HETEROCYCLES

Note | Regular issue | Vol. 81, No. 4, 2010, pp. 991-996
Received, 29th December, 2009, Accepted, 9th February, 2010, Published online, 10th February, 2010.
DOI: 10.3987/COM-09-11899

■ 12-epi-Fragilide G, a New Briarane-Type Diterpenoid from the Gorgonian Coral Ellisella robusta

Yu-Chia Chang, Tsong-Long Hwang, Sheng-Kai Huang, Li-Won Huang, Mei-Ru Lin, and Ping-Jyun Sung*

, National Museum of Marine Biology and Aquarium, 2 Houwan Road, Checheng, Pingtung, Taiwan 944, Taiwan, R.O.C.

Abstract

A new chlorinated briarane-type diterpenoid, 12-epi-fragilide G (1), was isolated from the gorgonian coral Ellisella robusta. The structure of 1 was elucidated by the interpretations of spectral data analysis and this compound was found to possess an s-cis diene moiety in its structure. Briarane 1 displayed inhibitory effects on elastase release by human neutrophils.

Previous studies on the gorgonians belonging to the genus Ellisella (family Ellisellidae), have resulted in the isolation of a series of novel natural products, including robustolides A–K, featuring with briarane carbon skeleton (3,8-cyclized cembranoid).1–6 During our further studies on the chemical constituents of a gorgonian coral Ellisella robusta, collected off Taiwan waters, a new chlorinated briarane, 12-epi-fragilide G (1) (Chart 1), which was found to possess an s-cis diene moiety, was isolated. In this paper, we reported the isolation, structure determination, and bioactivity of above new briarane 1.
12-epi-Fragilide G (1) was obtained as a white powder. The HRESIMS data established the molecular formula of 1 as C28H35ClO12, with m/z 621.1718 [(M+Na)+, calcd. 621.1715], indicating 11 degrees of unsaturation. The IR spectrum of 1 showed the presence of hydroxy (3464 cm–1), γ-lactone (1783 cm–1), and ester (1737 cm–1) groups. From the 13C NMR data of 1 (Table 1), a disubstituted olefin and an

