Rumphellols A and B, New Caryophyllene Sesquiterpenoids from a Formosan Gorgonian Coral, Rumphella antipathies

Two new marine-derived caryophyllene-type sesquiterpenoids, rumphellols A and B (1 and 2), were obtained from the gorgonian coral, Rumphella antipathies, collected off the waters of Taiwan. Although caryophyllene-type sesquiterpenes are rarely found in marine organisms, compounds of this type could be principal components of R. antipathies. The structures of new Compounds 1 and 2 were determined by analysis of their spectroscopic data, including 1D and 2D NMR experiments. Caryophyllene 1 and 2 were evaluated in terms of their anti-inflammatory activity by examining their inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.


Results and Discussion
Rumphellol A (1) was isolated as a colorless oil, and the molecular formula of this compound was determined to be C15H24O2 by high resolution electronspray ionization mass spectrum (HRESIMS) at m/z 237.1836 (calcd. for C15H24O2 + H, 237.1849). IR absorptions at νmax 3429 (broad) and 1724 cm −1 revealed the presence of hydroxy and carbonyl functionalities. The 13 C NMR spectrum of 1 showed 15 carbon signals (Table 1), which were assigned with the assistance of the distortionless enhancement by polarization transfer (DEPT) spectrum to four methyls, four sp 3 methylenes, two sp 3 methines, two sp 3 quaternary carbons (including an oxygenated quaternary carbon), an sp 2 methine and two sp 2 quaternary carbons (including a carbonyl). The 13 C resonances at δC 212.7 (C-5) demonstrated the presence of a ketonic carbonyl. From the 13 C NMR data, a trisubstituted olefin was deduced from the signals at δC 128.8 (C-3) and 138.4 (C-4). Comparison of the 13 C NMR and DEPT spectra with the molecular formula indicated that there must be an exchangeable proton, requiring the presence of a hydroxy group. Thus, the NMR data accounted for two degrees of unsaturation and required 1 to be a sesquiterpenoid with two rings. The 1 H NMR spectrum of 1 (Table 1) showed the presence of four methyl groups, including two methyls attached to a quaternary carbon (H3-14 and H3-15), a methyl attached to an oxygenated quaternary carbon (H3-13) and a vinyl methyl (H3-12). In addition, four pairs of aliphatic methylene protons (H2-2, H2-6, H2-7 and H2-10), two aliphatic methine protons (H-1 and H-9) and an olefin proton (H-3) were observed in the 1 H NMR spectrum of 1.    The gross structure of 1 and all of the 1 H and 13 C NMR data associated with the molecule were determined by 2D NMR studies, including 1 H-1 H COSY, heteronuclear multiple quantum correlation (HMQC) and HMBC experiments. The 1 H NMR coupling information in the 1 H-1 H COSY spectrum of 1 enabled identification of the C-10/C-9/C-1/C-2/C-3 and C-6/C-7 units ( Figure 1). These data (together with the HMBC correlations between H-1/C-8, C-9; H2-2/C-1, C-3, C-4, C-9; H-3/C-2, C-5; H2-6/C-4, C-5, C-7, C-8; H2-7/C-5, C-6, C-8, C-9; and H-9/C-1, C-2 (Table 1 and Figure 1)) established the connectivity from C-1 to C-9 within the nine-membered ring. The methyls attached at C-4 and C-8 were confirmed by the HMBC correlations between H3-12/C-3, C-4, C-5 and H3-13/C-7, C-8, C-9, respectively. The cyclobutane ring, which is fused to the nine-membered ring at C-1 and C-9, was elucidated by the 1 H-1 H COSY correlations between H-9 and H2-10 and by the HMBC correlations between H-1/C-11, H-9/C-10 and H2-10/C-1, C-8, C-9. These data, together with the HMBC correlations between H2-10/C-11, C-14, C-15; H3-14/C-1, C-10, C-11, C-15 and H3-15/C-1, C-10, C-11, C-14, unambiguously established the planar structure of 1.  The stereochemistry of 1 was elucidated from the interactions observed in a nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 2) and by the vicinal 1 H-1 H coupling constants. The trans geometries of H-1 and H-9 were indicated by a 9.2-Hz coupling constant between these two ring juncture protons, and H-9 and H-1 were assigned as α-and β-oriented protons, respectively, in 1. In the NOESY experiment, H-9 exhibited a correlation with H3-13, indicating that H-9 and Me-13 are located on the same face and can be assigned as α protons, since H-1 is β-oriented and H-9 did not show a correlation with H-1. Furthermore, H-3 showed an interaction with H3-12, revealing the Z geometry of the C-3/4 double bond in 1. Based on the above findings, the configurations of all chiral carbons of 1 were assigned as 1R*, 8R* and 9S*. Rumphellol B (2) was isolated as a colorless oil that gave a pseudomolecular ion [M + Na] + at m/z 289.2128 in the HRESIMS, indicating the molecular formula C17H30O2 (calcd. for C17H30O2 + Na, 289.2138) and implying three degrees of unsaturation. A broad IR absorption was observed at 3441 cm −1 , suggesting the presence of a hydroxy group in 2. The 13 C NMR and DEPT spectra of 2 (Table 2) showed 17 carbons, including four methyls, seven sp 3 methylenes (including an oxymethylene), three sp 3 methines (including an oxymethine) and three quaternary carbons (including an oxygenated quaternary carbon).

