Briarenols I—K, New Anti-inflammatory 8,17-Epoxybriaranes from the Octocoral Briareum excavatum (Briareidae)

Five 8,17-epoxybriaranes, including three new compounds—briarenols I–K (1–3), along with two known analogues, briaexcavatolide P (4) and briaexcavatin P (5), were isolated from the octocoral Briareum excavatum. The structures of briaranes 1–3 were elucidated by spectroscopic methods, including 1D and 2D NMR studies and (+)-HRESIMS. Briarane 4 exerted inhibition effects on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) release from RAW 264.7.


Introduction
Octocorals of the genus Briareum (family Briareidae) [1][2][3][4] are proven to be the most important source to produce briarane-type diterpenoids [5]. The compounds of this type are only found in marine invertebrates, particularly in octocorals and demonstrated a wide spectrum of bioactivities, such as anti-inflammatory activity [6] and cytotoxicity [7]. In our continuing research into the chemical constituents of an octocoral B. excavatum (Nutting 1911), which was distributed extensively in the coral reefs of Taiwan, have resulted in isolation of three previously unreported 8,17-epoxybriaranes-briarenols I-K (1-3) along with two known analogues, briaexcavatolide P (4) [8] and briaexcavatin P (5) [9],
The stereochemistry of 2 was elucidated from the NOE interactions observed in a NOESY experiment ( Figure 3) and by the vicinal 1 H− 1 H coupling constant analysis. In the NOESY spectrum, correlations were observed between H-10 with H-3 and H-12; and H-12 correlated with H-11, indicating that these protons should be α-oriented. H-14 gave a correlation with H3-15, confirming the β-orientation for this proton. H-2 showed a correlation with H-14, and a lack of coupling constant was detected between H-2/H-3, indicating the dihedral angle between H-2/H-3 is approximately 90° and the 2-hydroxy group was β-oriented. H-4 exhibited correlations with H-7 and 2-hydroxy proton, confirming the β-orientations for H-4 and H-7. H-9 was found to show correlations with H-11, H3-18, and H3-20, and from molecular models, H-9 and H3-18 should be placed on the α-and β-face, respectively. The Z-configuration of C-5/C-6 double bond was elucidated by a correlation between H-6 and H3-16. The NMR data of 2 were found to be similar to those of a known briarane, briaexcavatin P (5) [9]. It was found that the 2-acetoxy substituent in 5 was replaced by a hydroxy group in 2. By comparison of the proton and carbon chemical shifts, coupling constants, NOESY correlations, and rotation value of 2 with those of 5, the stereochemistry of 2 was confirmed to be the same as that of 5, and the configurations of the stereogenic centers of 2 were assigned as 1S,2R,3R,4R, 7S,8S,9S,10S,11R,12S,14S, and 17R (Supplementary Materials,. Briarane 3 (briarenol K) was found to have a molecular formula of C26H36O11 based on its (+)-HRESIMS peak at m/z 547.21514 (calculated for C26H36O11 + Na, 547.21498). Its absorption peaks in the IR spectrum showed ester carbonyl, γ-lactone, and broad OH stretching at 1739, 1780, and 3468 cm −1 , respectively. The 13 C NMR spectrum indicated that three esters and a γ-lactone were present, as carbonyl resonances were observed at δC 168.1, 170.2, 170.4, and 170.4, respectively (Table 1). The 1 H NMR data also indicated that presence of three acetate methyls at δH 2.22, 2.03, and 2.00 (each 3H  s; Table 2). It was found that the spectroscopic data of 3 were similar to those of a known briarane,  The inhibition effects of briaranes 1-5 on the release of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein from lipopolysaccharides (LPS)-stimulated RAW 264.7 were assessed. The results showed that briarane 4 reduced the release of iNOS and COX-2 to 35.37% and 54.61% at a concentration of 10 µ M, respectively ( Figure 5 and Table 3). Briarane 1 was found to be weaker than those of 4 in term of reducing the expression of iNOS and COX-2, indicating that the hydroxy group at C-16 in 1 reduced the activity.
