A new antiangiogenic C 24 oxylipin from the soft coral Sinularia numerosa Takahiro Yamashita a,z , Yoichi Nakao a,b, * , Shigeki Matsunaga a , Tsutomu Oikawa c , Yukimitsu Imahara d , Nobuhiro Fusetani a,e, * a Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan c Kanagawa University of Human Services, 1-10-1 Heisei-cho, Yokosuka, Kanagawa 238-8522, Japan d Wakayama Prefectural Museum of Natural History, 370-1, Funao, Kainan, Wakayama 642-0001, Japan e Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan article info Article history: Received 7 March 2008 Revised 20 September 2008 Accepted 31 October 2008 Available online 5 November 2008 Keywords: Angiogenesis inhibitor Tube formation Marine natural products Oxylipin abstract A new oxylipin, 15-hydroxy-tetracosa-6,9,12,16,18-pentaenoic acid (15-HTPE; 1) was isolated as an inhibitor of tube-formation from the soft coral Sinularia numerosa. Its structure was elucidated by means of spectral analysis and chemical degradation. 15-HTPE inhibited tube formation of EA.hy926 cells at the concentration of 20–40 lM. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Angiogenesis is a promising target for cancer chemotherapy 1,2 ; the first antiangiogenic agent Avastin Ò was approved in 2004 by FDA for treatment of colorectal and non-small cell lung cancers 3 and numbers of antiangiogenic agents are under clinical trials. 4 To discover antiangiogenic compounds, several assay systems have been developed. Among them, the tube-formation system using endothelial cells and collagen gel 5 is one of the most popular in vitro models, representing many features of in vivo angiogene- sis; activation of endothelial cells by the growth factors, degrada- tion of basement matrices, cell migration, and formation of tube- like structure of the primary blood vessels. In the course of our search for new antiangiogenic agents from marine invertebrates, we tested anti-tube-formation activity of 150 Japanese marine invertebrates by an assay system of the mod- ified in vitro tube-formation model 6 using EA.hy 926 cells, a per- manent human endothelial cell line. 7,8 Out of 10 active samples, the soft coral Sinularia numerosa collected in southern Japan showed remarkable inhibitory activity. Bioassay-guided isolation provided a new oxylipin, 15-hydroxy-tetracosa-6,9,12,16,18-pen- taenoic acid (15-HTPE; 1), along with the known 11-hydroxy-eico- sa-5,8,12,14-tetraenoic acid (11-HETE; 2), and 9-hydroxy- octadeca-6,10,12-trienoic acid (9-HOTE; 3). This paper deals with the isolation, structural elucidation, and biological activity of 1. 1: R =H 4: R = Me 2: R =H 5: R = Me 3: R =H 6: R = Me OH OR O OH OR O OH OR O 2. Results and discussion The combined MeOH and CHCl 3 extracts of the frozen sample (1.0 kg, wet weight) were partitioned between H 2 O and CHCl 3 ; the latter layer was further separated by the modified Kupchan procedure. 9 The CHCl 3 layer inhibiting tube formation was fur- ther separated by solvent partitioning with n-hexane/CH 2 Cl 2 / MeCN (10:3:7). The active lower layer was subjected to centrif- ugal partition chromatography (CPC) with the solvent system of n-heptane/EtOAc/MeCN/MeOH/H 2 O (6:6:1:4:3; mobile phase: upper layer, stationary phase: lower layer) to afford three active 0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2008.10.083 * Corresponding authors. Tel./fax: +81 3 5286 2568 (Y.N.), tel./fax: +81 138 40 8884 (N.F.). E-mail addresses: ayocha@waseda.jp (Y. Nakao), anobu@fish.hokudai.ac.jp (N. Fusetani). z Deceased author. Bioorganic & Medicinal Chemistry 17 (2009) 2181–2184 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc