configuration of 1 was determined using the single-crystal X-ray diffraction analysis. Compounds 1–3 belong to a class of amide derivatives that have not been found in nature before, sharing the same characteristic signals of the butyl moiety and amide group. These isolated compounds mentioned above were tested for the cytotoxic activity. Graphical Abstract