New 9-Hydroxybriarane Diterpenoids from a Gorgonian Coral Briareum sp. (Briareidae)

Six new 9-hydroxybriarane diterpenoids, briarenolides ZI–ZVI (1–6), were isolated from a gorgonian coral Briareum sp. The structures of briaranes 1–6 were elucidated by spectroscopic methods and by comparison of their spectroscopic data with those of related analogues. Briarenolides ZII (2) and ZVI (6) were found to significantly inhibit the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.


Results and Discussion
The molecular formula of a new briarane, briarenolide ZI (1), was determined as C 24 H 33 ClO 11 (eight degrees of unsaturation) by high-resolution electrospray ionization mass spectrum (HRESIMS) at m/z 555.16025 (calcd. for C 24 H 33 ClO 11 + Na, 555.16036). The IR of 1 showed absorptions at 1715, 1769 and 3382 cm´1, which were consistent with the presence of ester, γ-lactone and hydroxy groups. The 13 C NMR spectrum (Table 1) suggested that 1 possessed an exocyclic carbon-carbon double bond based on signals at δ C 138.6 (C-5) and 116.9 (CH 2 -16), which was confirmed by the 1 H NMR spectrum of 1 (Table 1), which showed two olefin proton signals at δ H 5.88 (1H, dd, J = 2.4, 1.2 Hz, H-16a) and 5.64 (1H,dd,J = 2.4,1.2 Hz,. Three carbonyl resonances at δ C 175. 3 (C-19), 173.4 and 169.3 (2ˆester carbonyls) revealed the presence of one γ-lactone and two ester groups in 1; two acetyl methyls (δ H 2.06, s, 2ˆ3H) were also observed. According to the overall unsaturation data, it was concluded that 1 was a diterpenoid molecule possessing four rings.  -11 and H-17, H 3 -18/C-19) permitted elucidation of the carbon skeleton (Table 1). HMBC correlations between H 2 -16/C-4, -5 and -6 indicated an exocyclic double bond at C-5, which was further confirmed by the allylic coupling between H 2 -16/H-6. HMBC correlations between H 3 -15/C-1, -2, -10 and -14 and H-2 and H-10/C-15, revealed that the ring junction C-15 methyl group was located at C-1. Furthermore, an HMBC correlation between H-2 (δ H 5.09) and the acetate carbonyl (δ C 173.4) revealed the presence of an acetate ester at C-2; and an HMBC correlation between a hydroxy proton (δ H 6.50) and C-4 oxygenated quaternary carbon suggested the presence of a hydroxy group at C-4. The C-4 hydroxy group was determined to be part of a hemiketal constellation on the basis of a characteristic carbon signal at δ C 96.7. 1 H-1 H COSY correlations between OH-9/H-9 and OH-12/H-12 suggested the presence of the hydroxy groups at C-9 and C-12. A carbon signal at δ C 81.8 (C-8) indicated 3 J-coupling with protons at δ H 2.23 (H-10), 1.33 (H 3 -18) and 2.73 (OH-9). Therefore, the remaining hydroxy and acetoxy groups had to be positioned at C-11 and C-14, respectively, as indicated by analysis of 1 H-1 H COSY correlations and characteristic NMR signal analysis. The intensity of the sodiated molecules [M + 2 + Na] + isotope peak observed in the ESIMS and HRESIMS spectra ([M + Na] + :[M + 2 + Na] + = 3:1) was evidence of the presence of one chlorine atom in 1. The methine unit at δ C 56.2 was more shielded than expected for an oxygenated carbon and was correlated to the methine proton at δ H 5.54 (H-6) in the heteronuclear multiple quantum coherence (HMQC) spectrum, and this proton signal was 3 J-correlated with H-7 (δ H 4.73) in the 1 H-1 H COSY spectrum, which proved that a chlorine atom was attached at C-6. These data, together with the HMBC correlations between H-17/C-9, - 18 and -19 and H 3 -18/C-8, -17 and -19, established the molecular framework of 1. The relative configuration of 1 was elucidated on the basis of a nuclear Overhauser effect spectroscopy (NOESY) experiment and by vicinal 1 H-1 H proton coupling constant analysis. Most naturally-occurring briarane natural products have Me-15 in the β-orientation and H-10 in the α-orientation [2][3][4][5][6], which were verified by the absence of a correlation between these two groups. In the NOESY experiment of 1 (Figure 2), H-10 correlated with H-2, H-9 and H 3 -20, indicating that these protons were situated