13-Epi-9-deacetoxyxenicin, a Cytotoxic Diterpene from the Soft Coral Asterospicularia laurae (Alcyonacea)

An investigation of the soft coral Asterospicularia laurae collected on the Great Barrier Reef, Australia, afforded the cytotoxic diterpene 13-epi-9-deacetoxyxenicin (1) in addition to the known metabolites 13-epi-9-deacetylxenicin (2) and gorgosterol. 13-Epi-9-deacetoxyxenicin readily underwent an autoxidation reaction in solution to afford a single product, the hydroperoxide 3. Structures, stereochemistry, and NMR assignments were established by high resolution NMR spectroscopy and by comparison with published data for known compounds.


Introduction
Asterospicularia laurae Utinomi is a moderately abundant soft coral species found in well lit areas of the Great Barrier Reef, especially on reef flats. Fabricius and Alderslade [1] have remarked upon the similarity between the polyp structure of Asterospicularia and those of the genera Xenia and Sympodium.
The chemistry of only one member of the genus Asterospicularia has been reported to date: Ksebati and Schmitz [2] reported in 1984 the isolation of 24-methyl-5α-cholestane-3β, 5, 6β, 22R, 24-pentol 6-acetate from Asterospicularia randalli. Here we report the isolation of diterpenes related to xenicin from A. laurae.
Xenicin was the first reported marine metabolite with the xenicane diterpenoid skeleton [3], a skeleton characterised by a 9-membered carbocyclic ring fused to a 6-membered oxygen-containing ring that is formally derived from cyclization of two aldehyde functions. Since the initial report, a large number of related metabolites have been isolated, with the 6-membered ring usually either formed from an enol-acetal or lactone linkage. The majority of these metabolites have been isolated from the soft coral genus Xenia [4][5][6][7][8][9][10][11], but others have been reported from the soft coral genera Anthelia [12,13], Alcyonium [13], and Capnella [14], from the blue coral Heliopora coerulea [15], and from gorgonians [16].

Results and Discussion
Chromatography of the dichloromethane extract of Asterospicularia laurae afforded gorgosterol, the new diterpenoid 13-epi-9-deacetoxyxenicin (1), and the known xenicane diterpenoid 13-epi-9-deacetylxenicin (2)   The formula of 1 was confirmed as C 26 H 36 O 7 by high resolution mass measurement of the [M+Na] + ion and the structure and stereochemistry were determined by comparison of its spectral data with those obtained for 13-epi-9-deacetylxenicin. The principal difference in the 1 H-NMR spectra was the absence for 1 of the allylic oxymethine signal that resonated at δ4.72 for (2). 1 H-and 13 C-NMR assignments (Table 1) were determined from gCOSY, gHMQC, and gHMBC NMR spectral data.
The stability of 1 in solution differed significantly from that of 13-epi-9-deacetylxenicin (2), as it readily underwent autoxidation (an Ene reaction, see Figure 1) to afford a single hydroperoxide product, while solutions of 13-epi-9-deacetylxenicin (2) were relatively stable.

O OH
The stereochemistry of the 8-hydroperoxy group in 3 is consistent with oxygen addition from the least hindered face of the alkene. It should be noted that single crystal x-ray structures of xenicane diterpenes like xenicin [3], indicate that the ∆ 7,8 bond lies orthogonal to the ring junction. This would be expected to prevent approach for endo addition to the olefinic bond. Indeed, the coupling constants (5.0 and 10.4 Hz) observed for H8 in 3 are consistent with exo addition, and a conformation similar to that determined for 13-epi-9-deacetylxenicin by single crystal x-ray crystallography [5a]. Further evidence for a conformation of 13-epi-9-deacetoxyxenicin with the ∆ 7,8 bond orthogonal to the ring junction (see Figure 2) is provided by the shielding of the 4a and 11a 1 H-NMR signals relative to those observed for the hydroperoxide (3).

General
Melting points were determined in a Stuart SMP1 apparatus and are uncorrected. Optical rotations were measured in CHCl 3 with a PolAAr 2001 polarimeter. Mass spectral data were determined on a Bruker BioAPEX 47e mass spectrometer operating in positive ion electrospray mode at the Australian Institute of Marine Science, Cape Ferguson. 1 H-NMR spectra were measured in CDCl 3 at 300 MHz and 13 C-NMR spectra at 75.5 MHz on a Varian Mercury NMR using residual solvent peaks for calibration. gCOSY, gHMQC and gHMBC spectra were obtained using the standard Varian pulse sequences optimised for 1 J= 140Hz, and 2,3 J= 8Hz. Merck t.l.c. grade silica gel (type 60) was used for column chromatography. All solvents used were freshly distilled.

Animal material
Samples of the soft coral Asterospicularia laurae were collected by hand using scuba (at a depth of 2-5m) at Old and Myrmidon Reefs (Great Barrier Reef, Australia), frozen immediately after collection and kept frozen until used. A voucher specimen (NTM C12570) has been deposited in the Museum and Art Gallery of the Northern Territory, Darwin.