CHO HO OH H H H Orostanal (1) 3 5 7 6 24 29 27 28 TETRAHEDRON LETTERS Tetrahedron Letters 42 (2001) 6349–6351 Pergamon Orostanal, a novel abeo-sterol inducing apoptosis in leukemia cell from a marine sponge, Stelletta hiwasaensis Tomofumi Miyamoto, a Kota Kodama, a Yuko Aramaki, a Ryuichi Higuchi a, * and Rob W. M. Van Soest b a Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi 3 -1 -1, Higashi -ku, Fukuoka 812 -8582, Japan b Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, PO Box 94766, 1090 GT Amsterdam, Netherlands Received 28 May 2001; accepted 13 July 2001 Abstract—A sterol derivative, orostanal, was obtained from a marine sponge of Stelletta hiwasaensis. Spectroscopic analysis and synthetic study revealed its structure as a novel 5(67)abeo-sterol. Orostanal and its synthetic analog induce apoptosis in human acute promyelotic leukemia cell. © 2001 Elsevier Science Ltd. All rights reserved. Apoptosis is defined as a physiological and pro- grammed cell death, which is distinguished from necro- sis. 1 Apoptosis plays an important role not only in the development but also in the maintenance of homeosta- sis. Abnormal control of apoptosis may play a role in the development and survival of cancer as well as other diseases. It is well known that natural anti-cancer drugs, etoposide and camptothecin, induce apoptosis in cancer cells, so that the substance which induces apop- tosis must be useful for human cancer chemotherapy. 2 While searching for biologically active substances from marine invertebrates, we isolated a novel sterol named orostanal (1) as an active constituent inducing apopto- sis, from a Japanese marine sponge, Stelletta hiwasaen - sis. In this paper, we will report on the isolation and structural determination of 1. S. hiwasaensis (wet weight 2.0 kg) was collected by hand at depths of 10 to 15 m off Oro Island, Fukuoka Prefecture, Japan, in 1997. 3 The n -hexane soluble frac- tion (7.86 g) of the acetone extract obtained from S. hiwasaensis showed chromatin condensation and DNA fragmentation at 33 g/mL against human acute promyelotic leukemia cell (HL-60). 4 Bioassay-guided separation of the active fraction by silica-gel chro- matography (CHCl 3 :MeOH/10:01:1), followed by reversed-phase column chromatography (95% MeOH/ H 2 O), and reversed-phase HPLC (100% MeOH) gave an active compound (1) (3.6 mg, 1.8×10 -4 % yield). Orostanal (1) 5 was obtained as an amorphous solid. The IR spectrum revealed absorption bands due to hydroxyl (3502 cm -1 ) and carbonyl (1715 cm -1 ). The 1 H, 13 C NMR and HSQC spectral data suggested the presence of one primary methyl, two secondary methyls, two tertiary methyls, ten methylenes, seven methines, two quaternary carbons, one oxygenated methine, one oxygenated quaternary carbon, one exo - methylene, and one aldehyde (Table 1). The HR-EI MS of 1 showed a molecular ion peak at m /z 444.3607 ( +0.4 mmu) corresponding to the molecular formula of C 29 H 48 O 3 . The 1 H– 1 H COSY, TOCSY, and HSQC– TOCSY spectra of 1 afforded three partial structures, A [C1C4], B[C6C12, C21, C23] and C[C25C27, C29]. These three partial structures and three quaternary carbones, two tertiary methyls, and exo -methylene were merged by the aid of the HMBC experiment as shown in Fig. 1. The relative stereochemistry except C-25 of 1 was assigned on the basis of the NOESY correlations, and the stereochemistry of C-25 was elucidated by compari- son of the 1 H NMR data with those of (25S )- and (25R )-24(28)-dehydroaplysterols as shown in Fig. 2. 6 * Corresponding author. Tel.: +81-92-642-6635; fax: +81-92-642-6639; e-mail: rhiguchi@phar.kyushu-u.ac.jp 0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S0040-4039(01)01278-3