RESEARCH FRONT CSIRO PUBLISHING Full Paper www.publish.csiro.au/journals/ajc Aust. J. Chem. 2010, 63, 895–900 Australin E Isolated from the Soft Coral Cladiella sp. Collected in Pohnpei Activates the Inositol 5-Phosphatase SHIP1 David E. Williams, A Ashraf Amlani, B Ariyanti S. Dewi, A Brian O. Patrick, A Leen van Ofwegen, C Alice L.-F. Mui, B and Raymond J. Andersen A,D A Departments of Chemistry and Earth & Ocean Sciences, University of British Columbia, Vancouver, B.C.V6T 1Z1, Canada. B Department of Surgery,The Jack Bell Research Centre, University of British Columbia, B.C. V6H 3Z6, Canada. C National Museum of Natural History, PO Box 9517, 2300 RA Leiden, The Netherlands. D Corresponding author. Email: randersn@interchange.ubc.ca Four new diterpenoids, australins E–H (1–4), have been isolated from the marine soft coral Cladiella sp. collected in Pohnpei. The structures of 1–4 were elucidated by analysis of their NMR and mass spectrometry data combined with an X-ray diffraction analysis of australin E (1). Australin E (1) activates the inositol 5-phosphatase SHIP1 in vitro. Manuscript received: 28 January 2010. Manuscript accepted: 18 March 2010. Introduction Aberrant activation of the phosphatidylinositol-3-kinase (PI3K) cell-signalling pathway contributes to many inflammatory diseases and cancers. [1] A key second messenger in the PI3K pathway is phosphatidylinositol-3,4,5-triphosphate (PI-3,4, 5-P 3 ), which is present in low amounts in unstimulated cells but is rapidly formed from PI-4,5-P 2 by PI3K in response to diverse extracellular stimuli. It has been proposed that one way to dampen aberrant PI3K signalling in hematopoietic cells would be to activate the inositol 5-phosphatase SHIP1, which is a negative regulator of the pathway that turns the signal off by converting the second messenger PI-3,4,5-P 3 to PI-3,4-P 2 . This proposal predicts that small-molecule activators of SHIP1 should have potential as drugs for the treatment of inflammatory disorders and cancers of the blood. [1] In 2005, we reported that the sponge meroterpenoid pelorol was an effective in vitro activator of SHIP1, and we synthesized the natural product and several analogues. [2] Pelorol, which has been isolated from several tropical sponges, [3,4] was the first small molecule, other than the endogenous activator PI-3,4-P 2 , known to activate SHIP1. [1a,2] The synthetic pelorol analogues 16A and MN100 (8) inhibited PI3K signalling in stimulated mast cells and macrophages, and showed promising in vivo anti- inflammatory activity in standard mouse models, thus providing proof-of-principle support for the use of SHIP1 activators as anti- inflammatory drugs. [1a] MN100 also showed significant in vitro cytotoxicity towards multiple myeloma cells. [1b] We have continued to screen our library of crude extracts of marine invertebrates using a kinetic enzyme assay [1,2] in an attempt to find additional small-molecule activators of SHIP1 that could act as drug leads. A crude extract of the soft coral Cladiella sp. (order: Alcyonacea, family: Alcyoniidae, genus: Cladiella) collected in Pohnpei showed promising activity in the SHIP1 assay. Bioassay-guided fractionation of the extract led to the identification of the four new diterpenoids australins E–H (1–4), that are related to australins A (5) and D (6) previ- ously isolated from the soft coral Cladiella australis collected in Taiwan. [5] Details of the isolation, structure elucidation, and biological activities of the diterpenoids 1–4 are presented below. Results and Discussion The soft coral Cladiella sp. was harvested by hand using SCUBA off Pohnpei and frozen for storage. [6] Lyophilized specimens of Cladiella sp. were exhaustively extracted with MeOH to give a crude extract that was active in the SHIP1 assay.The residue from the MeOH extract was partitioned between H 2 O and EtOAc. Fractionation of the EtOAc soluble materials via sequential application of Sephadex LH-20 chromatography, flash silica-gel column chromatography, and repetitive reversed-phase HPLC gave pure samples of the four new diterpenoids australins E–H (1–4) (Scheme 1). Australin E (1) was obtained as optically active colourless needles with a sharp melting point that gave a [M + Na] + ion in the HRESIMS at m/z 515.2979, which is appropriate for the molecular formula C 28 H 44 O 7 requiring seven sites of unsatura- tion. The 1 H/ 13 C/gCOSY/gHSQC/gHMBC NMR data obtained for 1 (Tables 1 and 2) identified resonances that could be assigned to 28 carbon atoms, thus consistent with the HRES- IMS data. Three methyl singlets (δ 1.00, 1.29, 1.60), one methyl doublet (δ 0.53 (J = 7.0 Hz)), and two methyl triplets (δ 0.79 (J = 7.4 Hz), 0.86 (J = 7.4 Hz)) were observed in the 1 H NMR spectrum of 1.As well as the six methyls, the presence of a ketone (δ 212.0 (C-9)), a trisubstituted olefin (δ 131.3 (C-11), 5.51 (H-12), 123.6 (C-12)), two quaternary carbinols (δ 81.3 (C-3), 84.5 (C-7)), two carbinol methines (δ 3.55 (H-2), 77.5 (C-2), 3.77 (H-6), 80.2 (C-6)), a carbinol methylene (δ 3.85/4.09 (H-19), 67.4 (C-19)), and two ester carbonyls (δ 171.5 (C-1 ′ ), 172.9 (C-1 ′ )) were apparent in the NMR data. Only a single © CSIRO 2010 10.1071/CH10053 0004-9425/10/060895