Animal Material
Specimens of the gorgonian coral, Rumphella antipathies (Nutting), were collected by hand using scuba equipment off the coast of Pingtung, Southern Taiwan. This organism was identified by comparison with previous descriptions [5]. A voucher specimen (Specimen No. NMMBA-TWGC-010) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation
Sliced bodies of the gorgonian R. antipathies (wet weight 402 g, dry weight 144 g) were extracted with a mixture of methanol (MeOH) and dichloromethane (CH2Cl2) (1:1) at room temperature. The extract was partitioned with ethyl acetate (EtOAc) and H2O. The EtOAc layer was separated by silica gel and eluted using n-hexane/EtOAc (stepwise, 25:1-pure EtOAc) to yield 29 fractions. Every fraction was checked using the 1 H NMR spectra. Fractions 12 and 17 were re-purified by normal-phase HPLC (NP-HPLC) using a mixture of CH2Cl2 and EtOAc as the mobile phase to afford 2 (15.0 mg, 9:1) and 1 (1.0 mg, 15:1), respectively.

Human Neutrophil Superoxide Anion Generation and Elastase Release
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide anion generation was carried out according to the procedures described previously [25,26]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. DPI (diphenyleneiodonium) and elastatinal were used as reference compounds in the anti-inflammatory test of the inhibitory effects on the generation of superoxide anions (IC50 = 3.26 μM) and the release of elastase (IC50 = 60.0 μM) by human neutrophils in response to fMet-Leu-Phe/Cytochalastin B (FMLP/CB) respectively. In the in vitro anti-inflammatory bioassay, the inhibitory effects on the generation of superoxide anion and the release of elastase by activated neutrophils were used as indicators. At a concentration of 10 μg/mL, for the significant activity of pure compounds, an inhibition rate ≥50% is required (inhibition rate ≤ 10%, not active; 20% ≥ inhibition rate ≥ 10%, weakly anti-inflammatory; 50% ≥ inhibition rate ≥ 20%, modestly anti-inflammatory).

Conclusions
Only one previous study has focused on the chemical components of the gorgonian coral, Rumphella aggregata [27]. The use of organic extracts from gorgonians belonging to the Rumphella genus in ecology and for medical use has also been reported [28,29]. In continuing studies of new substances from marine invertebrates collected off the waters of Taiwan, two new caryophyllene-type sesquiterpenoids, rumphellols A and B (1 and 2), were isolated from the gorgonian coral, Rumphella antipathies. The structures of new sesquiterpenoids 1 and 2 were elucidated on the basis of spectroscopic methods, and these two compounds were found to display inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils. The gorgonian coral, Rumphella antipathies, has been transplanted to culturing tanks located in the National Museum of Marine Biology and Aquarium, Taiwan, for extraction of additional natural products to establish a stable supply of bioactive material.