Spongian Diterpenoids from the Sponge Spongia (Heterofibria) sp. Ludmila P. Ponomarenko, ² Anatoly I. Kalinovsky, ² Shamil Sh. Afiyatullov, ² Michail A. Pushilin, ‡ Andrey V. Gerasimenko, ‡ Vladimir B. Krasokhin, ² and Valentin A. Stonik* ,² Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Prospect 100 let VladiVostoku, 159, VladiVostok 690022, Russian Federation, and Institute of Chemistry, Far East Branch of the Russian Academy of Sciences, Prospect 100 let VladiVostoku, 159, VladiVostok 690022, Russian Federation ReceiVed February 13, 2007 Five new (1, 2, 4-6) and one known (3) diterpenoid were isolated from the keratose sponge Spongia (Heterofibria) sp. Structures of these compounds and their absolute configurations were proposed on the basis of X-ray analysis of 1, its CD spectrum, and NMR and MS spectroscopic studies of 1-6. One of the new diterpenoids was shown to be 2(R),3- (S),4(S)-3,18-methylene-2-acetoxyspongia-13(16),14-diene (6), possessing a novel carbon skeleton system. Marine sponges of the genus Spongia (the family Spongiidae, the order Dictyoceratida) are a source of various terpenoid compounds belonging to the spongian and rearranged spongian series. 1 The first spongian diterpenoid, isogatholactone, was described by Minale and collaborators from the Mediterranean sponge Spongia officinalis in 1974. 2 Later a number of other related diterpenoids were isolated from S. officinalis, 3 S. zimocca, 4 S. matamata, 5 S. arabica, 6 and Spongia sp. 7 Spongian and related diterpenoids isolated from Spongia spp. have been reported to exhibit a wide spectrum of biological activites including cytot- oxicity, 3d,4,7b,c antibacterial properties, 3c and toxicity against some marine macroorganisms. 5a,b A keratose sponge identified as Spongia (Heterofibria) sp. 8 was collected near Suwarrow atoll (Northern Cook Islands) from a depth of 5 m during an expedition onboard the R/V “Akademic Oparin”. In this paper we report the isolation of five new (1, 2, 4-6) and one known spongian diterpenoid (3), previously found from an unidentified sponge and shown to be an inhibitor of the lyase activity of DNA polymerase . 9 Results and Discussion The ethanol-chloroform extract of the sponge was fractionated by silica gel flash column chromatography followed by preparative HPLC using an Ultrasphere-Si column to obtain 1, 2, and a fraction containing monoacetates 3-6. This fraction was further separated by reversed-phase HPLC using an Ultrasphere ODS column to yield individual compounds 3-6. Compound 1 was obtained as colorless crystals from an n-hex- ane-ethylacetate mixture. The structure and absolute stereochem- istry of 1 were established by spectroscopic analysis and a single- crystal X-ray diffraction study followed by CD spectroscopy. NMR spectra of 1 (Table 1) exhibited signals typical of a ,′-disubstituted furan ring in the furan-containing spongian diterpenoids isolated from sponges as well as from some nudibranchs 10,11 (δ C 136.9, 136.6, 135.1, and 119.4; δ H 7.06 q, J ) 1.5 Hz and 7.09 d, J ) 1.6 Hz) and signals of three angular methyl groups (δ H 1.15 s, 1.17 s, 1.25 s), one acetate group (δ H 2.02, δ C 170.9), one oxygen- bearing carbon (δ C 65.9 CH 2 ), and a ketone group (δ C 213.1). A quartet signal at δ H 7.06 was assigned to CH-16 due to coupling with a proton at C-12 in the COSY spectrum. Inspection of the 2D NMR data ( 1 H- 1 H COSY, HSQC, HMBC) and X-ray analysis (for details see Supporting Information) allowed us to assemble the structure as 19-acetoxyspongia-13(16),14-dien-3-one (1); the con- formations of rings A and B are chairs, while that of ring C is a half-chair. To distinguish between the two alternative enantiomeric struc- tures, the CD spectrum of 1 was recorded. It showed positive Cotton effects with [θ] 287 ) 0.15 × 10 4 and [θ] 222 ) 2.23 × 10 4 . Application of the octant rule indicated that 1 has the absolute configuration shown in Figure 1. This 4S,5R,8R,9R,10R-configu- ration for 1 is consistent with that for other spongian diterpenoids with established absolute stereochemistry. 1,7a,11 Compound 1 is an acetylated derivative of the previously known 19-hydroxyspongia-13(16),14-dien-3-one from the sponge Hyatella intestinalis. 12 Indeed, the comparison of 1 H and 13 C NMR spectra of the product obtained as a result of alkaline hydrolysis of 1 and those of 19-hydroxyspongia-13(16),14-dien-3-one indicated their identity, although their optical rotations were somewhat different ([R] 25 D +32.7 (c 0.22, CHCl 3 ) for the product obtained by us and [R] 21 D +18.8 (c 0.6, CHCl 3 ) in the literature 12 ). Comparison of the 1 H and 13 C NMR data of 2-6 with those of 1 (Table 1) revealed these compounds as belonging to the same class of spongian-based furanoditerpenes. It was suggested that all these metabolites shared with 1 the same absolute configurations in their polycyclic systems. The HREIMS of 2 displayed a molecular ion at m/z 402.2414; that along with the NMR data (Table 1) suggested the molecular formula C 24 H 34 O 5 (calculated m/z 402.2406). NMR spectra of 2 exhibited signals of the same furan moiety (Table 1): three methyl groups (δ H 0.95 s, 1.02 s, 1.21 s), two acetoxy groups (δ H 2.04, δ C 170.5, 21.2 and δ H 2.08, δ C 171.0, 21.1), and two oxygen-bearing * To whom correspondence should be addressed. Tel: 7-4232-311168. Fax: 7-4232-314050. E-mail: stonik@piboc.dvo.ru. ² Pacific Institute of Bioorganic Chemistry. ‡ Institute of Chemistry. 1110 J. Nat. Prod. 2007, 70, 1110-1113 10.1021/np070068t CCC: $37.00 © 2007 American Chemical Society and American Society of Pharmacognosy Published on Web 06/14/2007