13-Epi-9-deacetoxyxenicin, a Cytotoxic Diterpene from the Soft Coral Asterospicularia laurae (Alcyonacea)
Department of Chemistry, School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Qld., 4811, Australia
*
Author to whom correspondence should be addressed.
Mar. Drugs 2003, 1(1), 18-26; https://doi.org/10.3390/md101018
Submission received: 14 February 2003
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Accepted: 16 October 2003
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Published: 15 November 2003
Abstract
: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 [4–11], 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) [5a].
The formula of 1 was confirmed as C26H36O7 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 1H-NMR spectra was the absence for 1 of the allylic oxymethine signal that resonated at δ4.72 for (2). 1H- and 13C-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.
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 Δ7,8 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 Δ7,8 bond orthogonal to the ring junction (see Figure 2) is provided by the shielding of the 4a and 11a 1H-NMR signals relative to those observed for the hydroperoxide (3).
Cytotoxicity assays were carried out against a cultured suspension of P388D1 mouse lymphoma cells. 13-Epi-9-deacetoxyxenicin (1) exhibited an IC50 of 0.1 μg/ml, while the IC50 of 13-epi-9-deacetylxenicin (2) was approximately 1.0 μg/ml.
Conclusions
The soft coral Asterospicularia laurae from Old and Myrmidon Reefs, Great Barrier Reef, Australia contains the cytotoxic diterpenes 13-epi-9-deacetylxenicin and 13-epi-9-deacetoxyxenicin. 13-Epi-9-deacetoxyxenicin readily undergoes an autoxidation reaction to form a hydroperoxide.
Experimental
General
Melting points were determined in a Stuart SMP1 apparatus and are uncorrected. Optical rotations were measured in CHCl3 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. 1H-NMR spectra were measured in CDCl3 at 300 MHz and 13C-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 1J= 140Hz, and 2,3J= 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.
Extraction and Purification
The frozen coral was freeze dried and the dried material (206.1 g) was extracted with dichloromethane (3 X 1.5 L). Solvent removal afforded the dichloromethane extract (7.4 g, 3.6%). The residue was then twice extracted with methanol. The methanol extract was evaporated and partitioned between water and dichloromethane. Removal of the dichloromethane afforded the methanol lipophilic extract (1.6 g, 0.8%), which was combined with the dichloromethane extract. A portion (3.7 g) of the combined extract was repeatedly subjected to vacuum liquid chromatography on silica gel [17] eluted with dichloromethane-ethyl acetate, hexane-ethyl acetate and hexane-diethyl ether mixtures for successive columns to afford gorgosterol (39 mg, 0.038%), 13-epi-9-deacetoxyxenicin (1) (44 mg, 0.043%) and 13-epi-9-deacetylxenicin (2) (106mg, 0.103%). 13-Epi-9-deacetoxyxenicin (1) crystallised from cold hexane in prisms m.p. 86–87º, [α]D +63º (c. 0.06, CHCl3). 13-Epi-9-deacetylxenicin (2) crystallised from diethyl ether in prisms m.p. 150–151º, [α]D +51º (c. 0.11, CHCl3) {lit. m.p. 142–4º, lit [α]D +44º (c. 0.48, CH2Cl2) [5a]}. Solutions of 1 in dichloromethane or ether underwent autoxidation to afford a single product that was identified as 3 and obtained as an oil [α]D +48º (c. 0.09, CHCl3).
Spectral Data
13-Epi-9-deacetoxyxenicin (1):1H- and 13C-NMR (CDCl3): see Table 1. Observed HMBC correlations: 1-Acetate CH3 to 1-acetate CO; H3 to C1, C4, C4a, C12; H8 to C6, C7, C9, C10, C18; H12 to C3, C4, C4a, C13, C14, 12-OCO; 12-Acetate CH3 to 12-acetate CO; H13 to 13-OCO, C14; 13-Acetate CH3 to 13-acetate CO; H14 to C12, C13, C15, C16, C17; H16 to C14, C15, C17; H17 to C14, C15, C16; H18 to C6, C7, C8; H19a/H19b to C10, C11, C11a; IR (Nujol) cm−1: 1761, 1732, 1457, 1373, 1229, 1157, 1027, 959, 928; HRMS (Electrospray) for C26H36O7Na [MNa]+: Calcd 483.2353; Found 483.2341.
13-Epi-9-deacetylxenicin (2):1H- and 13C-NMR spectral data (see Table 1) was in agreement with the published data [5a].
Hydroperoxide (3):1H- and 13C-NMR: see Table 1. Observed HMBC correlations: 1-Acetate CH3 to 1-acetate CO; H3 to C1, C4, C4a, C12; H12 to C3, C4, C4a, C13, C14, 12-OCO; 12-Acetate CH3 to 12-acetate CO; H13 to 13-OCO, C14; 13-Acetate CH3 to 13-acetate CO; H14 to C16, C17; H16 to C14, C15, C17; H17 to C14, C15, C16; H18a/H18b to C6, C8; H19a/H19b to C10, C11a; HRMS (Electrospray) for C26H36O9Na [MNa]+: Calcd 515.2251; Found 515.2235.
Figure 1.
Autoxidation of an alkene - an Ene reaction
Figure 2.
Computer generated proposed conformation of 13-epi-9-deacetoxyxenicin showing the orthogonal arrangement of the C7-C8 bond relative to the C4a-C11a ring junction.
Figure 2.
Computer generated proposed conformation of 13-epi-9-deacetoxyxenicin showing the orthogonal arrangement of the C7-C8 bond relative to the C4a-C11a ring junction.
| Compound | 1 | 2 | 3 | |||
|---|---|---|---|---|---|---|
| Carbon | δ1H, mult., J(Hz) | δ13C | δ1H, mult., J(Hz) | δ13C | δ1H, mult., J(Hz) | δ13C |
| 1 | 5.87, d, 1.5 | 91.4 | 5.86, d, 1.8 | 91.7 | 6.08, d, 2.4 | 92.4 |
| 3 | 6.49, d, 1.8 | 141.7 | 6.49, d, 1.5 | 141.9 | 6.45, brs | 142.0 |
| 4 | 113.6 | 113.5 | 111.5 | |||
| 4a | 2.2, m | 37.1 | 2.33, brd, 13.8 | 36.6 | 2.44, m | 32.1 |
| 5 | 1.55, m; 1.95, m | 30.3 | 2.05, m; 1.97, m | 30.5 | 1.78, m; 2.32, m | 30.5 |
| 6 | 2.0 – 2.2, m, 2H | 39.6 | 2.2, m | 39.5 | 2.22, m; 2.38, m | 25.6 |
| 7 | 135.5 | 133.0 | 145.5 | |||
| 8 | 5.37, brt, 7.9 | 124.4 | 5.24, brd, 9.3 | 131.0 | 4.41, dd, 5.0, 10.4 | 90.0 |
| 9 | 2.25, m; 2.45, m | 24.9 | 4.72, brdd, 7.2, 6.0 | 67.7 | 2.03, m; 1.64, m | 26.5 |
| 10 | 2.15 – 2.25, m | 35.4 | 2.3, m; 2.6, m | 46.3 | 2.12–2.24, m | 30.7 |
| 11 | 151.3 | 147.2 | 146.6 | |||
| 11a | 1.95, m | 48.9 | 1.85, brs | 49.4 | 2.70, brt, 1.5 | 39.3 |
| 12 | 5.32, d, 6.6 | 74.9 | 5.31, d, 6.8 | 74.9 | 5.29, d, 7.5 | 74.8 |
| 13 | 5.68, dd, 6.6, 9.3 | 70.0 | 5.69, dd, 6.8, 9.3 | 69.9 | 5.59, dd, 7.5, 9.3 | 69.6 |
| 14 | 5.14, dsept, 9.3, 1.2 | 119.5 | 5.13, brd, 9.3 | 119.5 | 5.10, brd, 9.3 | 119.6 |
| 15 | 140.0 | 140.1 | 140.1 | |||
| 16 | 1.74, d, 1.2 | 25.8 | 1.74, brs | 25.8 | 1.73, brs | 25.9 |
| 17 | 1.75, d, 1.2 | 18.5 | 1.74, brs | 18.6 | 1.75, brs | 18.6 |
| 18 | 1.65, d, 0.8 | 16.7 | 1.64, brs | 17.7 | 5.34, brs; 5.41, brs | 119.0 |
| 19 | 4.78, d, 1.2; 4.86, brs | 112.9 | 4.56, brs; 4.84, brs | 115.1 | 4.79, brs, 4.87, brs | 112.1 |
| 1-OAc | 2.03, s | 20.9 | 2.02, s | 20.9 | 2.05, s | 21.0 |
| 169.5 | 169.5 | 169.4 | ||||
| 12-OAc | *2.02, s | 21.1 | 2.01, s | 21.1 | 2.01, s | 21.1 |
| 169.8 | 169.8 | 169.8 | ||||
| 13-OAc | *2.00, s | 21.1 | 2.00, s | 21.2 | 1.95, s | 21.0 |
| 169.8 | 170.0 | 169.9 | ||||
| 8-OOH | 8.17, brs | |||||
*12- and 13-acetate assignments for 1 may be interchanged.
Acknowledgements
We wish to thank Dr. Phil Alderslade from the Museum and Art Gallery of the Northern Territory, Fannie Bay, Darwin, for taxonomy, Mr. Rick Willis from A.I.M.S. for mass spectrometry, and Mr. Stephen Wright for cytotoxicity assays.
- Sample Availability: Samples are available from the authors.
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Bowden, B.F.; Cusack, B.J.; Dangel, A. 13-Epi-9-deacetoxyxenicin, a Cytotoxic Diterpene from the Soft Coral Asterospicularia laurae (Alcyonacea). Mar. Drugs 2003, 1, 18-26. https://doi.org/10.3390/md101018
AMA Style
Bowden BF, Cusack BJ, Dangel A. 13-Epi-9-deacetoxyxenicin, a Cytotoxic Diterpene from the Soft Coral Asterospicularia laurae (Alcyonacea). Marine Drugs. 2003; 1(1):18-26. https://doi.org/10.3390/md101018
Chicago/Turabian StyleBowden, Bruce F., Bernard J. Cusack, and Alissa Dangel. 2003. "13-Epi-9-deacetoxyxenicin, a Cytotoxic Diterpene from the Soft Coral Asterospicularia laurae (Alcyonacea)" Marine Drugs 1, no. 1: 18-26. https://doi.org/10.3390/md101018
