Bioactive Eunicellin-Based Diterpenoids from the Soft Coral Cladiella krempfi

Four new eunicellin-based diterpenoids, krempfielins A–D (1–4), along with two known compounds (5 and 6) have been isolated from a soft coral Cladiella krempfi. The structures of the new metabolites were elucidated by extensive spectroscopic analysis and by comparison with spectroscopic data of related known compounds. Compounds 5 and 6 were shown to exhibit cytotoxicity against a limited panel of cancer cell lines. Furthermore, compounds 2, 3, 5 and 6 were shown to exert significant in vitro anti-inflammatory activity against LPS-stimulated RAW264.7 macrophage cells.


Results and Discussion
Krempfielin A (1) was obtained as a colorless oil. The HRESIMS (m/z 505.2777 [M + Na] + ) of 1 established a molecular formula of C 26 H 42 O 8 , implying six degrees of unsaturation. The IR spectrum of 1 revealed the presence of hydroxyl and carbonyl groups from absorptions at 3445 and 1730 cm −1 , respectively. The 13 C NMR spectroscopic data of 1 exhibited 26 carbon signals (Table 1), which were assigned by the assistance of DEPT spectrum to six methyls (including one acetate methyl at δ C 21.9), six methylenes (including one sp 2 methylene at δ C 114.9), nine methines (including five oxymethines at δ C 90.5, 81.9, 78.8, 77.0 and 72.9), five quaternary carbons (including two sp 2 oxygenated quaternary carbons at δ C 172.3 and 170.5, two sp 3 oxygenated quaternary carbons at δ C 86.0 and 79.3, and one sp 2 quaternary carbon at δ C 143.7). The NMR data of 1 (Tables 1 and 2) showed the appearance of a terminal methylene group (δ C 114.9, CH 2 and 143.7, qC; δ H 5.20 brs), a isopropyl moiety (δ C 28.9, CH; 21.5, CH 3 ; and 16.6, CH 3 and δ H 1.85, m, 1H; 0.97, d, 3H, J = 6.6 Hz and 0.86, d, 3H, J = 6.6 Hz), one n-butyrate (δ C 172.3, qC; 37.3, CH 2 ; 18.3, CH 2 and 13.6, CH 3 ; and δ H 2.28, m, 2H; 1.65, m, 2H and 0.97, t, 3H, J = 7.2 Hz) and one acetate group (δ C 170.5, qC and 21.9, CH 3 and δ H 2.09, s, 3H), respectively. Analysis of HMQC correlations showed that proton signals appearing at δ H 2.34 (1H, m), 3.30 (1H, t, J = 5.7 Hz), 3.69 (1H, s), and 4.05 (1H, dd, J = 9.3, 5.7 Hz) were correlated to two ring juncture methine carbons at δ C 43.6 and 50.3 and two oxymethine carbons at δ C 90.5 and 81.9, respectively. This suggested the presence of a tetrahydrofuran structural unit. In addition, the 1 H-1 H COSY correlations of 1 assigned three isolated consecutive proton spin systems ( Figure 1). The molecular framework of 1 was further established by HMBC data ( Figure 1). Furthermore, H-12 (δ 5.44) and an acetate methyl exhibited HMBC correlations to the acetate carbonyl carbon (δ 170.5), revealing the location of an acetate at C-12. The location of a n-butyrate at C-3 was then deduced by the chemical shifts of C-3 (δ 86.0) and H 3 -15 (δ 1.47). From the above results, the structure of compound 1 was shown to be highly related to that of a known compound, litophynol B (5) [16].  The relative configuration of 1 was mostly confirmed to be the same as that of 5 by comparison of the chemical shifts of both compounds and was further confirmed by NOE correlations (Figure 2). Furthermore, one additional NOE correlation between H-10 with H-12 suggested that H-12 was β-oriented and the relative configuration of 1 was proposed as 1R*, 2R*, 3R*, 6S*, 7R*, 8S*, 9S*, 10R*, 12S*, and 14R*. The HRESIMS of krempfielin B (2) exhibited a [M + Na] + peak at m/z 461.2881 and established a molecular formula of C 25 H 42 O 6 , appropriate with five degrees of unsaturation. By comparison of the 1 H and 13 C NMR data of 2 with those of 5, it was found that they were very similar. However, a methoxyl group (δ H 3.36, 3H, s; δ C 56.95, CH 3 ) was observed in 2. In addition, the position of methoxyl group at C-6 was confirmed by the HMBC correlation of the methoxyl proton (δ H 3.36) to an oxymethine carbon (δ C 88.3, CH, C-6). A more detailed analysis of the 1 H and 13 C NMR spectroscopic data and correlations in the 1 H-1 H COSY and HMBC spectra led to the establishment of the gross structure of 2 ( Figure 1). The NOESY correlations of 2 ( Figure 2) also showed the stereochemistry similarity between compounds 2 and 5. All of the above information suggested that 2 was the 6-O-methyl derivative of 5.
