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. 2003 Nov 15;1(4):18–26.

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

Bruce F Bowden 1,*, Bernard J Cusack 1, Alissa Dangel 1
PMCID: PMC3783871

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.

Keywords: Cytotoxic, xenicane diterpene, soft coral, Asterospicularia

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].

graphic file with name marinedrugs-01-00018f3.jpg

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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.

Table 1.

NMR Assignments for 1–3 in CDCl3

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
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*

12- and 13-acetate assignments for 1 may be interchanged.

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.

Figure 1.

Figure 1

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Autoxidation of an alkene - an Ene reaction

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).

Figure 2.

Figure 2

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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.

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.

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.

Footnotes

Sample Availability: Samples are available from the authors.

References

  • 1.Fabricius K., Alderslade P. Soft Corals and Sea Fans. Australian Institute of Marine Science; Townsville: 2001. pp. 136–137. [Google Scholar]
  • 2.Ksebati M. B., Schmitz F. J. 24ɛ-Methyl-5α-cholestane-3β, 5, 6β, 22R, 24-pentol 6-acetate: new polyhydroxylated sterol from the soft coral Asterospicularia randalli. Steroids. 1984;43:639–649. doi: 10.1016/0039-128x(84)90026-6. [DOI] [PubMed] [Google Scholar]
  • 3.Vanderah D. J., Steudler P. A., Ciereszko L. S., Schmitz F. J., Ekstrand J. D., van der Helm D. Marine Natural Products. Xenicin: a diterpenoid possessing a nine-membered ring form the soft coral Xenia elongata. J. Am. Chem. Soc. 1977;99:5780–5784. doi: 10.1021/ja00459a040. [DOI] [PubMed] [Google Scholar]
  • 4.(a) Groweiss A., Kashman Y. Xeniculin. Xeniaphyllenol and xeniaphyllenol oxide new diterpenoids from the soft coral Xenia macrospiculata. Tetrahedron Lett. 1978;(25):2205–2208. [Google Scholar]; (b) Kashman Y., Groweiss A. Xeniolide-A and -B; two new diterpenoids from the soft coral Xenia macrospiculata. Tetrahedron Lett. 1978;(48):4833–4836. [Google Scholar]; (c) Kashman Y., Groweiss A. New diterpenoids from the soft corals Xenia macrospiculata and Xenia obscuronata. J. Org. Chem. 1980;45:3814–3824. [Google Scholar]; (d) Groweiss A., Kashman Y. Eight new Xenia diterpenoids from three soft corals of the Red Sea. Tetrahedron. 1983;20:3385–3396. [Google Scholar]
  • 5.(a) Braekman J. C., Daloze D., Tursch B., Declercq J. P., Germain G., Van Meerssche M. Chemical studies of marine invertebrates 39: Three novel diterpenes from the soft coral Xenia novae-Britanniae. Bull. Soc. Chim. Belg. 1979;88:71–77. [Google Scholar]; (b) Bowden B. F., Coll J. C., Ditzel E., Mitchell S. J., Robinson W. T. Studies of Australian soft corals XXVIII. The structure determination of two new diterpenes from the genus Xenia (Alcyonacea) Aust. J. Chem. 1982;35:997–1002. [Google Scholar]; (c) Bowden B.F., Coll J.C., Engelhardt L.M., Heaton A., White A.H. Studies of Australian soft corals XLI. Structure determination of a new Sesquiterpene from Xenia novae-britanniae and an investigation of a Xenia species. Aust. J. Chem. 1987;40:1483–1489. [Google Scholar]
  • 6.(a) Lelong H., Ahond A., Chiaroni A., Poupat C., Riche C., Potier P., Pousset J., Pousset M., Laboute P., Menou J. L. Invertebres marins du lagon Neo-Caledonien, VIII. Metabolites terpeniques de Xenia membranacea. J. Nat. Prod. 1987;50:203–210. [Google Scholar]; (b) Almourabit A., Ahond A., Chiaroni A., Poupat C., Riche C., Potier P., Laboute P., Menou J.-L. Marine Invertebrates from New Caledonia IX. Havannachlorhydrines, four new diterpenoid metabolites from Xenia membranacea have been isolated. J. Nat. Prod. 1988;51:282–292. [Google Scholar]; (c) Almourabit A., Gillet B., Ahond A., Beloeil J.-C., Poupat C., Potier P. Marine Invertebrates from New Caledonien sea 11. Havannahine, and three desoxyhavannahines isolated from the soft coral Xenia membranacea. J. Nat. Prod. 1989;52:1080–1087. [Google Scholar]; (d) Almourabit A., Ahond A., Poupat C., Potier P. Invertebres marins du lagon Neo-Caledonien, XII. Isolement et etude structurale de nouveaux diterpenes extraits de L’Alcyonaire Xenia membranacea. J. Nat. Prod. 1990;53:894–908. [Google Scholar]
  • 7.König G. M., Coll J. C., Bowden B. F., Gulbis J. M., MacKay M. F., LaBarre S. C., Laurent D. The structure determination of a Xenicane diterpene from Xenia garciae. J. Nat. Prod. 1989;52:294–299. [Google Scholar]