) of 1. The stereochemistry of 1 was deduced from an NOESY experiment ( Figure 2) and biogenetic considerations. The NOE correlations of H-10/H-11, H-10/H-12, and H-11/H-12 indicated that these protons were situated on the same face of the structure and were assigned as the α protons since the C-15 methyl is the β-substituent at C-1. The NOE correlation between H 3 -15 and H-14 implied that H-14 had a β-orientation. H-3 exhibited a correlation with H-10, and, as well as a lack of coupling constants were detected between H-2/H-3 and H-3/H-4, indicating the dihedral angles between H-2/H-3 and H-3/H-4 were approximately 90 • and the 2-acetoxy, 3-hydroxy, and 4-n-butyroxy groups were β-, β-, and α-oriented, respectively. A correlation from H-4 to H-7, suggested that H-7 was β-oriented. The Z-configuration of C-5/6 double bond was confirmed based on the fact that the C-6 olefinic proton (δ H 5.53) correlated to one of the C-16 hydroxymethyl protons (δ H 4.04). H-9 was found to correlate with H-11, H 3 -18, and H 3 -20. From a consideration of molecular model, H-9 was found to be reasonably close to H-11, H 3 -18, and H 3 -20, thus, H-9 should be placed on the α face, and Me-18 was β-oriented in the γ-lactone moiety, and the 8,17-epoxy group should be α-oriented. It was found that the NMR signals of 1 were similar to those of a known briarane, briaexcavatolide P (4) (Figure 1) [8], except that the signals corresponding to the Me-16 vinyl methyl in 4 were replaced by signals for a hydroxymethyl group in 1. Additionally, as briaranes 1-5 were isolated along with a known briarane, excavatolide B (6) [6,10,11] from the same target organism, B. excavatum, and the absolute configuration of 6 was determined by a single-crystal X-ray diffraction analysis [6,11]. Therefore, on biogenetic grounds to assume that briaranes 1-5 had the same absolute stereochemistry as that of 6, tentatively, and the configurations of stereogenic carbons of 1 were determined as 1R,2R,3S,4R,7S,8S, 9S,10S,11R,12S,14S, and 17R (Supplementary Materials, Figures S1-S10).
Briarenol J (2) 12 + Na, 563.20990). The IR spectrum showed bands at 3483, 1779, and 1727 cm −1 , consistent with the presence of hydroxy, γ-lactone, and ester groups, respectively, in 2. From the 13 C and DEPT data ( Table 2), one trisubstituted double bond was deduced from the signals of two carbons at δ C 139.3 (C-5) and 124.3 (CH-6). A methyl-containing tetrasubstituted epoxy group was confirmed from the signals of two oxygenated quaternary carbons at δ C 69.9 (C-8) and 61. 8 (C-17), and from the chemical shift of a tertiary methyl (δ H 1.66, 3H, s; δ C 10.3, CH 3 -18; Tables 1 and 2). Four carbonyl resonances at δ C 170. 9, 170.0, 169.5, and 169.2 in the 13 C spectrum confirmed the presence of a γ-lactone and three esters. All the esters were identified as acetates by the presence of three methyl singlet resonances in the 1 H NMR spectrum at δ H 2.32, 2.16, and 2.14, respectively.