on the same face; they were assigned as α protons, as C-15 methyl was β-oriented at C-1. The oxymethine proton H-14 was found to exhibit a response with H 3 -15, but not with H-10, revealing that H-14 was β-oriented. H-12 correlated with each of the C-13 methylene protons and H 3 -20, but not with H-10, indicating that H-12 was β-oriented and was positioned on the equatorial direction in the cyclohexane ring by modeling analysis. H-17 exhibited correlations with H-9 and H-7 and was also found to be reasonably close to H-9 and H-7 by modeling analysis; thus, H-17 could therefore be placed on the β face in 1, and H-7 was β-oriented. One of the C-3 methylene protons (δ H 3.73) displayed a correlation with H 3 -15; therefore, it was assigned as the H-3β proton, and the other was assigned as H-3α (δ H 1.46). H-6 displayed correlations with H-3β and H-7, which confirmed that this proton was in the β-orientation, and the oxygen bridge between C-4 and C-8 was found to be α-oriented by modeling analysis. Based on the aforementioned results, the structure, including the relative configuration, of 1 was elucidated unambiguously. Int. J. Mol. Sci. 2016, 17, 79 4 of 13 with H-9 and H-7 and was also found to be reasonably close to H-9 and H-7 by modeling analysis; thus, H-17 could therefore be placed on the β face in 1, and H-7 was β-oriented. One of the C-3 methylene protons (δH 3.73) displayed a correlation with H3-15; therefore, it was assigned as the H-3β proton, and the other was assigned as H-3α (δH 1.46). H-6 displayed correlations with H-3β and H-7, which confirmed that this proton was in the β-orientation, and the oxygen bridge between C-4 and C-8 was found to be α-oriented by modeling analysis. Based on the aforementioned results, the structure, including the relative configuration, of 1 was elucidated unambiguously. Briarenolide ZII (2) was isolated as a white powder and had a molecular formula of C28H38O10 on the basis of HRESIMS at m/z 557.23552 (calcd. for C28H38O10 + Na, 557.23572). Carbonyl resonances in the 13 C NMR spectrum of 2 ( Table 2) at δC 173.0, 170.7, 170.4 and 169.9 demonstrated the presence of a γ-lactone and three other esters in 2. It was found that the NMR signals of 2 were similar to those of a known briarane analogue, excavatolide F (7) [7] (Figure 1), except that the signals corresponding to the 9-acetoxy group in 7 were replaced by signals for a hydroxy group in 2. The correlations from a NOESY experiment of 2 also revealed that the stereochemistry of this metabolite was identical to that of 7. Thus, briarenolide ZII (2) was found to be the 9-O-deacetyl derivative of 7.  Briarenolide ZII (2) was isolated as a white powder and had a molecular formula of C 28 H 38 O 10 on the basis of HRESIMS at m/z 557.23552 (calcd. for C 28 H 38 O 10 + Na, 557.23572). Carbonyl resonances in the 13 C NMR spectrum of 2 ( Table 2) at δ C 173.0, 170. 7, 170.4 and 169.9 demonstrated the presence of a γ-lactone and three other esters in 2. It was found that the NMR signals of 2 were similar to those of a known briarane analogue, excavatolide F (7) [7] (Figure 1), except that the signals corresponding to the 9-acetoxy group in 7 were replaced by signals for a hydroxy group in 2. The correlations from a NOESY experiment of 2 also revealed that the stereochemistry of this metabolite was identical to that of 7. Thus, briarenolide ZII (2) was found to be the 9-O-deacetyl derivative of 7.
Briarenolide ZIII (3) had a molecular formula C 24 H 32 O 10 as deduced from HRESIMS at m/z 503.18858 (calcd. for C 24 H 32 O 10 + Na, 503.18877). The IR spectrum of 1 showed three bands at 3444, 1779 and 1732 cm´1, which were in agreement with the presence of hydroxy, γ-lactone and ester groups. Carbonyl resonances in the 13 C NMR spectrum of 3 at δ C 171. 8, 170.7 and 170.6 revealed the presence of a γ-lactone and two esters (Table 3). Both esters were identified as acetates by the presence of two acetyl methyl resonances in the 1 H (δ H 2.01, 1.98, each 3Hˆs) and 13 C (δ C 21.1, 21.1) NMR spectra (Table 3).
It was found that the NMR data of 3 were similar to those of a known briarane analogue, 2β-acetoxy-2-(debutyryloxy)-stecholide E (8) [1] (Figure 1), except that the signals corresponding to the 4-hydroxy group in 3 were not present in 8. A correlation from the NOESY signals of 3 showed that H-4 correlated with H-2, but not with H 3 -15, indicating that the hydroxy group at C-4 was β-oriented. The results of 1 H-1 H COSY and HMBC correlations fully supported the positions of functional groups, and hence, briarenolide ZIII (3) was found to be the 4β-hydroxy derivative of 8.  Briarenolide ZIV (4) was obtained as a white powder, and the molecular formula of 4 was determined to be C 28 H 40 O 11 (9˝of unsaturation) by HRESIMS at m/z 575.24645 (calcd. for C 28 H 40 O 11 + Na, 575.24628). The IR spectrum of 4 showed three bands at 3444, 1778 and 1732 cm´1, consistent with the presence of hydroxy, γ-lactone and ester carbonyl groups. Carbonyl resonances in the 13 C NMR spectrum of 4 showed signals at δ C 173. 9, 173.2, 170.8 and 170.4, which revealed the presence of a γ-lactone and three esters in 4 (Table 4), of which, two of the esters were identified as acetates based on the presence of two acetyl methyl resonances in the 1 H NMR spectrum of 4 at δ H 1.97 (2ˆ3H, s) ( Table 4). The other ester was found to be an n-butyrate group based on 1 H NMR studies, which revealed seven contiguous protons (δ H 0.94, 3H, t, J = 7.2 Hz; 1.65, 2H, sextet, J = 7.2 Hz; 2.23, 2H, t, J = 7.2 Hz). According to the 1 H and 13 C NMR spectra, 4 was found to have a γ-lactone moiety (δ C 173.9, C-19) and a trisubstituted olefin (δ C 145.4, C-5; 121.6, CH-6; δ H 5.32, 1H, d, J = 8.8 Hz, H-6). The presence of a tetrasubstituted epoxide that contained a methyl substituent was established based on the signals of two oxygenated quaternary carbons at δ C 71.8 (C-8) and 63.7 (C-17) and confirmed by the proton signals of a methyl singlet at δ H 1.51 (3H, s, H 3 -18). Thus, from the NMR data, five degrees of unsaturation were accounted for, and 4 was identified as a tetracyclic compound. From the 1 H-1 H COSY spectrum of 4 (Table 4), three different structural units, including C-2/-3/-4, C-6/-7 and C-9/-10/-11/-12/-13/-14, were identified. From these data and the HMBC correlation results (Table 4), the connectivity from C-1 to C-14 could be established. A methyl attached at C-5 was confirmed by an allylic coupling between H 3 -16/H-6 and by the HMBC correlations between H 3 -16/C-4, -5 and -6. The C-15 and C-20 methyl groups were identified as being positioned at C-1 and C-11 from the HMBC correlations between H 3 -15/C-1, -2, -10, -14 and H 3 -20/C-10, -11, -12, respectively. Furthermore, the acetate esters positioned at C-2 and C-14 were established by the HMBC correlations between δ H 4.97 (H-2) and 4.70 (H-14) and the acetate carbonyls at δ C 170.4 and 170.8, respectively. The location of an n-butyrate group in 4 was verified by an HMBC correlation between H-12 (δ H 4.83) and the n-butyrate carbonyl carbon (δ C 173.2) ( Table 4). These data, together with the HMBC correlations between H 3 -18/C-8, -17 and -19, established the main molecular framework of 4. The NMR data of 4 were found to be similar to those of a known briarane, excavatolide Z (9) [8] (Figure 1), except that the signals corresponding to the 4-hydroxy group in 4 were not present in 9, and an 11β-hydroxy group was found in 9. The correlations from NOESY signals of 4 ( Figure 3) also showed that the relative configurations of most chiral centers of 4 were similar to those of 9. H-10 exhibited interactions with H-2 and H-11, and H-2 correlated with H-4, indicating that the hydroxy group at C-4 and the methyl group at C-11 were β-oriented; additionally, briarenolide ZIV (4) was found to be the 4β-hydroxy-11-dehydroxy-11β-methyl derivative of 9.   