Iris missouriensis Nutt. (Iridaceae) is indigenous to North America and widely distributed along the west coast. The whole plant has been extensively used by American Indians for medicinal purposes (1). Prompted by the discovery of potential antitumor compounds from other Iris species (e.g., irisquinone) (2), we have similarly investigated I. missouriensis. In earlier studies, using the roots of I. missouriensis, two novel quinones (irisoquin and deoxyirisoquin) (3), a novel triterpene (missourin) (4), and two cytotoxic triterpenes (isoiridogermanal and zeorin) (4) were isolated and characterized. In continuing our search for potential anticancer drugs from this plant, mangiferin, 7β-hydroxystigmasterol, 7β-hydroxysitosterol, 7-oxostigmasterol, 7-oxositosterol, and betulinic acid were isolated. The structures of these compounds were determined by spectral analyses and confirmed by comparison with authentic samples or published data. In addition, a novel triterpene was isolated and given the trivial name missouriensin (1). The mass spectrum showed 1 to have a molecular ion (m/z 484) corresponding to C32H52O3. Furthermore, a close structural relationship between 1 and zeorin acetate (3) was suggested by comparison of 13C-nmr spectra, and the 1H-nmr angular methyl signals of 1-4. As indicated by the 1H-nmr and 13C-nmr spectra, only seven angular methyls were present in 1 as opposed to the eight methyl signals demonstrated by compounds 2 (zeorin) and 3. The two singlets at δ 1.16 and 1.12, corresponding to the C29 and C30 methyls of 3, were absent in the 1H-nmr spectrum of 1. However, the 1H-nmr spectrum of 1 revealed two singlets at δ 4.88 and 5.03 (each integrating for one proton) and a broad singlet at δ 1.85 (3H), suggesting the presence of a terminal methylene moiety and one olefinic methyl group. The presence of this vinylidene group was further supported by ir absorption bands at 3100, 1650, and 880 cm-1. These spectral data suggested a terminal methylene was situated at the C22-C29 position. The ir spectrum of 1 also showed strong absorption bands at 3485 and 1735 cm-1 that are associated with hydroxy and ester groups, respectively. Also, the presence of an acetate group was indicated by the broad singlet at δ 2.02 (3H). The location of the acetate group at the 6 position of compound 1 was confirmed by the doublet of a triplet signal (1H) of the methine proton with coupling constants of 11 and 7.2 Hz at δ 5.21 (4). The 21β-H signal of zeorin acetate (3) and its dehydration product (4) appeared as quartets at δ 2.22 and 2.86, respectively. Thus, the absence of this signal in the 1H-nmr of compound 1 suggested that the OH group was located at the 21β position. In order to confirm the structure of 1, chemical conversion of zeorin was performed as shown in Scheme 1. Dehydration of zeorin acetate (3) with POCl3 afforded a mixture of two compounds (4 and 5) (5), that were separated by chromatography over AgNO3 impregnated silica gel utilizing n-hexane-C6H6 (5:1) as the eluent. Oxidation of compound 4 over SeO2 gave one major product, and this was in every aspect identical to compound 1. Hence, the structure of missouriensin (1) was established as 6α-acetoxyl-21β-hydroxylhop-22(29)-ene. Evaluation of the cytotoxic potential of these isolates with cultured P-388 cells showed that only 7β-hydroxystigmasterol and 7β-hydroxysitosterol were active, having ED50 values of 0.1 and 0.2 μg/ml, respectively.