The mushroom Stropharia rugosoannulata is called saketsubatake in Japanese, and wine-cap stropharia in English. It belongs to the family Strophariaceae, which is widespread in northern temperate zones throughout the world. It is edible and is cultivated for food. During screening for anti-endoplasmic-reticulum (ER) stress and anti-methicillin-resistant Staphylococcus aureus (MRSA) effects of the extracts of various mushrooms, we found activity in the extract of this mushroom. ER stress induces apoptotic pathways with signaling between the ER and mitochondria. By triggering apoptosis on neural cells, the stress is a major cause of degenerative diseases such as Alzheimers disease. [1,2] MRSA has developed resistance to most antibiotics and is one of the most prevalent pathogens in nosocomial infections. Therefore, anti-ER-stress and anti-MRSA substances are urgently required. Recently we reported that several active steroids were isolated from this mushroom. [3] In further search for bioactive compounds from the mushroom, we discovered four novel steroids having a very unique and unprecedented carbon skeleton. Herein, we describe the isolation, structure determination, and biological activity of the compounds from the mushroom.Fresh fruiting bodies of S. rugosoannulata were extracted with EtOH and then with acetone. After the solutions were combined and concentrated, they were partitioned between n-hexane and H 2 O, CHCl 3 and H 2 O, and then EtOAc and H 2 O. The hexane-soluble residue was fractionated by repeated chromatography. As a result, four novel compounds (1-4), which were named strophasterols A, B, C, and D, were isolated (Scheme 1).Strophasterol A (1) was obtained as white crystals. Its molecular formula was determined to be C 28 H 44 O 4 by HRESIMS with m/z 467.3100 [M+Na] + (calcd for C 28 H 44 NaO 4 467.3137), thus indicating the presence of seven degrees of unsaturation in the molecule. The planar structure of 1 was elucidated by interpretation of the NMR spectra, including DEPT, COSY, HMBC, and HMQC data. The DEPT experiment indicated the presence of six methyl, eight methylene, and eight methane groups, as well as six quaternary carbon atoms. In the NMR spectra of 1, typical signals of a sterol corresponding to two hydroxymethines [C3: d H = 3.94 ppm (m), d C = 68.2 ppm; C6: d H = 4.84 ppm (m), d C = 63.0 ppm], four doublet methyls [C2'CH 3 : d H = 0.97 ppm (d, J = 6.7 Hz), d C = 20.8 ppm; C4''CH 3 : d H = 0.72 ppm (d, J = 6.7 Hz), d C = 16.4 ppm; C2''CH 3 : d H = 0.74 ppm (d, J = 6.7 Hz), d C = 15.6 ppm; C3''CH 3 : d H = 0.83 ppm (d, J = Scheme 1. Structures of strophastrerols A-