exocyclic carbon-carbon double bond were deduced from the signals of four carbons at δC 142.1 (s, C-5), 132.6 (d, CH-4), 130.4 (d, CH-3), and 115.2 (t, CH2-16). Five carbonyl resonances at δC 174.6 (s, C-19), 170.3, 170.0, 169.9, and 169.3 (4×s, ester carbonyls), confirmed the presence of a γ-lactone and four esters in 1. In the 1H NMR spectrum of 1 (Table 1), four acetyl methyls (δH 2.11, 2.10, 2.05, and 2.02, each 3H×s) were observed. Thus, the NMR data accounted for seven degrees of unsaturation and requiring 1 to be tetracyclic. An exocyclic epoxy group was confirmed from the signals of two oxygenated carbons at δC 57.3 (s, C-11) and 49.2 (t, CH2-20), and further supported by the proton chemical shifts of H2-20 (δH 2.77, 1H, dd, J = 4.0, 1.2 Hz, H-20a; 2.65, 1H, d, J = 4.0 Hz, H-20b). Moreover, a methyl singlet (δH 1.17, 3H, s, H3-15), a methyl doublet (δH 1.25, 3H, d, J = 7.2 Hz, H3-18), two aliphatic methine protons (δH 3.84, 1H, br s, H-10; 2.85, 1H, q, J = 7.2 Hz, H-17), a pair of aliphatic methylene protons (δH 2.27, 1H, m; 2.01, 1H, m; H2-13), five oxymethine protons (δH 5.70, 1H, d, J = 9.6 Hz, H-2; 5.18, 1H, d, J = 2.4 Hz, H-9; 4.98, 1H, dd, J=2.8, 2.8 Hz, H-14; 4.52, 1H, dd, J = 3.2, 2.8 Hz, H-12; 4.16, 1H, d, J = 4.0 Hz, H-7), four olefin protons (δH 6.88, 1H, d, J = 15.6 Hz, H-4; 6.01, 1H, dd, J = 15.6, 9.6 Hz, H-3; 5.34, 1H, s, H-16a; 5.26, 1H, s, H-16b), a chlorinated methine proton (δH 5.07, 1H, d, J = 4.0 Hz, H-6), and a hydroxy proton (δH 3.08, 1H, br s, OH-8) were observed in the 1H NMR spectrum of 1.
The gross structure of 1 was determined by 2D NMR studies, including 1H–1H COSY, HMQC, and HMBC experiments. From the 1H–1H COSY spectrum of 1 (Table 1), it was possible to establish the separate spin systems that map out the proton sequences from H-2/H-3, H-3/H-4, H-6/H-7, and H-9/ H-10. These data, together with the HMBC correlations between H-2/C-1, -3, -4; H-3/C-5; H-4/C-2, -3, -6; H-6/C-4, -5, -7, -8; H-7/C-6, -9; H-9/C-1, -8, -10; and H-10/C-1, -8, -9 (Table 1), established the connectivity from C-1 to C-10 in a ten-membered ring. An exocyclic double bond at C-5 was confirmed by the HMBC correlations between H-16a/C-4, -6; H-16b/C-4, -5, -6; H-4/C-16; and H-6/C-16; and further confirmed by the 1H–1H COSY correlation between H-4 and H-16a (by allylic coupling). The cyclohexane ring, which is fused to the ten-membered ring at C-1 and C-10, was elucidated by the HMBC correlations between H-2/C-14; H-9/C-11; H-10/C-11, -20; and one proton of C-13 methylene (H-13α)/C-1. The epoxy group positioned at C-11/20 was confirmed by the connectivity between H-20b and C-11, -12. The C-15 methyl group was positioned at C-1 from the HMBC correlations between H3-15/C-1, -2, -10, -14; H-2/C-15; and H-10/C-15. In addition, the HMBC correlations also revealed that three acetoxy groups should attach at C-2, C-9, and C-12, respectively (Table 1). The hydroxy proton signal at δH 3.08 was revealed by its HMBC correlations to C-7, -8, and C-9, indicating its attachment to C-8, an oxygen-bearing quaternary carbon at δC 82.8. Thus, the remaining acetoxy group should be positioned at C-14, as indicated by analysis of the 1H–1H COSY correlations and characteristic NMR signals analysis. These data, together with the 1H–1H COSY correlation between H-17 and H3-18 and the HMBC correlations between H-17/C-8, -9, -18, -19 and H3-18/C-8, -17, -19, unambiguously established the molecular framework of 1.
In a previous study, the 13C chemical shifts of exocyclic 11,20-epoxy groups in briarane derivatives were summarized, that while the 13C NMR data for C-11 and C-20 were appeared at δC 55–61 and 47–52 ppm, respectively, the epoxy group was α-oriented (11R*) and the cyclohexane ring should be existed in chair conformation.7 Based on the above observations, the configuration of 11,20-epoxy group in 1 (δC 57.3, s, C-11; 49.2, t, CH2-20) should be α-oriented and the cyclohexane ring was existed in a chair conformation.
The relative stereochemistry of 1 was elucidated mainly from the interactions observed in a NOESY experiment (Figure 1) and by the vicinal 1H–1H coupling constants. As per convention while analyzing the stereochemistry of briarane-type natural products, H-10 and H3-15 were assigned to the α and β face, anchoring the stereochemical analysis because no correlation was observed between H-10 and H3-15. In the NOESY experiment of 1, H-10 gave correlations to H-2, H-9, OH-8, and H3-18, suggesting that these protons were located on the same face and assigned as α protons, since C-15 methyl is the β-substituent at C-1. H-14 was found to exhibit responses with H-2, H-13α/β, and H3-15, but not with H-10, revealing the β-orientation of this proton. In addition, H-12 was found to correlate with H-13α/β and one proton of C-20 methylene (δH 2.27, H-20a), indicating the C-12 acetoxy group was α-oriented. H-7 exhibited correlations with H-6 and H-17, suggesting that these protons were positioned on the β face in 1. The trans geometry of C-3/4 double bond was indicated by a 15.6 Hz coupling constant between H-3 (δH 6.01) and H-4 (δH 6.88). Moreover, the olefin proton H-3 showed a correlation with H3-15, but not with H-2; and H-4 showed responses with H-2 and OH-8, demonstrating the E-configuration of Δ3,4 and established the conjugated s-cis diene moiety in 1. Based on the above findings, the structure of 1 was established, and the configurations of all chiral centers of 1 are assigned as 1R*, 2S*, 6S*, 7R*, 8R*, 9S*, 10S*, 11R*, 12R*, 14S*, and 17R*. By comparison the related spectral data with those of a known briarane analogue, fragilide G (2) (Chart 1), which was isolated from a Taiwanese gorgonian coral Junceella fragilis.8 The structure of 1 was found to be the 12-epi-compound of fragilide G (2) and should be named as 12-epi-fragilide G.

It has to be noted here that a briarane containing an s-cis diene moiety as presented in 1, is unprecedential. To the best of knowledge, only three briarane metabolites, robustolide J, fragilides B, and fragilide G,6,8,9 were found to possess the functional group of this type. 12-epi-Fragilide G (1) is the fourth example which possessing an s-cis diene moiety in structure. Brarane 1 displayed 61.4% inhibitory effect on elastase release by human neutrophils at 10 μg/mL.

EXPERIMENTAL
General Experimental Procedures. Optical rotation values were measured with a JASCO P-1010 digital polarimeter. Infrared spectra were obtained on a VARIAN DIGLAB FTS 1000 FT-IR spectrophotometer. The NMR spectra were recorded on a VARIAN MERCURY PLUS 400 FT-NMR at 400 MHz for 1H and 100 MHz for 13C, in CDCl3, respectively. Proton chemical shifts were referenced to the residual CHCl3 signal (δH 7.26 ppm). 13C NMR spectra were referenced to the center peak of CDCl3 at δC 77.1 ppm. ESIMS and HRESIMS data were recorded on a BRUKER APEX II mass spectrometer. Column chromatography was performed on silica gel (230–400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F254 (0.25 mm, Merck) and spots were visualized by spraying with 10% H2SO4 solution followed by heating. HPLC was performed using a system composed of a HITACHI L-7100 pump, a HITACHI L-7455 photo diode array detector, a RHEODYNE 7725 injection port, and a normal phase semi-preparative column (Hibar 250×10 mm, LiChrospher Si 60, 5 µm).
Animal Material. Specimens of Ellisella robusta were collected by hand using scuba gear off the southern Taiwan coast. This organism was identified by comparison with previous descriptions.10–12 A voucher specimens was deposited in the National Museum of Marine Biology & Aquarium (NMMBA), Taiwan.
Extraction and Isolation. The freeze-dried and minced material of E. robusta (wet weight 1909 g, dry weight 830 g) was extracted with a mixture of MeOH and CH2Cl2 (1:1). The residue was partitioned between EtOAc and H2O. The EtOAc layer was separated by silica gel and eluted using hexane/EtOAc to yield 28 fractions. Fraction 10 was separated on silica gel and eluted using CH2Cl2/EtOAc (10:1–pure EtOAc) to yield 14 fractions, 10A–10N. Fractions 10F and 10G were combined and purified by normal phase HPLC, using a mixture of CH2Cl2/acetone to afford 1 (1.2 mg, 13:1).
12-epi-Fragilide G (1): white powder; mp 238–240 °C; [α]23D –102 (c 0.05, CHCl3); IR (neat) νmax 3464, 1783, 1737 cm–1; 13C NMR (CDCl3, 100 MHz) and 1H NMR (CDCl3, 400 MHz) data, see Table 1; ESIMS m/z 621 (M+Na)+; HRESIMS m/z 621.1718 (Calcd for C28H3535ClO12+Na, 621.1715).
Human Neutrophil Elastase Release. Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Elastase release were carried out according to the procedures described previoulsy.13,14 Briefly, the elastase release experiment was performed using MeO-Suc-Ala- Ala-Pro-Valp-nitroanilide as the elastase substrate.

