Cytotoxic and Anti-Inflammatory Eunicellin-Based Diterpenoids from the Soft Coral Cladiella krempfi

Five new eunicellin-based diterpenoids, krempfielins E–I (1–5) and seven known compounds (6–12) were isolated from the organic extract of a Taiwanese soft coral Cladiella krempfi. The structures of the new metabolites were elucidated on the basis of extensive spectroscopic analysis. Metabolites 5, 6, 10 and 12 were shown to exhibit cytotoxicity against a limited panel of cancer cell lines. Furthermore, compounds 6 and 10 could potently inhibit the accumulation of the pro-inflammatory iNOS protein, and 6 and 12 could significantly reduce the expression of COX-2 protein in LPS-stimulated RAW264.7 macrophage cells.


Results and Discussion
The new metabolite krempfielin E (1) showed the molecular ion peak [M + Na] + at m/z 487.2669 in the HRESIMS and established a molecular formula of C 26 H 40 O 7 , implying seven degrees of unsaturation. The IR absorptions bands at  max 3421 and 1732 cm −1 revealed the presence of hydroxy and ester carbonyl functionalities. The 13 C NMR spectrum measured in CDCl 3 showed signals of twenty-six carbons (Table 1) which were assigned by the assistance of the DEPT spectrum to four methyls (including one acetate methyl δ C 21.0), seven sp 3 methylenes, two sp 2 methylenes, eight sp 3 methines (including four oxymethines), one sp 3 and four sp 2 quaternary carbons (including two ester carbonyls).The NMR spectroscopic data of 1 (Tables 1 and 2) showed the presence of two 1,1-disubstituted double bonds (δ C 118.1 CH 2 , 112.0 CH 2 , 152.2 C, and 145.2 C; δ H 5.51 s, 5.22 s, 4.81 s, and 4.65 s). Two ester carbonyls (δ C 172.5 and 171.2) were assigned from the 13 C NMR spectrum and their signals were correlated with the methylene protons (δ H 2.10, 2H, m) of an n-butyrate and protons of an acetate methyl (δ H 2.07 s, 3H), respectively. Therefore, the remaining three degrees of unsaturation identified 1 as a tricyclic molecule. The 1 H-1 H COSY and HMBC correlations ( Figure 1) were further used for establishing the molecular skeleton of 1. The COSY experiment assigned three isolated consecutive proton spin systems. Above evidences and the analysis of HMBC spectrum ( Figure 1) suggested that 1 is an eunicellin-based diterpenoid. Furthermore, the acetoxy group attaching at C-19 was confirmed by the HMBC correlations from oxymethylene [δ H 3.94 (H 2 -19)] and acetate methyl protons (δ H 2.07) to the ester carbonyl carbon appearing at δ 171.2 (C). Thus, the remaining one n-butyryloxy group had to be positioned at C-3, an oxygen-bearing quaternary carbon resonating at δ 84.4 ppm. On the basis of above analysis, the planar structure of 1 was established.     Krempfielin F (2) was found to have the molecular formula C 24 H 36 O 6 and seven degrees of unsaturation, as indicated from the HRESIMS (m/z 443.2408 [M + Na] + ). The 13 C NMR spectrum of 2 showed signals of twenty-four carbons (Table 1), which were characterized by the DEPT spectrum as six methyls, five methylenes (including one sp 2 methylene), eight methines (including three oxygenated carbons), and five quaternary carbons (including one ketone carbonyl, two ester carbonyls, and one sp 2 quaternary carbon of an olefinic group). The presence of two acetoxy groups was indicated by the 1 H NMR signals ( Table 2)    Comparison of the NMR data of 3 with those of 2 revealed the replacement of one acetoxy group (δ H 2.18, 3H, s; δ C 169.6, C and 22.4, CH 3 ) in 2 by an n-butyryloxy group in 3 (δ H 0.99, 3H, t, J = 7.5 Hz; 1.68, 2H, m; 2.28, 2H, m; and δ C 172.5, C; 37.3, CH 2 ; 18.4, CH 2 and 13.7, CH 3 ), and an additional hydroxy group substituted at C-8 of 3 that downfielded H-8 to δ H 3.77 and C-8 to δ C 78.5 ppm. The placement of the n-butyryloxy group at C-3 was confirmed by the HMBC experiment which showed a correlation between H-2 and the carbonyl carbon (δ C 172.5 C) of this n-butyryloxy group. The NOE correlations of 3 also showed that the stereochemistry of this metabolite is identical with that of 2 excepted for the presence of the α-oriented hydroxy group at C-8.
The HRESIMS of krempfielin H (4) exhibited a [M + Na] + ion peak at m/z 443.2408, appropriate for a molecular formula of C 24 H 36 O 6 . By analysis of 2D NMR spectra, including 1 H-1 H COSY, HMQC, and HMBC, compound 4 was shown to possess the same molecular framework as that of 2. Furthermore, it was found that the NMR data of 4 were very similar to those of 2 (Tables 1 and 2), suggesting that 4 might be an isomer of 2. From NOESY spectrum, it was found that the β-oriented H-10 showed NOE interactions with both H-7 and H-8β (δ H 1.87), while H-8β showed NOE interactions with H-7, indicating the β-orientation of H-7. This inferred the α-orientation of methyl substituent at C-7. Further analysis of other NOE interactions revealed that 4 possessed the same relative configuration sat C-1, C-2, C-3, C-9, C-10, C-12 and C-14 as those of 2. Therefore, 4 was found to be the C-7 epimer of 2.
The related metabolite, krempfielin I (5), had a molecular formula of C 30 H 48 O 8 as indicated by the HRESIMS (m/z 559.3243, [M + Na] + ) and NMR data (Tables 1 and 2). The 13 C NMR spectrum of 5 revealed the appearance of three ester carbonyls (δ C 173.3, 173.1 and 171.6), which were correlated with protons of two methylenes (δ H 2.55, 2.25, m, each 2H; and δ C 36.7 and 36.5) of two n-butyrates and the methyl protons (δ H 2.09 s, 3H and δ C 21.4) of one acetate group, respectively. The planar structure of 5 was established by 1 H-1 H COSY and HMBC correlations (Figure 2). The HMBC connectivities from H-2 (δ H 3.63 br s, 1H) and H-8 (δ H 5.27 d, 1H, J = 9.2 Hz) to two carbonyl carbons δ C 173.3 (C) and 173.1 (C) determined the positions of the two n-butyrates at C-8 and C-3. Also, the location of an acetate group at C-6 was supported by the HMBC connectivities from both of the acetate methyl protons (δ H 2.09 s, 3H) and oxygenated methine proton (δ H 5.71 d, 1H, J = 5.6 Hz) to the carbon resonating at δ C 171.6 (C). The relative configuration of 5 was confirmed by analyzing the key NOE correlations (Figure 3).
The cytotoxicity of the diterpenoids 1-12 against the growth of five human carcinoma cells A549, BT483, H1299, HepG2, SAS and one human normal cell line BEAS2B was studied ( Table 3). The results showed that 1-4, 7-9 and 11 are not cytotoxic toward the above cancer and normal cells. Compounds 5, 6, 10 and 12 exhibited cytotoxicity toward the above five cancer cell lines and the human normal cell line; 10, being the most cytotoxic. The in vitro anti-inflammatory effects of compounds 1-12 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 4). At a concentration of 10 μM, compounds except 2, 4, and 11 were found to significantly reduce the expression of iNOS protein, relative to the control cells stimulated with LPS only. Among them, 6 and 10 could potently reduce the levels of iNOS protein to 6.4 ± 0.8% and 12.8 ± 2.9%, respectively. Compounds 6 and 12 also effectively reduced COX-2 expression (52.5 ± 8.0% and 48.1 ± 10.8%, respectively) in the same LPS-stimulated cells. These results revealed that n-butyryloxy group at C-3 could significantly enhance the cytotoxic and anti-inflammatory activities in eunicellin-type compounds. Overall, compounds 6, 10 and 12 exhibited interesting cytotoxic and anti-inflammatory activity and could become lead compounds in the future drug development.

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 were obtained with a Bruker APEX II mass spectrometer. NMR spectra were recorded either on a Varian UNITY INOVA-500 FT-NMR, a Varian 400MR 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 a ODS column (25.0 × 21.2 mm, 5 μm).

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 (specimen no. 200806CK) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.