Bioactive Cembranoids from the Soft Coral Sinularia crassa

Eight new cembranoids, crassarines A–H (1–8) were isolated from the Formosan soft coral Sinularia crassa. Compounds 1–3 represent the rare cembranoids with a 1,12-oxa-bridged tetrahydrofuran ring, while 4 and 5 are the firstly discovered 1,11-oxa-bridged tetrahydropyranocembranoids. The absolute configuration of 6 was determined using the Mosher’s method. Compounds 6 and 8 were found to significantly inhibit the expression of both pro-inflammatory iNOS and COX-2 proteins at 10 μM, respectively, while compounds 4–8 were found to be non-cytotoxic toward the selected human liver cancer cells.


Introduction
Soft corals were proven to be a rich source of terpenoids [1]. We previously have isolated a series of bioactive cembrane- [2][3][4] and norcembrane- [5][6][7][8] diterpenoids from the Formosan soft corals of the genus Sinularia. Although this genus has been well studied regarding bioactive constituents, previous investigations on an Indian soft coral Sinularia crassa (Tixier-Durivault, 1951) had resulted in the isolation of only a sphingosine and a steroid possessing anti-inflammatory [9,10] and 5α-reductase inhibitiory activities [11], respectively. This prompted us to investigate the bioactive compounds from the Formosan soft coral S. crassa and the present study has led to the isolation of eight new cembranoids, crassarines A-H (1-8, see Chart 1) from the ethanolic extract of this organism. The structures of these compounds have been established by extensive spectroscopic analysis and chemical method. The anti-inflammatory activity of 1-8 to inhibit up-regulation of the pro-inflammatory iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) proteins in LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage cells and the cytotoxicity of compounds 4-8 against a panel of cancer cell lines including human liver carcinoma (HepG2 and HepG3), human breast carcinoma (MCF-7 and MDA-MB-231), and human lung carcinoma (A-549) were evaluated in order to discover bioactive natural products.

Results and Discussion
The HRESIMS of crassarine A (1) exhibited a pseudomolecular ion peak at m/z 361.2353 [M + Na] + , consistent with a molecular formula of C 20 H 34 O 4 , appropriate for four degrees of unsaturation. The IR spectrum of 1 showed a broad absorption band at 3461 cm −1 and a strong absorption band at 1698 cm −1 , implying the presence of hydroxy and carbonyl groups. The latter was identified as a ketone functionality from the carbon resonance at δ 211.8 (Table 1). In addition, carbon resonances at δ 133.3 (CH) and 134.3 (CH) were attributed to the presence of an 1,2-disubstituted double bond. The above functionalities accounted for two of the four degrees of unsaturation, suggesting a bicyclic structure in 1. By interpretation of 1 Figure 1). According to the downfield-shifted carbon chemical shifts at δ 88.1 (C-1, C), 75.0 (C-11, CH), and 85.7 (C-12, C) [12] as well as the HMBC correlations from H 3 -20 to C-11, C-12, and C-13 and H 3 -16 (or H 3 -17) to C-17 (or C-16), C-15, and C-1, an ether linkage between C-1 and C-12 forming a tetrahydrofuran (THF) ring and a hydroxy group at C-11 were assigned for 1. The location of C-6 ketone was suggested from the carbon resonances of the adjacent methylenes at δ 53.3 (C-5) and 51.6 (C-7). This was further confirmed by the HMBC correlations from both H 2 -7 and H 2 -5 to C-6. In addition, the HMBC correlations from H 3 -18 to C-3, C-4, and C-5 helped to locate the C-2/C-3 double bond and a hydroxy group at quaternary C-4 (δ 71.4). Hence, the planar structure of 1, a cembranoid possessing a 1,12-bridged tetrahydrofuran ring, was established as shown in Figure 1. Table 1. 13 C NMR spectroscopic data of compounds 1−8.