The molecular formula C 26 H 42 O 7 with six degrees of unsaturation was assigned to krempfielin C (3) from its HRESIMS data (m/z 489.2829 [M + Na] + ). The NMR spectroscopic data of 3 (Tables 1 and 2) showed the presence of one acetoxy group (δ C 171.9, qC and 21.4, CH 3 ; and δ H 2.09 s, 3H) and one n-butyryloxy group (δ C 172.4, qC; 37.4, CH 2 ; 18.4, CH 2 and 13.6, CH 3 ; and δ H 2.34 m, 1H; 2.31 m, 1H; 1.67 m, 2H and 0.99 t, 3H, J = 7.2 Hz). NMR data of 3 showed similarities with those of 5, except for the presence of an acetoxyl group at C-6 of 3 that downfielded H-6 to δ H 5.72 and C-6 to δ C 82.2 ppm. These observations could be further confirmed by the correlations observed in the 2D NMR (including 1 H-1 H COSY, HMBC and NOESY) experiments of 3 (Figures 1 and 2). Krempfielin D (4) was isolated as a colorless oil with a molecular formula C 27 H 44 O 8 which possesses six units of unsaturation, as indicated by HRESIMS (m/z 519.2934). The 1 H and 13 C NMR spectral data of 4 (Tables 1 and 2) revealed that the structure of metabolite 4 should be similar to that of 1, as the NMR spectral data of 4 are almost identical with those of 1 except for the presence of a methoxyl group (δ H 3.36, 3H, s) in 4. Also, the 13 C NMR spectrum of 4 showed the same number of methylene, methine, and quaternary carbons as that of 1, except for the presence of a methoxyl carbon, which showed a signal at δ C 56.8 (qC). Furthermore, the methoxyl protons gave an HMBC cross-peak with an oxymethine carbon (δ 87.4, CH), indicating the presence of the methoxyl group at C-6 in 4. The stereochemistry of 4 was confirmed by comparison of the NMR data and NOE correlations of both 1 and 4.
The cytotoxicity of the diterpenoids 1-6 against five human carcinoma cell lines A549, H1299, BT483, HepG2, SAS and one human normal cell line BEAS2B was evaluated by the MTT assay. It was found that only 5 showed activity against the proliferation of H1299 and BT483 cancer cells (ED 50 values of 18.1 ± 1.5, and 13.2 ± 1.1 μg/mL), and 6 exhibited cytotoxicity toward A549, BT483 and SAS cancer cell lines (ED 50 values of 15.8 ± 2.0, 8.5 ± 1.0 and 14.3 ± 1.8 μg/mL), respectively. Furthermore, 5 and 6 were found to be non-cytotoxic toward the normal cell BEAS2B. In the present study, the in vitro anti-inflammatory effects of compounds 1-6 were also tested by examining the inhibitory activity of these compounds toward the LPS-induced up-regulation of pro-inflammatory proteins, iNOS and COX-2 in RAW264.7 macrophage cells (Figure 3). At a concentration of 10 μM, compounds 2-6 were found to significantly reduce the levels of iNOS protein, relative to the control cells stimulated with LPS only. However, these metabolites did not effectively reduce the expression of COX-2 protein. (n = 6). Relative intensity of the LPS alone stimulated group was taken as 100%. * Significantly different from LPS alone stimulated group (* P < 0.05). a stimulated with LPS; b stimulated with LPS in the presence of 1-6 (10 μM).

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. ESIMS and HRESIMS were obtained with a Bruker APEX II mass spectrometer. The NMR spectra were recorded in CDCl 3 either on a Varian UNITY INOVA-500 FT-NMR, a Varian 400MR FT-NMR or a Bruker AMX-300 FT-NMR. Silica gel (Merck, 230-400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical TLC. High performance liquid chromatography was performed on a Hitachi L-7100 HPLC apparatus with an ODS column (250 × 21.2 mm, 5 mm).

Animal Material
C. krempfi was collected by hand using scuba off the coast of Penghu islands of Taiwan in June 2008, at a depth of 5-10 m, and stored in a freezer until extraction. A voucher sample was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.