Discovery of New Eunicellins from an Indonesian Octocoral Cladiella sp.

Two new 11-hydroxyeunicellin diterpenoids, cladieunicellin F (1) and (–)-solenopodin C (2), were isolated from an Indonesian octocoral Cladiella sp. The structures of eunicellins 1 and 2 were established by spectroscopic methods, and eunicellin 2 was found to be an enantiomer of the known eunicellin solenopodin C (3). Eunicellin 2 displayed inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils. The previously reported structures of two eunicellin-based compounds, cladielloides A and B, are corrected in this study.


Results and Discussion
Cladieunicellin F (1) was isolated as a colorless oil and the molecular formula for this compound was determined using HRESIMS to be C 20 H 34 O 3 (four degrees of unsaturation) (m/z 345.2404 [M + Na] + , calculated for 345.2406). Comparison of the 13 C NMR and DEPT data with the molecular formula indicated that there must be two exchangeable protons, which required the presence of two hydroxyl groups. This deduction was supported by a broad absorption in the IR spectrum at 3414 cm −1 . The 13 C NMR data for 1 confirmed the presence of twenty carbon signals (Table 1), which were characterized by DEPT as four methyls, an sp 2 methylene, six sp 3 methylenes, six sp 3 methines (including two oxymethines), two sp 3 oxygenated quaternary carbons, and an sp 2 quaternary carbon. Based on the 1 H and 13 C NMR spectra ( were indicative of the two methyls of an isopropyl group. A tertiary methyl group bonded to an oxygenated carbon was evident from the singlet signal at  H 1.24 (3H, s, H 3 -17). Thus, from the reported data, the proposed skeleton of 1 was suggested to be an eunicellin-based diterpenoid with three rings.    3 -17/C-11, permitted the elucidation of the carbon skeleton. An exocyclic carbon-carbon double bond at C-7 was confirmed by the HMBC correlations between H 2 -16/C-6, -7, -8; H-6/C-16; and H-8b/C-16 and further supported by the allylic coupling between H-8b/H 2 -16. The presence of the C-2/3 epoxide group was confirmed by the HMBC correlations between H-1/C-2, -3; H-2/C-1, -3, -10, -15; H 2 -4/C-2, -3; and H 3 -15/C-2, -3, -4. Thus, the remaining hydroxyl groups should be positioned at C-6 and C-11, as indicated by the key 1 H-1 H COSY correlations and characteristic NMR signals, although the hydroxyl protons for OH-6 and OH-11 were not observed in the 1 H NMR spectrum of 1.
The relative configuration of 1 was elucidated from the interactions observed in a NOESY experiment ( Figure 2 Furthermore, H-6 correlated with two protons of the C-9 methylene and Me-15. Consideration of molecular models found that H-6 was reasonably close to H 2 -9 and Me-15 when H-6 was -oriented in 1. Based on the above findings, the structure of 1, including its relative configuration, was established, and the chiral centers for 1 were assigned as 1R*, 2S*, 3S*, 6R*, 10R*, 11R*, and 14R*. To the best of our knowledge, cladieunicellin F (1) is the first eunicellin derivative possessing a C-2/3 epoxy group.  Eunicellin 2 was isolated as a colorless oil, and the molecular formula of this compound was determined using HRESIMS to be C 20 H 34 O 2 (m/z 329.2455 [M + Na] + , calculated for 329.2456). Thus, four degrees of unsaturation were determined for 2. Detailed analysis of the NMR data showed that the data for 2 were similar to those of a known eunicellin analogue, solenopodin C (3) (Figure 1), which was isolated from the gorgonian Solenopodium stechei [8]. However, the optical rotation value of 2  Table 2) were assigned using 2D NMR data analysis and comparison to the NMR data of 3. The proton chemical shifts for C-8, C-9, C-12, and C-13 methylene protons and the carbon chemical shifts for C-1, C-4, C-12, and C-14 of compound 3 should be revised (Table 2). In a previous study, we reported the isolation and structure determination of two eunicellins, cladielloides A (4) and B (5) (Figure 3) [3]. However, based on detailed spectral data analysis, we found that the structures for these two compounds should be revised. 1D and 2D NMR spectral data analysis, particularly 1 H-1 H COSY and HMBC experiments, of cladielloide A ( Table 3), showed that the main carbon skeleton of cladielloide A was established correctly. However, in the HMBC experiment for cladielloide A, key correlations between H-4 ( H 5.14) and an ester carbonyl at  C 171.4 (s, C-1') and between H-2' ( H 4.86) and two ester carbonyls at  C 171.4 (s, C-1') and 171.1 (s, acetate carbonyl) were detected, and these findings indicated that the 2'-acetoxybutyrate group should be positioned at C-4. Thus, the remaining hydroxyl groups are attached at C-3 and C-6 in cladielloide A, respectively. Furthermore, in the NOESY spectrum of cladielloide A, H-6 ( H 4.21) correlated with H 2 -5 ( H 2.97 and 1.75) and H 2 -8 ( H 2.35), but no correlation was found between H-6 and H 3 -15. Consideration of molecular models found that H-6 was reasonably close to H 2 -5 and H 2 -8 when it was -oriented. Based on the above findings, the structure, including the relative configuration, of cladielloide A should be revised as presented in eunicellin 6. Cladielloide B was found by HRESIMS to be an isomer of cladielloide A [3]. These two compounds were found to possess the same planar structure by NMR data analysis (Tables 3 and 4). In the NOESY experiment of cladielloide B, H-6 ( H 4.66) exhibited correlations with H 3 -15 ( H 1.33); a proton of C-5 methylene ( H 2.48); and a proton of C-8 methylene ( H 2.65), indicating that the 6-hydroxyl group in cladielloide B should be -oriented as presented as eunicellin 7. Based on the above findings, the structures of cladielloides A and B should be revised as structures 6 and 7, respectively. The authors apologize for any inconvenience caused by these errors.         The in vitro anti-inflammatory effects of eunicellins 1 and 2 were tested. Eunicellin 2 displayed significant inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils at a concentration of 10g/mL (Table 5).

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 and 100 MHz for 1 H and 13 C spectra, respectively, in CDCl 3 at 25 °C . Proton chemical shifts were referenced to the residual CHCl 3 signal ( H 7.26 ppm). 13 C NMR spectra were referenced to the center peak of CDCl 3 at  C 77.1 ppm.
ESIMS and HRESIMS data 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. HPLC was performed using a system comprised of a HITACHI L-2130 pump, a HITACHI photodiode array detector L-2455, and a RHEODYNE 7725 injection port. A reverse phase column (Polaris 5 C18-A 250 × 10.0 mm, Varian, silica gel 60, 5 m) was used for HPLC.

Animal Material
The octocoral Cladiella sp. was collected and imported legitimately by the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan from Indonesia in 2004. The material was stored in a freezer until extraction procedures were applied. A voucher specimen (NMMBA-IND-SC-001) was deposited in the NMMBA, Taiwan. This organism was identified by comparison with previous descriptions [10,11].