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The E geometry for the C-2/C-3 double bond was deduced from a 16.0 Hz coupling constant (Table 1) between H-2 and H-3. The relative configuration of 1 was determined by the interpretation of NOE correlations (Figure 2). The NOE correlations between H 3 -20/H 3 -16 (or H 3 -17), H-11/H-13a (δ H 2.61), H-11/H-8, and H 3 -20/H 2 -13 suggested the 1S*,8S*,11R*,12S* configuration as depicted in Figure 2. In addition, the NOE correlations observed for H-2 with both H-15 and H 3 -18 and for H 3 -18 with H-3 suggested the 4S* configuration. In order to understand the orientation of 4-OH and 11-OH, the pyridine-induced solvent shifts were measured [13,14]. The significant differences of chemical shifts (Δδ = δ CDCl 3 − δ C 5 D 5 N ) due to pyridine-induced deshielding effect of hydroxy group were observed for H-7a (Δδ = −0.93 ppm), H 3 -20 (Δδ = −0.24 ppm), and H-13a (Δδ = −0.63 ppm) ( Table 2), suggesting that 4-OH is close to H-7a, and the 11-OH is not only close to H-13a but also gauche-oriented to H 3 -20, as shown in Figure 2. To determine the absolute configuration, we applied the Mosher's method on 1. However, we were unable to prepare the corresponding Mosher esters of 1 by usual reaction conditions [3,4]. This might be due to the steric hindrance of THF ring adjacent to C-11.    13 C NMR spectroscopic data of 2 were close to those of 1. A comparison of NMR spectroscopic data of 2 with those of 1 indicated that 2 possesses an acetoxy group [δ C 170.9 (C), δ C 21.0 (CH 3 ); δ H 2.09], which was suggested to be attached at C-11 due to the downfield-shifted proton resonance at δ H 4.08 (1H, br d, J = 10.5 Hz, H-11) in comparison with the relevant case of 11-OH analogue 1 (δ H 3.24, 1H, br d, J = 9.6 Hz, H-11). The structure elucidation of 2 was accomplished by an extensive analysis of its 2D NMR correlations, which led to the establishment of its planar structure, as shown in Figure 1. Except for the C-11 substituent and the THF ring in both compounds 1 and 2, the differences were observed for the chemical shifts of protons and carbons around the C-4 asymmetric center, in particular those of H 3 -18 (δ H 1.37 and δ C 28.9 for 1; δ H 1.25 and δ C 29.8 for 2). These observations suggested that the configuration at C-4 in 2 should be opposite to that in 1. Moreover, 1 and 2 shared the same NOE correlations around asymmetric centers C-1, C-8, C-11, and C-12. To confirm the above elucidation, 1 was acetylated to obtain 1a, which displayed different 1 H NMR spectrum to that of 2 (see Experimental). Consequently, 2 was determined to be the 4-epi-11-O-acetyl derivative of 1. The 13 C and 1 H NMR spectral data of 3 are very similar to that of 2 (Tables 1 and 2); however, 1 H NMR spectrum of 3 showed a singlet at δ 8.18 which correlates with carbon signal at δ 160.9 in the HSQC spectrum, indicating the presence of a formyloxy group at C-11 in 3. On the basis of the above data, 3 was identified as the 11-O-formyl derivative of 2. Literature review showed that this is the first cembranoid with a formyloxy group.
Crassarine D (4) possesses the same molecular formula as that of 1. The 13 C NMR data (Table 1) of 4 were mostly similar to those of 1, except for those of sp 3 oxygenated carbons, suggesting that they vary mainly in the heterocyclic ring. The upfield shift for H-11 from δ 3.24 (1H, br d, J = 9.6 Hz) in 1 to δ 3.02 (1H, d, J = 8.8 Hz) in 4 indicates that an ether linkage should be located between C-1 and C-11 to form a tetrahydropyran (THP) ring. The HMBC correlation from H-11 to C-1 (δ 77.5, C) confirmed the presence of this THP ring in 4, rather than the THF ring in 1. The detailed analysis of the correlations observed in the COSY, HMBC, and HSQC spectra further assigned all the spectroscopic data and established the planar structure of 4 ( Figure 1). The E geometry of C-2/C-3 double bond was also deduced from the coupling constant (16. , and a trisubstituted epoxide [δ H 2.87 (1H, dd, J = 7.6, 6.0 Hz); δ C 59.5 (C) and 57.0 (CH)] were also evident. Above NMR signals suggested 6 to be the 1,3-diene cembranoid with an epoxy group [15]. The 