Rumphellaones B and C, New 4,5-Seco-Caryophyllane Sesquiterpenoids from Rumphella antipathies

Two new 4,5-seco-caryophyllane sesquiterpenoids, rumphellaones B (1) and C (2), which were found to possess unprecedented γ-lactone moieties, were obtained from the gorgonian coral Rumphella antipathies. The structures of 1 and 2 were elucidated by spectroscopic methods and compound 2 was found to display modest inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils at a concentration of 10 μg/mL.

gorgonian coral Rumphella antipathies. The structures of 1 and 2 were elucidated by

Introduction
The chemical constituents of gorgonian corals of the genus Rumphella, which are widely distributed in the subtropical and tropical waters of the Indo-Pacific Ocean have been investigated for ecological and medical uses [1][2][3]. As part of our ongoing investigation into the isolation of new substances from marine invertebrates collected in the waters of Taiwan, an intersection of the Kuroshio and Oyashio currents, the chemical constituents of an organic extract of the gorgonian coral Rumphella antipathies (Scheme 1) which displayed meaningful signals in NMR studies were studied. Previous chemical investigations on R. antipathies yielded a series of caryophyllane-type sesquiterpenoid analogues, including kobusone [4], isokobusone [5], rumphellolides A-I [6][7][8][9], and rumphellatins A-D [10][11][12], which mostly possess a bicyclo[7.2.0] carbon skeleton. Moreover, in an our previous study, the first 4,5-seco-caryophyllane derivative, rumphellaone A [13], was isolated from R. antipathies. In further studies on this interesting organism, two new 4,5-seco-caryophyllane derivatives, rumphellaones B (1) and C (2), were isolated. In this paper, we describe the isolation, structure determination and anti-inflammatory properties of compounds 1 and 2 (Scheme 1). Scheme 1. The gorgonian coral Rumphella antipathies and the structures of rumphellaones B (1), C (2) and A (3).  the presence of γ-lactone and α,β-unsaturated ketone groups. The 13 C-NMR and DEPT spectra of 1 (Table 1) showed that this compound has 15 carbons, including four methyls, three sp 3 methylenes, two sp 3 methines, two sp 2 methines and four quaternary carbons (including an oxygenated quaternary carbon, an ester carbonyl and a ketone carbonyl). From the 13 C-NMR data, three degrees of unsaturation were accounted for and 1 must thus be a compound with two rings. From the 1 H-1 H COSY experiment of 1 (Table 1 and Figure 1), it was possible to establish the spin systems that map out the proton sequences from H 2 -10/H-9/H-1/H-2/H-3 and H 2 -6/H 2 -7, which were assembled with the assistance of an HMBC experiment (Table 1 and Figure 1) Based on the consideration of molecular formula, an oxygen atom had to be placed between the C-5 carbonyl carbon (δ C 176.7) and the C-8 oxygenated quaternary carbon (δ C 86.6) to form a γ-lactone moiety. The relative configuration of 1 was established by an analysis of interactions that were found in the NOESY experiment ( Figure 2) and by vicinal 1 H-1 H coupling constant analysis. Due to the α-orientation of H-9, a large coupling constant was found between H-9 and H-1 (J = 9.2 Hz), indicating that H-1 has a β-orientation. H-1 showed a correlation with the tertiary methyl Me-15 suggesting that H-1 and H 3 -15 are located on the same face. Me-13 showed an interaction with H-9 and by comparison the NMR data of C-8 oxygenated quaternary carbon (δ C 86.6) and Me-13 (δ H 1.25, 3H, s; δ C 24.6) with those of a similar analogue, rumphellaone A (3) (δ C 87.2, C-8; δ H 1.31, 3H, s; δ C 24.9, CH 3 -13) [13], indicating that Me-13 was α-oriented at C-8. The trans geometry of the C-2/3 double bond was indicated by a 16.0 Hz coupling constant between H-2 (δ H 6.77) and H-3 (δ H 6.09). Based on the above findings, the configurations of all chiarl carbons of 1 were assigned to be 1R*, 8S* and 9S*.    (Table 2), including three methyls, six methylenes (including an oxymethylene), two methines and four quaternary carbons (including an oxygenated quaternary carbon and two carbonyls). Thus, from the 13 C-NMR data, two degrees of unsaturation was accounted for, and 2 must have two rings. The 1 H-NMR spectrum of 2 showed that all three methyl groups are isolated. In addition, six pairs of aliphatic methylene protons and two aliphatic methine protons were observed in the 1 H-NMR spectrum of 2 (Table 2). It was found that the spectral data (IR, 1 H and 13 C-NMR) of 2 were similar to those of a known analogue, rumphellaone A (3) [13]. However, the 1 H and 13 C-NMR spectra revealed that the signals corresponding to the C-13 methyl group in 2 disappeared and were replaced by those of an additional hydroxymethyl group. Thus, compound 2 was found to be the 13-hydroxy derivative of 3 with the structure as described by formula 2. The in vitro anti-inflammatory effects of compounds 1 and 2 were examined and 2 displayed modestly inhibitory effects on the generation of superoxide anions (inhibition rate = 24.7%) and the release of elastase (inhibition rate = 21.1%) by human neutrophils in response to FMLP/CB at a concentration of 10 μg/mL.

Animal Material
Specimens of the gorgonian coral Rumphella antipathies (Nutting) were collected by hand using scuba equipment off the coast of Pingtung, Southern Taiwan. 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) are extracted with a mixture of methanol (MeOH) and dichloromethane (CH 2 Cl 2 ) (1:1) at room temperature. The extract was partitioned between ethyl acetate (EtOAc) and H 2 O and the EtOAc layer was subjected to 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 21 and 27 were re-purified by normal phase HPLC (NP-HPLC) using a mixture of n-hexane and acetone as the mobile phase to afford 1 (5.0 mg, 3:1) and 2 (3.4 mg, 2:1), respectively.

Generation of Superoxide Anions and Release of Elastase by Human Neutrophils
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [14,15]. 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-Valp-nitroanilide as the elastase substrate. 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. For 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
The gorgonian coral R. antipathies, collected off the waters of Taiwan, has proven to be a rich source of caryophyllane-and clovane-type sesquiterpenoids. In our continuing investigation on the chemical constituents of R. antipathies, two new 4,5-secocaryophyllane derivatives, rumphellaones B (1) and C (2), were isolated. It is noteworthy to mention that metabolites 1 and 2 represent the second and third 4,5-secocaryophyllane derivative containing a γ-lactone moiety, respectively, and compound 2 was found to display modestly inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils.