Simplexins P–S, Eunicellin-Based Diterpenes from the Soft Coral Klyxum simplex

Four new eunicellin-based diterpenes, simplexins P–S (1–4), and the known compound simplexin A (5), have been isolated from the soft coral Klyxum simplex. The structures of the new metabolites were determined on the basis of extensive spectroscopic analysis, particularly 1D and 2D NMR experiments. Compounds 1 and 3–5 were shown to exhibit cytotoxicity against a limited panel of cancer cell lines, 3 being the most cytotoxic.


Results and Discussion
Simplexin P (1) was obtained as a white powder. Its molecular formula C 26 H 42 O 7 was determined by the HRESIMS (m/z 489.2827 [M + Na] + ) was which deduced six degrees of unsaturation. The IR absorptions bands at ν max 3255 and 1717 cm -1 revealed the presence of hydroxy and ester carbonyl functionalities. The 13 C NMR spectrum measured in CDCl 3 showed signals of 26 carbons (Table 1) which were assigned as six methyls, six sp 3 methylenes, one sp 2 methylene, eight sp 3 methines (including four oxymethines), two sp 3 and three sp 2 quaternary carbons (including two ester carbonyls) by DEPT. In the 13 C NMR spectrum of 1, two carbonyl resonances at δ 172.6 and 170.2 ppm confirmed the presence of two ester groups. In the 1 H NMR spectrum of 1 (Table 2), one acetate methyl (δ 2.12) and one n-butyryloxy [δ 0.92 (3H, t, J = 7.5 Hz), 1.60 (2H, m), and 2.13 (2H, m)] groups were observed. Moreover, two 1 H NMR singlet signals at δ 5.13 and 5.46 revealed the presence of one olefinic methylene. In addition, the diagnostic signals at δ 4.17 and 3.58 implied the presence of an ether linkage between C-9 and C-2. On the basis of the above results and by the assistance of 1 H-1 H COSY and HMBC experiments (Figure 1), the molecular framework of 1 could be established as an eunicellin-type skeleton. Furthermore, the acetoxy group positioned at C-12 was confirmed by HMBC correlations from oxymethine [δ 4.89 (H-12)] and acetate methyl (δ 2.12) to the ester carbonyl carbon at δ 170.2 (C). Thus, the remaining one n-butyryloxy group was located at C-3, an oxygen-bearing quaternary carbon resonating at δ 84.4 ppm. On the basis of the above analysis, the planar structure of 1 was established unambiguously.  The relative configuration of 1 was determined by analysis of NOE correlations observed in the NOESY spectrum (Figure 2), which showed NOE interactions between H-1 and H-10, revealing they were both β-oriented. Also, correlations between H-2 and both H 3    Compound 2, simplexin Q, was assigned as the molecular formula C 26 H 42 O 7 from its HRESIMS data, appropriate for six degrees of unsaturation. 1 H and 13 C NMR spectral data of 2 (Tables 1 and 2) also showed the presence of one acetoxy group (δ C 170.0, C; 22.4, CH 3 ; δ H 2.00, 3H, s) and one n-butyryloxy group (δ C 172.6, C; 37.4, CH 2 ; 18.6, CH 2 ; 13.6, CH 3 ; δ H 2.15, 2H, m; 1.59, 2H, m; 0.94, 3H, t, J = 7.5 Hz). Comparison of the NMR data of 2 with those of the known compound simplexin A (5) [12], revealed that the only difference was the presence of an oxymethine (δ H 3.90; δ C 66.8) at C-13 in 2, instead of the methylene (δ H 2.27 and 1.44; δ C 18.1) in 5 arising from the substitution of a hydroxy moiety at C-13 in 2, instead of a methylene moiety at the same carbon in 1. Furthermore, the molecular framework was also established by 1 H-1 H COSY and HMBC experiments (Figure 1). The relative configuration of 2, deduced using a NOESY spectrum, is similar to that of 5. In addition, H-13 was found to exhibit a NOE correlation with H-1 but not with H-14, revealing the α-orientation of the hydroxyl group at C-13. Therefore, the structure of 2 was found to possess the (1R*, 2R*, 3R*, 6S*, 9R*, 10S*, 11R*, 13R*, 14R*)-configuration. Simplexin R (3), isolated as a white powder, was assigned a molecular formula C 24 H 38 O 8 from high resolution ESIMS analysis. The presence of the acetate groups was indicated by IR absorption at 1733 cm -1 , 1 H NMR signals (Table 2) at δ 1.95 (s, 3H) and 2.01 (s, 3H) and 13 C NMR (Table 1) signals at δ 22.3 (CH 3 ), 22.4 (CH 3 ), 169.9 (C) and 170.0 (C). The NMR spectral data of 3 showed the presence of a 1,1-disubstituted carbon-carbon double bond (δ C 118.2, CH 2 and 145.6, C; δ H 5.35, s and 5.46, s) and a hydroperoxy proton (δ H 7.78, s). Comparison of the NMR data of 3 with those of 2 revealed the replacement of the n-butyryloxy moiety at C-3 and the hydroxy group at C-6 in 2 by the acetoxy and the hydroperoxy groups in 3, respectively. The relative configuration of 3 was determined mainly by the assistance of the NOESY experiment. The NOE correlations of 3 indicated that 3 possessed the same configurations for each chiral center as those of 2.
Simplexin S (4) showed the pseudomolecular ion peak [M + Na] + at m/z 403.2463 in the HRESIMS and the molecular formula was determined as C 22 H 36 O 5 . NMR spectroscopic data of 4 (Tables 1 and 2) showed the presence of one acetoxy group (δ C 170.3, C; 22.6, CH 3 ; δ H 2.01, 3H, s). Comparison of the NMR data of 4 with those of 5 revealed that the only difference between both compounds arises from the replacement of the hydroxy group at C-3 in 4 by one n-butyryloxy moiety in 5. The NOESY spectrum indicated that 4 was found to possess the (1R*, 2R*, 3R*, 6S*, 9R*, 10S*, 11R*, 14R*)-configuration.

General Experimental Procedures
Melting points were determined using a Fisher-Johns melting point apparatus and were uncorrected. Optical rotations were measured on a JASCO DIP-1000 digital 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 on a Varian Unity INOVA 500 FT-NMR at 500 MHz for 1 H and 125 MHz for 13 C in CDCl 3 . Si gel 60 (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 Merck Hibar Si-60 column (250 × 21 mm, 7 µm).

Animal Material
Klyxum simplex (230 g, wet wt), was collected by hand using scuba off the coast of Dongsha Atoll, in September, 2006, at a depth of 11 m, and stored in a freezer until extraction. A voucher sample (specimen No. 20060901-1) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

Cytotoxicity Testing
Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of compounds 1-5 were performed using the Alamar Blue assay [14,15].

Conclusions
In previous studies, a series of new eunicellin-based diterpenoids were isolated from the cultured and wild-type soft corals Klyxum simplex. Our continued investigation on the chemical constituents of wild-type soft coral K. simplex has again led to the isolation of four new eunicellin-based diterpenoids, simplexins P-S. Simplexin R (3) exhibited significant cytotoxicity against CCRF-CEM and MOLT 4 cells, and moderate to weak cytotoxicity against K-562 and T47D cells. Also, compounds 1, 4 and 5 exhibited moderate to weak cytotoxicity toward CCRF-CEM and MOLT 4 cell lines. Besides our research, many recent studies have showed the versatile structures and bioactivities of eunicellin-type compounds [16][17][18][19][20][21]. These studies along with the new simplexins described here suggest that eunicellin-type compounds, in particular 3, are worthy of further biomedical investigation.