Novel Caryophyllane-Related Sesquiterpenoids with Anti-Inflammatory Activity from Rumphella antipathes (Linnaeus, 1758)

Two previously undescribed caryophyllane-related sesquiterpenoids, antipacids A (1) and B (2), with a novel bicyclo[5.2.0] core skeleton, and known compound clovane-2β,9α-diol (3), along with rumphellolide L (4), an esterified product of 1 and 3, were isolated from the organic extract of octocoral Rumphella antipathes. Their structures, including the absolute configurations were elucidated by spectroscopic and chemical experiments. In vivo anti-inflammatory activity analysis indicated that antipacid B (2) inhibited the generation of superoxide anions and the release of elastase by human neutrophils, with IC50 values of 11.22 and 23.53 μM, respectively, while rumphellolide L (4) suppressed the release of elastase with an IC50 value of 7.63 μM.

Antipacid B (2) was isolated as a colorless colloid that showed a sodiated adduct ion peak in (+)-HRESIMS at m/z 261.1468 [M + Na] + , which accounted for the molecular formula, C 14 H 22 O 3 (calcd. for C 14 H 22 O 3 + Na, 261.1467), with 4 degrees of unsaturation. The spectroscopic data of 2 resembled those of 1 ( Table 1). The one-dimensional (1D) and two-dimensional (2D) NMR spectra revealed that the signals corresponding to the propanoic acid moiety in 1 were replaced by those of an acetic acid in 2 ( Figure 3). Therefore, 2 was assigned as having a structure with the same stereochemistry as 1 because of the stereogenic carbons that 2 had in common with 1 by correlations observed in the NOESY spectrum ( Figure   Compound 3 was identified by comparison of its spectroscopic data with those of clovane-2β,9αdiol, which had been previously isolated from terrestrial plants Dipterocarpus pilosus [34], Salvia canariensis [35], Viguiera excelsa [36], Viguiera linearis [37], and Sindora sumatrana [38]. This was the first occasion in which this metabolite was obtained from a marine source.   (Table 2), including six methyls, twelve methylenes, five methines (including two oxymethines), five sp 3 quaternary carbons, an ester carbonyl, and a ketonic carbonyl. Therefore, 4 was identified as having five rings.  Figure 5), and these findings together with the results of key HMBC correlations shown in Figure 5 confirmed the carbon skeleton of 4. An HMBC correlation between H-2´(δ H 4.83), an oxymethine proton, and the C-5 ester carbonyl carbon (δ C 173.6) was found, which proved the existence of an ester linkage in 4. It was found that the NMR data were similar to those of 1 and 3, and this compound was proven to be the dehydrated product of 1 and 3. Due to the absolute configurations of 1 and 3 having been determined, the absolute configurations of the stereogenic carbons of 4 were assigned as (1R,8S,9S,1´S,2´S,5´S,8´R,9´R) (Supplementary Materials, Figures S17-S23).
The proposed biogenetic pathway of sesquiterpenoids 1-4 is outlined in Scheme 1. The ringopening reaction might be rationally derived from (8R,9R)-isocaryolane-8,9-diol [30] (the numbering system used in reference [30] was different to that in this study), which had also been isolated from R. antipathes [21], and might subsequently, under oxidation, produce the carbon skeletons of 1 and 2. The in vitro anti-inflammatory effects of 1-4 were assessed (Table 3). Antipacid B (2) displayed inhibitory effects on the generation of superoxide anions and the release of elastase by human neutrophils (IC 50 = 11.22 and 23.53 µM, respectively). Antipacid A (1) did not show activity, implying that the presence of a large substituent at C-8 weakens the activity in comparison with the structure and anti-inflammatory activities of 2. Although 1 and 3 were not active, rumphellolide L (4), the dehydrated product of 1 and 3 with esterification, showed activity in inhibiting the release of elastase (IC 50 = 7.63 µM). Table 3. Inhibitory effects of sesquiterpenoids 1-4 on superoxide anion generation and elastase release by human neutrophils in response to N-Formyl-l-methionyl-l-leucyl-l-phenylalanine/ Cytochalasin B (fMLF/CB).

General Experimental Procedures
Optical rotations were recorded on a JASCO-P1010 polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). IR spectra were obtained on a Varian Diglab FTS 1000 FT-IR spectrometer (Varian Inc., Palo Alto, CA, USA). NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C) (Varian Inc.) using the residual CHCl 3 (δ H 7.26 ppm) and CDCl 3 (δ C 77.1 ppm) signals as internal references for 1 H and 13 C NMR, respectively.

Animal Material
The octocoral R. antipathes (Linnaeus, 1758) was collected by hand by self-contained underwater breathing apparatus (SCUBA) divers off the coast of South Taiwan in May 2004. The samples were stored in a −20 • C freezer until used for extraction. Identification of the species of this organism was performed by comparison as described in previous studies [39,40]. A voucher specimen (no.: NMMBA-TWGC-010) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Superoxide Anion Generation and Elastase Release by Human Neutrophils
The proinflammatory suppression assay was employed to assess the activities of isolated compounds 1-4 against the generation of superoxide anions and the release of elastase by human neutrophils according to the protocols described in the literature [41].

Conclusions
The current work illustrated the anti-neutrophilic inflammatory properties of caryophyllanerelated sesquiterpenoids, and two metabolites with novel structures, antipacids A and B (1 and 2), clovane-2β,9α-diol (3), and rumphellolide L (4), an esterified product of 1 and 3, were isolated from R. antipathes. Compound 2 displayed inhibitory effects on the generation of superoxide anions and the release of elastase, and 4 showed activity in suppressing the release of elastase. These results indicated a structural-dependent specificity of C-8 in 1, 2, and 4 in neutrophilic targets, which will motivate future research examining this specificity, as well as clarify the molecular mechanisms of the active leads.