New Furanocembranoids from Briareum violaceum

Three new furanocembranoids—briaviodiol F (1) and briaviotriols A (2) and B (3)—along with a known analogue, briaviodiol A (4), were obtained from a cultured-type octocoral Briareum violaceum. The structures of cembranoids 1–3 were elucidated by using spectroscopic methods. In vitro study demonstrated that compounds 2 and 4 exerted inhibition effects on inducible nitric oxide synthase (iNOS) release from RAW 264.7, a macrophage cell line that originated from a mouse monocyte macrophage, stimulated with lipopolysaccharides.

Based on NOESY correlations and further information provided by MM2 forcefield calculations [11], the relative stereochemistry of 1 with the stable conformation is shown in Figure 2 (Supplementary Figures S1-10). When H-9 was α-oriented in 1, a correlation between H-9 and H3-19 was observed, suggesting that these protons were on the α-face, and H-8 was β-oriented. H-8
The 1  The presence of a vinyl methyl group on C-4 was supported by HMBC from H 3 -18 to C-3, C-4, C-5; H-3α (δ H 2.78) to C-18 and H-5 to C-18. Furthermore, HMBC from OH-13 to C-13, C-14 and OH-14 to C-1, C-13, C-14 suggested the existence of hydroxy groups at C-13 and C-14, respectively. Therefore, the methoxy group was on C-2, since the HMBC spectrum exhibited a correlation between the singlet at δ H 3.39 (OMe) and C-2 (δ C 109.5). Taking into account the molecular formula, the remaining oxygen atom must be part of the tetrahydrofuran ring located between C-9 and C-12.
Based on NOESY correlations and further information provided by MM2 forcefield calculations [11], the relative stereochemistry of 1 with the stable conformation is shown in Figure 2 (Supplementary Figures S1-10). When H-9 was α-oriented in 1, a correlation between H-9 and H 3 -19 was observed, suggesting that these protons were on the α-face, and H-8 was β-oriented. H-8 correlated with H-13, and the hydroxy proton OH-13 correlated with H 3 -20, suggesting that the hydroxy group at C-13 and the Me-20 at C-12 were α-oriented. H-14 exhibited a NOESY correlation with H-13, and no coupling constant was detected between H-13 and H-14 in the 1 H NMR spectrum, implying that the dihedral angle located between H-13 and H-14 was about 90 • , and the 14-hydroxy group was β-oriented. Correlations between H-5 and H-3, and H-14 and H-3 (δ H 2.78) suggested that this proton is α, and the proton at δ H 3.04 is 3β. Additionally, the proton signal of a methoxy group displayed NOESY correlations with both H-3α/β, which indicated that the methoxy group at C-2 was α-oriented. H 3 -18 was found to show a NOESY correlation with H-3β, but not with H-5, and H-5 was shown to be correlated with H-3α and H-14, which suggested an E-configuration of the C-4/5 double bond. The aforementioned results enabled establishment of the relative configuration of 1, and therefore its stereogenic carbons were assigned as 2R*,8R*,9S*,12R*,13S*, 14R*. correlated with H-13, and the hydroxy proton OH-13 correlated with H3-20, suggesting that the hydroxy group at C-13 and the Me-20 at C-12 were α-oriented. H-14 exhibited a NOESY correlation with H-13, and no coupling constant was detected between H-13 and H-14 in the 1 H NMR spectrum, implying that the dihedral angle located between H-13 and H-14 was about 90°, and the 14-hydroxy group was β-oriented. Correlations between H-5 and H-3, and H-14 and H-3 (δH 2.78) suggested that this proton is α, and the proton at δH 3.04 is 3β. Additionally, the proton signal of a methoxy group displayed NOESY correlations with both H-3α/β, which indicated that the methoxy group at C-2 was α-oriented. H3-18 was found to show a NOESY correlation with H-3β, but not with H-5, and H-5 was shown to be correlated with H-3α and H-14, which suggested an E-configuration of the C-4/5 double bond. The aforementioned results enabled establishment of the relative configuration of 1, and therefore its stereogenic carbons were assigned as 2R*,8R*,9S*,12R*,13S*, 14R*.     Briaviotriol A (2) was found to have the molecular formula C 21 H 32 O 7 , as established by (+)-HRESIMS at m/z 419.20377 (calcd. for C 21 H 32 O 7 + Na, 419.20402). The 1 H and 13 C NMR spectra of 2 were very similar to those of 1. Comparison between the 1 H and 13 C NMR data of 2 ( Table 2) and those of 1 suggested that the double bond is located between C-4 and C-18 in 2 instead of C-4 and C-15 in 1. HMBC from H 2 -18 to C-3, C-4, C-5; and from H 2 -3 and H-5 to C-18, corroborated the existence of an exocyclic double bond at C-4. In the HSQC spectrum, an oxymethine carbon (δ C 69.1) correlated with the methine proton (δ H 4.57), and this proton had 3 J-correlations with H 2 -6 (δ H 1.81, 1H, m and 1.93, 1H, m) in the 1 H-1 H COSY spectrum, demonstrating that a hydroxy group was attached to C-5. The stereochemistry of 2 was established from the correlations observed in the NOESY spectrum ( Figure 3 and Supplementary Figures S2-10). In addition, in the NOESY spectrum H-9 was correlated with H 3 -19, which suggested that these protons were positioned on the same face and were assigned as α protons, as H-8 was β-oriented. H-13 correlated with H-8 and H-14, but no coupling between H-13 and H-14 was observed, demonstrating that the hydroxy groups at C-13 and C-14 were αand β-oriented, respectively. Correlations between an oxygen-bearing methyl (δ H 3.26) and H-13 suggested that the C-2 methoxy group was situated on the β face. Additionally, correlation between H-5 and H-8 supported a β-orientation of H-5. Based on the aforementioned results, the relative configurations of the stereogenic carbons of 2 were determined as 2S*,5S*,8R*, 9S*,12R*,13S*,14R*.    Table 3) with those of 1 (δ H 5.30, 1H, dd, J = 8.0, 5.6 Hz; δ C 135.1, CH-5; δ C 127.1, C-4) ( Table 1), as well as a NOESY correlation between H-5 and H 3 -18, indicated the Z-configuration of the C-4/5 double bond (Figure 4 and Supplementary Figure S3-10). Furthermore, the HSQC spectrum showed that an oxymethine carbon (δ C 68.7) was correlated with a methine proton (δ H 4.86; H-6), and this proton exhibited 3 J-correlations with the olefinic proton H-5 (δ H 5.29) and H 2 -7 (δ H 1.34, 1H, m; 1.80, 1H, m) in the COSY spectrum, which confirmed a hydroxy group at C-6. As H-6 showed a NOESY correlation with H-3β, this suggested that the C-6 hydroxy group was α-oriented. Based on a NOESY experiment (Figure 4 and Supplementary Figures S3-10), 3 was identified to have the stereogenic centers 2R*,6S*,8R*,9S*,12R*,13S*,14R*. Since 3 has never been previously reported, it was named briaviotriol B.  Using an in vitro pro-inflammatory suppression assay, the effects of 1-4 on the release of iNOS protein from lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells were assessed. First, alamar blue cell viability assessment revealed that 1-4 did not have significant cytotoxic effects in RAW 264.7 cells. The results of the in vitro pro-inflammatory suppression assay showed that 2 and 4 at 10 μM suppressed the release of iNOS to 67.7 and 61.9%, respectively, when compared with results of the cells stimulated with only LPS (Table 4). Compound 1 showed no suppression effect on iNOS release.