The stereochemistry of 2 was elucidated from the NOE interactions observed in a NOESY experiment ( Figure 3) and by the vicinal 1 H− 1 H coupling constant analysis. In the NOESY spectrum, correlations were observed between H-10 with H-3 and H-12; and H-12 correlated with H-11, indicating that these protons should be α-oriented. H-14 gave a correlation with H 3 -15, confirming the β-orientation for this proton. H-2 showed a correlation with H-14, and a lack of coupling constant was detected between H-2/H-3, indicating the dihedral angle between H-2/H-3 is approximately 90 • and the 2-hydroxy group was β-oriented. H-4 exhibited correlations with H-7 and 2-hydroxy proton, confirming the β-orientations for H-4 and H-7. H-9 was found to show correlations with H-11, H 3 -18, and H 3 -20, and from molecular models, H-9 and H 3 -18 should be placed on the αand β-face, respectively. The Z-configuration of C-5/C-6 double bond was elucidated by a correlation between H-6 and H 3 -16. The NMR data of 2 were found to be similar to those of a known briarane, briaexcavatin P (5) [9]. It was found that the 2-acetoxy substituent in 5 was replaced by a hydroxy group in 2. By comparison of the proton and carbon chemical shifts, coupling constants, NOESY correlations, and rotation value of 2 with those of 5, the stereochemistry of 2 was confirmed to be the same as that of 5, and the configurations of the stereogenic centers of 2 were assigned as 1S,2R,3R,4R, 7S,8S,9S,10S,11R,12S,14S, and 17R (Supplementary Materials, Figures S11-S20).
Molecules 2020, 25, x FOR PEER REVIEW 6 of 9 β-oriented. The locations of the functional groups were further confirmed by other HMBC and COSY correlations (Figure 4), and hence briarenol K was assigned the structure of 3. The NOESY spectrum exhibited a correlation from H-10 to H-12, further supporting that H-12 was α-oriented and the stereogenic centers of 3 were assigned as 1S,2S,7S,8S,9S,10S,11S,12S,14S, and 17R, by the correlations observed in a NOESY spectrum ( Figure 4) and this compound was found to be the 12-epimer of briareolide B (7) [12] (Supplementary Materials, Figures S21-S30). The inhibition effects of briaranes 1-5 on the release of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein from lipopolysaccharides (LPS)-stimulated RAW 264.7 were assessed. The results showed that briarane 4 reduced the release of iNOS and COX-2 to 35.37% and 54.61% at a concentration of 10 µ M, respectively ( Figure 5 and Table 3). Briarane 1 was found to be weaker than those of 4 in term of reducing the expression of iNOS and COX-2, indicating that the hydroxy group at C-16 in 1 reduced the activity.  Table 3. Effects of briaranes 1-5 on LPS-induced pro-inflammatory iNOS and COX-2 protein expression in macrophages.
The inhibition effects of briaranes 1-5 on the release of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein from lipopolysaccharides (LPS)-stimulated RAW 264.7 were assessed. The results showed that briarane 4 reduced the release of iNOS and COX-2 to 35.37% and 54.61% at a concentration of 10 µM, respectively ( Figure 5 and Table 3). Briarane 1 was found to be weaker than those of 4 in term of reducing the expression of iNOS and COX-2, indicating that the hydroxy group at C-16 in 1 reduced the activity.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 9 β-oriented. The locations of the functional groups were further confirmed by other HMBC and COSY correlations (Figure 4), and hence briarenol K was assigned the structure of 3. The NOESY spectrum exhibited a correlation from H-10 to H-12, further supporting that H-12 was α-oriented and the stereogenic centers of 3 were assigned as 1S,2S,7S,8S,9S,10S,11S,12S,14S, and 17R, by the correlations observed in a NOESY spectrum ( Figure 4) and this compound was found to be the 12-epimer of briareolide B (7) [12] (Supplementary Materials, Figures S21-S30). The inhibition effects of briaranes 1-5 on the release of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein from lipopolysaccharides (LPS)-stimulated RAW 264.7 were assessed. The results showed that briarane 4 reduced the release of iNOS and COX-2 to 35.37% and 54.61% at a concentration of 10 µ M, respectively ( Figure 5 and Table 3). Briarane 1 was found to be weaker than those of 4 in term of reducing the expression of iNOS and COX-2, indicating that the hydroxy group at C-16 in 1 reduced the activity.  Table 3. Effects of briaranes 1-5 on LPS-induced pro-inflammatory iNOS and COX-2 protein expression in macrophages.