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Note | Regular issue | Vol. 78, No. 6, 2009, pp. 1563-1567
Received, 22nd December, 2008, Accepted, 29th January, 2009, Published online, 2nd February, 2009.
DOI: 10.3987/COM-08-11636
Rumphellolide H, a New Natural Caryophyllane from the Gorgonian Rumphella antipathies

Tsong-Long Hwang, Yin-Di Su, Wan-Ping Hu, Li-Fan Chuang, and Ping-Jyun Sung*

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

A new natural caryophyllane-type sesquiterpenoid, rumphellolide H (1), which possesses a tetrahydropyran moiety in structure, was isolated from the gorgonian coral Rumphella antipathies. The structure of 1 was established by spectral data analysis and this metabolite showed moderate inhibitory effects on elastase release by human neutrophils.

Previous chemical investigations on Rumphella antipathies have yielded a series of interesting caryophyllane derivatives, including kobusone,1 isokobusone,2 rumphellatins A–D,35 and rumphellolides A–G.6,7 We describe herein the isolation, structure determination, and bioactivity of a new natural sesquiterpenoid, rumphellolide H (1), a caryophyllane derivative that possesses a tetrahydropyran moiety, from the further studies on R. antipathies.

Rumphellolide H (1) was isolated as a colorless oil. The molecular formula for 1 was determined to be C15H26O3 by HRESIMS at m/z 277.1782 (C15H26O3+Na, calcd, 277.1780). Comparison of the 1H NMR (Table 1) and DEPT data with the molecular formula indicated that there must be two exchangeable protons, requiring the presence of two hydroxy groups, and this deduction was supported by a broad absorption in the IR spectrum at 3424 cm1. From the 13C NMR data of 1 (Table 1), no olefinic carbon and carbonyl carbon was observed. Thus, caryophyllane 1 must be tricyclic. In the 13C NMR spectrum of 1, the signals for four downfield carbons appeared between δC 69–79 ppm, including an oxymethylene (δC 69.5, t, CH2-13), an oxymethine (δC 70.5, d, CH-5), two oxygenated quaternary carbons (δC 78.1, s, C-4; 75.6, s, C-8), and 11 aliphatic sp3 carbon signals (a quaternary carbon, two methines, five methylenes, and three methyls) were observed. The 1H NMR spectrum showed that all three methyl groups are isolated (δH 1.21, 3H, s, H3-12; 1.02, 2×3H, s, H3-14 and H3-15). Furthermore, five pairs of aliphatic methylene protons (H2-2, H2-3, H2-6, H2-7, H2-10), two aliphatic methine protons (H-1, H-9), an oxymethine proton (H-5), and a pair of hydroxymethyl protons (H2-13) were observed in the 1H NMR spectrum of 1.

Examination of the coupling information in the 1H–1H COSY spectrum of 1 enabled identification of the C-1/2/3, C-5/6/7, C-9/10, and C-1/9 units (Figure 1). These data, together with the HMBC correlations, established the carbon skeleton of 2 (Table 1 and Figure 1). A methyl attached at C-4 was confirmed by the HMBC correlations between H3-12/C-3, -4, -5 and H2-3/C-12. The hydroxymethyl group positioned at C-8 was confirmed by the connectivity between the downfield methylene protons (δH 3.26 and 3.17) and C-7, -8, and C-9. Based on the above findings, only one oxygen atom has not been assigned, and this atom had to be positioned between C-4 and C-8 (an ether bridge), there being two oxygenated quaternary carbons were observed in the 13C NMR spectrum of 1.

The relative configurations of five chiral centers at C-1, C-4, C-5, C-8, and C-9 in 1 were elucidated by analysis of NOESY interactions (Figure 2) and vicinal 1H–1H coupling constants. The trans geometry of H-1 (δH 1.91, ddd, J=10.4, 10.0, 1.8 Hz) and H-9 (δH 2.04, ddd, J=10.0, 7.2, 5.2 Hz) is indicated by an 10.0 Hz coupling constant between these two ring junction protons, and H-9 and H-1 were assigned as α- and β-oriented, respectively. In the NOESY experiment of 1, H-5 showed correlations with H-1, H3-12, and one proton of C-3 methylene (δH 1.70), indicating that these protons (H-1, H-5, H3-12, and H-3β) are located on the same face of the molecule and assigned as β-protons. Furthermore, H3-12 exhibited responses with H2-3, but not with H-1, suggesting the C-12 methyl should be placed on the equatorial direction in the tetrahydropyran ring. Furthermore, H2-13 showed responses with H-9, suggesting the hydroxymethyl group attached at C-8 was α-oriented. On the basis of the above findings, the structure 1 was established and the configurations of the chiral centers of 1 were assigned as 1R*, 4S*, 5R*, 8S*, 9S*.
The compounds of caryophyllane-type natural products exist widely in terrestrial plant;
8 however, caryophyllane analogues were shown to be rarely found in marine organisms.912 Caryophyllane 1 was first obtained as a synthetic product,13,14 however, there has been no report of 1 (rumphellolide H) obtained from any natural source. In biological activity testing, caryophyllane 1 was found to show 32.7% inhibitory effect on human neutrophil elastase release at 10 µg/mL.

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, respectively, in CDCl3. Proton chemical shifts were referenced to the residual CHCl3 signal (δH 7.26 ppm) and 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 using 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 by using a system comprised of a HITACHI L-7100 pump, a HITACHI photo diode array detector L-7455, and a RHEODYNE 7725 injection port. A semi-preparative column (Hibar 250–25 mm, LiChrospher Si 60, 5 µm, MERCK) was used for HPLC.
Animal Material. Specimens of Rumphella antipathies were collected in May 2004, off the southern coast of Taiwan. This organism was identified by comparison with previous description.15 A voucher specimen was deposited in the NMMBA, Taiwan.
Extraction and Isolation. A freeze-dried and minced sample of R. antipathies (wet weight 402 g, dry weight 144 g) was extracted with a mixture of MeOH and CH2Cl2 (1:1). The extract was partitioned between hexane and 9:1 MeOH–H2O. The MeOH–H2O layer was diluted to 1:1 MeOH–H2O and partitioned against CH2Cl2. The CH2Cl2 layer was separated on silica gel and eluted using hexane/EtOAc (stepwise, 0–100% EtOAc) to yield fractions A–D. Fraction B was separated on silica gel and eluted using CH2Cl2/acetone (stepwise, 20:1–1:1) to yield fractions B1–B18. Fraction B8 was repurifed by normal-phase HPLC, using mixtures of hexane and acetone as a mobile phase to afford 1 (0.9 mg, 5:1).
Rumphellolide H (1): colorless oil; [α]D –1 (c 0.04, CHCl3); IR (KBr) νmax 3424 cm1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; ESIMS m/z 277 (M+Na)+; HRESIMS m/z 277.1782 [(M+Na)+, calcd for C15H26O3+Na, 277.1780]. The related physical (optical rotation value) and spectral (1H NMR) data of natural product 1 are in full agreement with those reported previously.13,14
Human Neutrophil Elastase Release. Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Elastase release was carried out according to the procedures described previously.16 Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp- nitroanilide as the elastase substrate.

This research was supported by grants from the TCRC, NMMBA (97100107 and 971001101); APORC, NSYSU (96C031702); NDHU; and NSTPBP, National Science Council (NSC 97-2323-B-291-001 and 95-2320-B-291-001-MY2), Taiwan, awarded to P.-J.S.


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