General Experimental Procedures
The JEOL NMR spectrometer (model ECZ400S, Tokyo, Japan) was used to record the spectra with the solvent peak of CHCl3 (δH 7.26 ppm) and CDCl3 (δC 77.1 ppm) as internal references for 1 H NMR and 13 C NMR, respectively. ESIMS and HRESIMS were obtained from the Bruker mass spectrometer with 7 Tesla magnets (model: SolariX FTMS system) (Bremen, Germany). Column chromatography, IR spectra and optical rotation were performed according to our earlier research [10].

Animal Material
Specimens of B. violaceum used for this study were collected in December 2016 from the cultivation tank (capacity = 270 tons) at the National Museum of Marine Biology and Aquarium Compound 4 was identified as briaviodiol A (Figure 1), by comparison of its 1 H and 13 C NMR data with those in the literature [9].
Using an in vitro pro-inflammatory suppression assay, the effects of 1-4 on the release of iNOS protein from lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells were assessed. First, alamar blue cell viability assessment revealed that 1-4 did not have significant cytotoxic effects in RAW 264.7 cells. The results of the in vitro pro-inflammatory suppression assay showed that 2 and 4 at 10 µM suppressed the release of iNOS to 67.7 and 61.9%, respectively, when compared with results of the cells stimulated with only LPS (Table 4). Compound 1 showed no suppression effect on iNOS release.

General Experimental Procedures
The JEOL NMR spectrometer (model ECZ400S, Tokyo, Japan) was used to record the spectra with the solvent peak of CHCl 3 (δ H 7.26 ppm) and CDCl 3 (δ C 77.1 ppm) as internal references for 1 H NMR and 13 C NMR, respectively. ESIMS and HRESIMS were obtained from the Bruker mass spectrometer with 7 Tesla magnets (model: SolariX FTMS system) (Bremen, Germany). Column chromatography, IR spectra and optical rotation were performed according to our earlier research [10].

Animal Material
Specimens of B. violaceum used for this study were collected in December 2016 from the cultivation tank (capacity = 270 tons) at the National Museum of Marine Biology and Aquarium (NMMBA) in Southern Taiwan. For its identification, this coral species was compared to reliable sources published earlier [1,2]. A voucher specimen was deposited in the NMMBA (voucher no.: NMMBA-CSC-005).

Molecular Mechanics Calculations
The molecular models were generated by implementing the MM2 force field [11] in ChemBio 3D Ultra software (ver. 12.0) which was created by CambridgeSoft (PerkinElmer, Cambridge, MA, USA).

In Vitro Anti-Inflammatory Assay
The pro-inflammatory suppression assay was performed using a murine macrophage cell line, RAW 264.7, which was purchased from the American Type Culture Collection (ATCC cell line no. TIB-71; Manassas, VA, USA). Untreated or LPS-induced RAW 264.7 cells were used to determine the anti-inflammatory activities of cembranoids 1-4 by assessing the inhibition of pro-inflammatory iNOS release from macrophage cells. The iNOS protein levels were measured by using western blotting