Briarenolide ZV (5) was obtained as a white powder and had the molecular formula C24H30O10, as determined by HRESIMS at m/z 505.20460 (calcd. for C24H30O10 + Na, 505.20442) (10° of unsaturation). The IR spectrum of 5 showed bands at 3445, 1770 and 1732 cm −1 , consistent with the presence of hydroxy, γ-lactone and ester carbonyl groups. Comparison of the 1 H and distortioneless enhancement by polar transfer (DEPT) spectra with the molecular formula revealed that there must   -9, H-11 C-1, -2, -8, -9, -11, -12, -14, -15, - Briarenolide ZV (5) was obtained as a white powder and had the molecular formula C 24 H 30 O 10 , as determined by HRESIMS at m/z 505.20460 (calcd. for C 24 H 30 O 10 + Na, 505.20442) (10˝of unsaturation). The IR spectrum of 5 showed bands at 3445, 1770 and 1732 cm´1, consistent with the presence of hydroxy, γ-lactone and ester carbonyl groups. Comparison of the 1 H and distortioneless enhancement by polar transfer (DEPT) spectra with the molecular formula revealed that there must be three exchangeable protons, requiring the presence of three hydroxy groups. In addition, it was found that the spectral data (IR, 1 H and 13 C NMR) of 5 (Table 5) were similar to those of a known briarane, excavatolide Z (9) [8] (Figure 1), except that 9 exhibited signals representing an n-butyrate substitution, which were replaced by a hydroxy group in 5. The results of 1 H-1 H COSY and HMBC correlations fully supported the positions of functional groups, and hence, briarenolide ZV (5) was found to be the 12-O-debutyryl derivative of 9.
The new briarane, briarenolide ZVI (6), had a molecular formula of C 26 H 36 O 11 as determined by HRESIMS at m/z 547.21473 (calcd. for C 26 H 36 O 11 + Na, 547.21498). Thus, nine degrees of unsaturation were therefore determined for the molecule of 6. In addition, the spectral data (IR, 1 H and 13 C NMR) ( Table 6) of 6 were found to be similar to those of a known briarane, excavatolide E (10) [9] (Figure 1). However, the NMR spectra revealed that the signals representing the C-4 methylene group in 10 were replaced by those of an additional acetoxy group. In the NOESY experiment of 6, H-10 gives correlations to H-2, H-9, H-11 and H-12, but not to H 3 -15 and H 3 -20, and H-2 was found to show a correlation with H-4, indicating that these protons (H-2, H-4, H-9, H-10, H-11 and H-12) are located on the same face of the molecule and assigned as α-protons, since the C-15 and C-20 methyls are the β-substituents at C-1 and C-11, respectively. The signal of H 3 -20 showed a correlation with H 3 -18, indicating that H 3 -18 and 8,17-epoxide group were βand α-oriented, respectively, in the γ-lactone ring in 6. H-4 correlated with H-2, but not with H-7 and H 3 -15, indicating that H-7 was β-oriented. H-14 was found to exhibit nuclear Overhauser effect (NOE) responses with H-2 and H 3 -15, but not with H-10, revealing the β-orientation of this proton. Thus, based on the above findings, Compound 6 was found to be the 4β-acetoxy derivative of 10, with a structure as described by Formula 6. Furthermore, the chemical shifts for H 3 -18 in briaranes 4, 5 and 6 were found to appear at δ H 1.51, 1.68 and 1.57, respectively, indicating that the 11β-hydroxy group in 5 led to a downfield chemical shift for H 3 -18. In an in vitro anti-inflammatory activity assay, Western blot analysis was used to evaluate the upregulation of the pro-inflammatory cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. At a concentration of 10 µM, briarenolides ZII (2) and ZVI (6) were found to significantly reduce the levels of iNOS to 47.2% and 55.7%, respectively, in comparison to the control cells stimulated with LPS only (Figure 4 and Table 7). By using trypan blue staining, it was observed that briarenolides ZI-ZVI (1-6) did not induce significant cytotoxicity in RAW264.7 macrophage cells.  -3, -4, -10, -14, -15, acetate carbonyl 3 3.16 dd (15.6, 12.8); 1.91 m 37. 6, 5.01 dd (12.8,5.6) 72.7,   2.20 dd (4.8,2.2) 41.6,3.6) 67.0,  In an in vitro anti-inflammatory activity assay, Western blot analysis was used to evaluate the upregulation of the pro-inflammatory cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. At a concentration of 10 μM, briarenolides ZII (2) and ZVI (6) were found to significantly reduce the levels of iNOS to 47.2% and 55.7%, respectively, in comparison to the control cells stimulated with LPS only (Figure 4 and Table 7). By using trypan blue staining, it was observed that briarenolides ZI-ZVI (1-6) did not induce significant cytotoxicity in RAW264.7 macrophage cells.  The relative intensity of the LPS-stimulated group was taken to be 100%. Band intensities were quantified by densitometry and are indicated as the percentage change relative to that of the LPS-stimulated group. Briarenolides ZII (2) and ZVI (6) and DEX significantly inhibited LPS-induced iNOS protein expression (<60%) in macrophages. The experiments were repeated three times (* p < 0.05, significantly different from the LPS-stimulated group). The relative intensity of the LPS-stimulated group was taken to be 100%. Band intensities were quantified by densitometry and are indicated as the percentage change relative to that of the LPS-stimulated group. Briarenolides ZII (2) and ZVI (6) and DEX significantly inhibited LPS-induced iNOS protein expression (<60%) in macrophages. The experiments were repeated three times (* p < 0.05, significantly different from the LPS-stimulated group).

General Experimental Procedures
Melting points were determined using a Fargo apparatus (Panchum Scientific Corp. Kaohsiung, Taiwan), and the values were uncorrected. Optical rotation values were measured with a Jasco P-1010 digital polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). IR spectra were obtained with an FT-IR spectrophotometer (Digilab FTS 1000; Varian Inc., Palo Alto, CA, USA); peaks are reported in cm´1. NMR spectra were recorded on a 400-MHz Varian Mercury Plus NMR spectrometer (Varian Inc.) using the residual CHCl 3 signal (δ H 7.26 ppm) as the internal standard for 1 H NMR and CDCl 3 (δ C 77.1 ppm) for 13 C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system (Bruker, Bremen, Germany). Column chromatography was performed using 230-400 mesh silica gel (Merck, Darmstadt, Germany). TLC was carried out on precoated 0.25 mm-thick Kieselgel 60 F 254 (Merck); spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. Normal-phase HPLC (NP-HPLC) was performed using a system equipped with a Hitachi L-7110 pump (Hitachi Ltd., Tokyo, Japan), a Hitachi L-7455 photodiode array detector and an injection port (7725; Rheodyne LLC, Rohnert Park, CA, USA). A semi-preparative normal-phase LiChrospher column (Hibar 250 mmˆ10 mm, Si 60, 5 µm, Merck) was used for HPLC. Reverse-phase HPLC (RP-HPLC) was performed with a system equipped with a Hitachi L-7100 pump, a Hitachi L-2455 photodiode array detector, a Rheodyne 7725 injection port and a 25 cmˆ10 mm Polaris 5 C-18-A column (5 µm; Varian Inc., Palo Alto, CA, USA).

Animal Material
Specimens of Briareum sp. were collected by hand by scuba divers in an area off the coast of southern Taiwan in July 2011 and stored in a freezer. A voucher specimen was deposited in the National Museum of Marine Biology & Aquarium (NMMBA-TW-SC-2011-77) [10][11][12][13][14].

Conclusions
Gorgonian corals belonging to the genus Briareum are proven to be rich sources of briarane-type compounds. Briarenolides ZII (2) and ZVI (6) are potentially anti-inflammatory compounds for future development [18,19]. This interesting species was transplanted to culture tanks located in the NMMBA, for extraction of natural material to establish a stable supply of bioactive substances.