  • 8.Miyamoto T., Takenaka Y., Yamada K., Higuchi R. Bioactive diterpenoids from Octocorallia, 2. Deoxyxeniolide B, a novel ichthyotoxic diterpenoid from the soft coral Xenia elongata. J. Nat. Prod. 1995;58:924–928. doi: 10.1021/np50120a017. [DOI] [PubMed] [Google Scholar]
  • 9.(a) Iwagawa T., Amano Y., Hase T., Shiro M. New xenia diterpenoids from a soft coral, Xenia species. Chem. Lett. 1995:695–696. [Google Scholar]; (b) Iwagawa T., Masuda T., Okamura H., Nakatani M. New xenia diterpenoids from a Xenia species of a soft coral. Tetrahedron. 1996;52:13121–13128. [Google Scholar]; (c) Iwagawa T., Amano Y., Hase T., Shiro M. New xenia diterpenoids from a soft coral, Xenia species. Tetrahedron. 1995;51:11111–11118. [Google Scholar]; (d) Iwagawa T., Amano Y., Kawasaki J., Hase T. New xenia diterpenes isolated from the soft coral, Xenia florida. J. Nat. Prod. 1998;61:1513–1515. doi: 10.1021/np9802116. [DOI] [PubMed] [Google Scholar]; (e) Iwagawa T., Nakamura K., Hirose T., Okamura H., Nakatani M. New xenicane diterpenoids isolated from the acetone extract of the soft coral, Xenia florida. J. Nat. Prod. 2000;63:468–472. doi: 10.1021/np990470a. [DOI] [PubMed] [Google Scholar]
  • 10.Vervoort H. C., Fenical W. Antibacterial diterpenoids from an undescribed soft-coral of the genus Xenia. Nat. Prod. Lett. 1995;6:49–55. [Google Scholar]
  • 11.Miyaoka H., Nakano M., Iguchi K., Yamada Y. Three new xenicane diterpenoids from Okinawan coral of the genus Xenia. Tetrahedron 1999, 55, 12977–12982; (b) Miyaoka, H., Mitome, H., Nakano, M., Yamada, Y. Xeniaoxolane: a new xenicane-type diterpenoid from the Okinawan soft coral Xenia species; absolute configurations of xeniaoxolane, xeniolide-A and xenialactol. Tetrahedron. 2000;56:7737–7740. [Google Scholar]
  • 12.(a) Coval S. J., Scheuer P. J., Matsumoto G. K., Clardy J. Two new xenicin diterpenoids from the octocoral Anthelia edmonsoni. Tetrahedron. 1984;40:3823–3828. [Google Scholar]; (b) Green D., Carmely S., Benayahu Y., Kashman Y. Antheliolide A and B: two new C24-acetoacetylated diterpenoids of the soft coral Anthelia glauca. Tetrahedron Lett. 1988;29:1605–1608. [Google Scholar]
  • 13.Rudi A., Ketzinel S., Goldberg I., Stein Z., Kashman Y. Antheliatin and zahavins A and B, three new cytotoxic xenicane diterpenes from two soft corals. J. Nat. Prod. 1995;58:1581–1586. [Google Scholar]
  • 14.Hooper G. J., Davies-Coleman M. T. New metabolites from the South African soft coral Capnella thyrsoidea. Tetrahedron. 1995;51:9973–9984. [Google Scholar]
  • 15.Tanaka J., Ogawa N., Liang J., Higa T., deNys R., Bowden B. F., Carroll A. R., Coll J. C., Bernardinelli G., Jefford C. W. Helioxenicins A-C: diterpenes from the blue coral Heliopora coerulea. Tetrahedron. 1994;50:9989–9996. [Google Scholar]
  • 16.(a) Schwartz R.E., Scheuer P. J., Zabel V., Watson W. H. The coraxeniolides, constituents of the pink coral Corallium sp. Tetrahedron. 1981;37:2725–2733. [Google Scholar]; (b) D’Ambrosio M., Guerriero A., Pietra F. Arboxeniolide-1, a new, naturally occurring xeniolide diterpenoid from the gorgonian Paragorgia arborea of the Crozet Is. (S. Indian Ocean) Z. Naturforsch. 1984;39:1180–1183. [Google Scholar]; (c) Fusetani N., Asano M., Matsunaga S., Hashimoto K. Acalycixeniolides, norditerpenoids which inhibit cell division of starfish eggs, from gorgonian Acalycigorgia inermis. Tetrahedron Lett. 1987;28:5837–5840. [Google Scholar]; (d) Hokama S., Tanaka J., Higa T., Fusetani N., Asano M., Matsunaga S., Hashimoto K. Ginamalline, a new norditerpene with allene functionality from four gorgonians of the genus Acalycigorgia. Chem. Lett. 1988:855–856. [Google Scholar]; (e) Stonik V.A., Makar’eva T.N., Dmitrenok A.S. New diterpenoid of the xenicane series from the gorgonian Paragorgia arborea. Khim. Prir. Soedin. 1990:125–126. [Google Scholar]; (f) Ochi M., Kataoka K., Tatsukawa A., Kotsuki H., Shibata K. Acalycigorgins A, B and C, three new biologically active diterpenoids from the gorgonian Acalycigorgia sp. Heterocycles. 1993;36:41–44. [Google Scholar]; (g) Ochi M., Kataoka K., Tatsukawa A., Kotsuki H., Shibata K. Biologically active xenicane diterpenoids from the gorgonian Acalycigorgia sp. Heterocycles. 1994;38:151–158. [Google Scholar]; (h) Fusetani N., Asano M., Matsunaga S., Hashimoto K. Acalycixeniolides, novel norditerpenes with allene functionality from two gorgonians of the genus Acalycigorgia. Tetrahedron. 1989;45:1647–1652. [Google Scholar]
  • 17.Coll J.C., Bowden B.F. The Application of vacuum liquid chromatography to the separation of terpene mixtures. J. Nat. Prod. 1986;49:934–936. [Google Scholar]

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