Bioactive Compounds from a Gorgonian Coral Echinomuricea sp. (Plexauridae)

A new labdane-type diterpenoid, echinolabdane A (1), and a new sterol, 6-epi-yonarasterol B (2), were isolated from a gorgonian coral identified as Echinomuricea sp. The structures of metabolites 1 and 2 were elucidated by spectroscopic methods. Echinolabdane A (1) possesses a novel tetracyclic skeleton with an oxepane ring jointed to an α,β-unsaturated-γ-lactone ring by a hemiketal moiety, and this compound is the first labdane-type diterpenoid to be obtained from marine organisms belonging to the phylum Cnidaria. 6-epi-Yonarasterol B (2) is the first steroid derivative to be isolated from gorgonian coral belonging to the genus Echinomuricea, and this compound displayed significant inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.


Introduction
The search for new natural products from marine organisms has been remarkably successful, and gorgonian corals have been proven to be rich sources of interesting natural terpenoid derivatives [1,2]. In a previous study, two sesquiterpenoid phenols, (7S,10R)-(+)-10,11-epoxycurcuphenol and (+)-curcuphenol [3], were isolated from the Formosan gorgonian coral Echinomuricea sp. (family Plexauridae). In continuation of our search for new natural substances from marine invertebrates collected off the waters of Taiwan at the intersection point of the Kuroshio current and the South China Sea surface current, we have further isolated a new labdane-type diterpenoid, echinolabdane A (1), and a new steroid derivative, 6-epi-yonarasterol B (2), from Echinomuricea sp. In this paper, we describe the isolation, structural characterization and bioactivity of new compounds 1 and 2 ( Figure 1).
The methine unit at δ C 100.9 (CH-16) was more shielded than expected for an oxygenated C-atom and was correlated with the methine proton at δ H 6.07 (H-16) in the HMQC spectrum, and this proton showed a 2 J-correlation with C-13 and showed 3 J-correlations with C-8, C-14 and C-15 in the HMBC spectrum, and was concluded to be a part of a hemiketal moiety.  The relative configuration of 1 was elucidated mainly from a NOESY spectrum as being compatible with that of 1 offered by computer modeling (Figure 3) [4], in which the close contacts of atoms in space calculated were consistent with the NOESY correlations. In the NOESY analysis of 1, the correlations of H-9 with H-5 and H-16, but not with H 3 -19 and H 3 -20, indicated that these protons (H-5, H-9 and H-16) were situated on the same face, and these were assigned as α protons, since the C-19 and C-20 methyls are β-substituents at C-8 and C-10, respectively. The Z-configuration of the C-13/14 double bond was elucidated from a correlation between H-14 (δ H 5.82) and H-12b (δ H 2.91). From the above evidence, the relative configurations of the chiral carbons of 1 were assumed to be 5S*, 8R*, 9R*, 10S* and 16R*. On the basis of the above findings, the structure of 1 was elucidated.
The configuration of two chiral centers (C-20 and C-24) in the side chain of 2 was elucidated by comparison of 13  suggested that H-6 was an axial hydrogen. This result further supported that the 6-acetoxy was α-oriented in 2. Due to the fact that coupling pattern of H-11 in 2 appeared as a broad singlet in the 1 H NMR spectrum of 2, it is difficult to elucidate the relative stereochemistry of the 11-hydroxy group in 2 by vicinal coupling constant analysis; however, H-11 showed significant correlations with H-8, Me-18 and Me-19 in the NOESY analysis of 2, which suggested that the 11-hydroxy group in 2 was α-oriented.    The in vitro anti-inflammatory effects of compounds 1 and 2 were tested (Table 3). 6-epi-yonarasterol B (2) was found to show significant inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrophotmeter; peaks are reported in cm −1 . The NMR spectra were recorded on a Varian Mercury Plus 400 or on a Varian Inova 500 NMR spectrometer. Coupling constants (J) are given in Hz. 1 H and 13 C NMR assignments were supported by 1 H-1 H COSY, HMQC, HMBC and NOESY experiments. ESIMS and HRESIMS 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 F 254 (0.25 mm, Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. Normal phase HPLC was performed using a system comprised of a Hitachi L-7100 pump, a Hitahci L-7455 photodiode array detector, a Rheodyne injection port and a normal phase column (Hibar 250 × 10 mm, Merck, silica gel 60, 5 μm). Reverse phase HPLC was performed using a system comprised of a Hitachi L-7100 pump, a Hitahci L-2455 photodiode array detector, a Rheodyne injection port and a reverse phase column (Polaris 5 C18-A 250 × 10 mm, Varian, silica gel 60, 5 μm).

Animal Material
Specimens of the gorgonian coral Echinomuricea sp. were collected by hand using scuba equipment off the coast of southern Taiwan and stored in a freezer until extraction. This organism was identified by comparison with previous descriptions [8,9]. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation
The freeze-dried and minced material of Echinomuricea sp. (wet weight 1.68 kg, dry weight 428 g) was extracted with a mixture of methanol (MeOH) and dichloromethane (1:1). The residue was partitioned with ethyl acetate (EtOAc) and H 2 O. The EtOAc layer was partitioned between MeOH and n-hexane. The n-hexane layer was separated by silica gel and eluted using n-hexane/EtOAc/MeOH to yield 21 fractions A-U. Fraction L was separated on silica gel and eluted using n-hexane/EtOAc (stepwise, 50:1-pure EtOAc) to yield 16 fractions, L1-L16. Fraction L8 was purified by normal-phase HPLC using a mixture of n-hexane and EtOAc (8:1) as the mobile phase to afford compound 1 (0.9 mg). Fraction R was chromatographed on silica gel and eluted using n-hexane/EtOAc (stepwise, 1:1-pure EtOAc) to yield fractions R1-R13. Fraction R7 was separated by normal-phase HPLC using a mixture of n-hexane and acetone (4:1) as the mobile phase to afford 14 fractions R7A-R7N. Fraction R7M was further purified by reverse-phase HPLC using a mixture of methanol and H 2 O (85:15) to yield 2 (0.7 mg).