11,12-epoxy group was assigned by the HMBC correlations from H 3 -20 to C-11, C-12, and C-13 and H 2 -14 to C-1, C-2, and C-13 ( Figure 1). The COSY cross peaks of H 2 -10/H-11 and H 2 -10/H-9 as well as the HMBC correlations from H 3 -19 to C-7, C-8, and C-9 assigned the hydroxy group at C-9 (δ C 75.3, CH). These findings and the detailed COSY and HMBC correlations established the planar structure of 6, as shown in Figure 1. The relative configuration of 6 was determined by the interpretation of NOESY spectrum. The crucial NOE correlations (Figure 2) between H-2/H 3 -18, H-2/H-15, and H-9/H-7 indicated the E geometry for all double bonds and suggested a s-trans geometry for the 1,3-diene. NOE correlations between H-11/H-3, H-11/H-14a, and H-3/H-14a showed that these protons should be pointed toward the core of 14-membered ring. Furthermore, the absence of NOE correlation between H-11 and H 3 -20 and the presence of correlation between H-9 and H 3 -20 suggested the 9S*,11S*,12S* configuration, as depicted in Figure 2. The absolute configuration of 6 was determined by the application of Mosher's method [16,17]. The (S)-and (R)-MTPA esters of 6 (6a and 6b, respectively) were prepared using the corresponding (R)-and (S)-MTPA chloride, respectively. The determination of chemical shift differences for the protons neighboring C-9 led to the assignment of the 9S configuration in 6 ( Figure 3). Thus, the absolute configuration of 6 was determined as 9S, 11S, 12S. The HRESIMS data of crassarine G (7) revealed a molecular formula of C 20 H 32 O 2 , the same as that of 6. The IR spectrum of 7 disclosed the presence of hydroxy group (ν max 3434 cm −1 ). A comparison of the NMR spectroscopic data of 7 (Tables 1 and 2) with those of 6 revealed that the hydroxy-containing methine (C-9) in 6 was replaced by a sp 3 methylene in 7. It was also found that resonances appropriate for H 3 -19 in 6 were absent from the 1 H and 13 C NMR spectra of 7 and replaced by signals for a hydroxymethyl group [δ H 3.93 and 3.89 (each 1H, d, J = 12.0 Hz); δ C 59.4 (CH 2 )]. Careful inspection of the 2D NMR spectra of 7 confirmed the above elucidation.  [18]. This furan moiety and the trisubstituted double bond were found to be conjugated according to the downfield-shifted proton resonance of H-2 at δ 5.95 (1H, s) [18]. This was further confirmed by the HMBC correlations from H-2 to C-1, C-3, C-14, and C-15, H 3 -18 to C-3, C-4, and C-5, and H-5 to C-3, C-4, and C-6. The above data together with the detailed inspection of the COSY and HMBC correlations of 8 established its planar structure (Figure 1). The relative configuration of 8 was determined mainly by the assistance of the NOESY experiment. The key NOE correlations between H-2 and both H-15 and H 3 -18 indicated an E geometry of C-1/C-2 double bond ( Figure 2). The trans epoxy group was deduced by the NOE correlations between H-11/H-13b and H 3 -20/H-13a. In addition, H-8 showed an NOE correlation with H 3 -20, instead of H-11, suggesting the 8S*,11S*,12S* configuration for 8.
The anti-inflammatory activity of diterpenoids 1-8 against the accumulation of pro-inflammatory iNOS and COX-2 proteins in RAW264.7 macrophage cells stimulated with LPS was evaluated using immunoblot analysis. At a concentration of 10 µM (Figure 4), 8 was found to significantly reduce the levels of iNOS protein (35.8 ± 10.7%), compared with the control cells stimulated with LPS only. At the same concentration, 6 could reduce COX-2 expression (65.6 ± 6.2%) by LPS treatment. Cytotoxicity of diterpenoids 4-8 against HepG2, HepG3, MCF-7, MDA-MB-231, and A-549 cancer cell lines was also evaluated. The results showed that the tested compounds were found to be inactive (IC 50 > 20 μM) toward the above cancer cell lines after 72 h exposure.

General
The

Animal Material
The soft coral Sinularia crassa was collected by hand using scuba off the coast of Sansiantai, Taitung county, Taiwan, in July 2008, at a depth of 10 m, and was stored in a freezer (−20 °C). This soft coral was identified by one of the authors (C.-F.D.). A voucher specimen (Specimen No. SST-03) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.