ACKNOWLEDGEMENTS
This research was supported by grants from the NMMBA (981001101, 99200321, and 99200322); APORC, NSYSU (96C031702); NDHU; and NSTPBP, National Science Council (NSC 98-2323-B- 291-001 and NSC 98-2320-B-291-001-MY3), Taiwan, awarded to P.-J.S.

References

1. C. Tanaka, Y. Yamamoto, M. Otsuka, J. Tanaka, T. Ichiba, G. Marriott, R. Rachmat, and T. Higa, J. Nat. Prod., 2004, 67, 1368. CrossRef
2. P.-J. Sung, W.-T. Tsai, M. Y. Chiang, Y.-M. Su, and J. Kuo, Tetrahedron, 2007, 63, 7582. CrossRef
3. Y.-M. Su, T.-Y. Fan, and P.-J. Sung, Nat. Prod. Res., 2007, 21, 1085. CrossRef
4. P.-J. Sung, M. Y. Chiang, W.-T. Tsai, J.-H. Su, Y.-M. Su, and Y.-C. Wu, Tetrahedron, 2007, 63, 12860. CrossRef
5. P.-J. Sung, W.-T. Tsai, M.-R. Lin, Y.-D. Su, C.-H. Pai, H.-M. Chung, J.-H. Su, and M. Y. Chiang, Chem. Lett., 2008, 37, 88. CrossRef
6. T.-L. Hwang, M.-R. Lin, W.-T. Tsai, H.-C. Yeh, W.-P. Hu, J.-H. Sheu, and P.-J. Sung, Bull. Chem. Soc. Jpn., 2008, 81, 1638. CrossRef
7. J.-H. Sheu, Y.-P. Chen, T.-L. Hwang, M. Y. Chiang, L.-S. Fang, and P.-J. Sung, J. Nat. Prod., 2006, 69, 269. CrossRef
8. P.-J. Sung, S.-H. Wang, M. Y. Chiang, Y.-D. Su, Y.-C. Chang, W.-P. Hu, C.-Y. Tai, and C.-Y. Liu, Bull. Chem. Soc. Jpn., 2009, 82, 1426. CrossRef
9. P.-J. Sung, Y.-P. Chen, Y.-M. Su, T.-L. Hwang, W.-P. Hu, T.-Y. Fan, and W.-H. Wang, Bull. Chem. Soc. Jpn., 2007, 80, 1205. CrossRef
10. F. M. Bayer, Proc. Biol. Soc. Wash., 1981, 94, 902.
11. F. M. Bayer and M. Grasshoff, Senckenbergiana Biol., 1994, 74, 21.
12. K. Fabricius and P. Alderslade, Soft Corals and Sea Fans–A Comprehensive Guide to the Tropical Shallow-Water Genera of the Central-West Pacific, the Indian Ocean and the Red Sea, Australian Institute of Marine Science, Queensland, Australia, 2001, pp. 224–225.
13. T.-L. Hwang, Y.-C. Su, H.-L. Chang, Y.-L. Leu, P.-J. Chung, L.-M. Kuo, and Y.-J. Chang, J. Lipid Res., 2009, 50, 1395. CrossRef
14. T.-L. Hwang, G.-L. Li, Y.-H. Lan, Y.-C. Chia, P.-W. Hsieh, Y.-H. Wu, and Y.-C. Wu, Free Radic. Biol. Med., 2009, 46, 520. CrossRef