11β,20β-Epoxybriaranes from the Gorgonian Coral Junceella fragilis (Ellisellidae).

Two 11,20-epoxybriaranes, including a known compound, juncenolide K (1), as well as a new metabolite, fragilide X (2), have been isolated from gorgonian Junceella fragilis collected off the waters of Taiwan. The absolute configuration of juncenolide K (1) was determined by single-crystal X-ray diffraction analysis for the first time in this study and the structure, including the absolute configuration of briarane 2 was established on the basis of spectroscopic analysis and compared with that of model compound 1. One aspect of the stereochemistry of the known compound 1 was revised. Briarane 2 was found to enhance the generation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) release from RAW 264.7 cells.

juncenolide K 1 2 Figure 1. The structures of juncenolide K and its revised structure (1) and fragilide X (2). Compound 1 was isolated as a colorless prism that showed a sodiated adduct ion [M + Na] + at m/z 513.20949 in the (+)-high-resolution electrospray ionization mass spectrum (HRESIMS) analysis. The result revealed that 1 had a molecular formula of C26H34O9 (calculated for C26H34O9 + Na, 513.20950) (unsaturation degrees = 10). The NMR chemical shifts for 1 and its proton

Results and Discussion
Compound 1 was isolated as a colorless prism that showed a sodiated adduct ion [M + Na] + at m/z 513.20949 in the (+)-high-resolution electrospray ionization mass spectrum (HRESIMS) analysis. The result revealed that 1 had a molecular formula of C 26 H 34 O 9 (calculated for C 26 H 34 O 9 + Na, 513.20950) (unsaturation degrees = 10). The NMR chemical shifts for 1 and its proton coupling data are identical to those reported for juncenolide K [13] (Table 1). Juncenolide K was initially assigned possessing an 11α,20α-epoxy configuration, and the cyclohexane ring was reported to exist with a chair conformation, but on the basis of our study of juncenolide K by a single-crystal X-ray diffraction analysis ( Figure 2) and spectroscopic analysis (Table 1 and Figure 3) (Supplementary Materials, Figures S1-S14), it appears that the 11,20-epoxy group in 1 was found to be 11β,20β-oriented and 1 possesses a cyclohexane ring in twist-boat form. The X-ray structure shows the twist-boat conformation of the cyclohexane ring in 1 and the Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram ( Figure 2) showed that the absolute configurations of the stereogenic centers of 1 are 1S,2S,7S,9S,10S,11S and 14S (Flack parameter x = 0.07(5)).

Results and Discussion
The stereochemistry of 2 was determined by NOE correlations observed in a NOESY experiment ( Figure 4) and possible biogenetic considerations. The NOE correlations of H-10/H-2, H-10/OH-8, H-10/H-9, and H-10/H-20b indicated that these protons are situated on the same face of the structure and were assigned as the α protons since the C-15 methyl is the βsubstituent at C-1. Meanwhile, correlations of H3-15/H-12 and H3-15/H-14 indicated that H-12 and H-14 were β-oriented, and the cyclohexane ring may exhibit a twist-boat conformation. The NOESY spectrum showed a correlation from H-6 to H3-16, revealing the Z geometry of the C-5/6 double bond. H3-18 exhibited correlations to OH-8 and H-9, suggesting the α-orientation of Me-18 at C-17. H-7 displayed a correlation with H-17, which confirmed that these two protons were β-oriented at C-7 and C-17, respectively. As briarane 2 was isolated along with 1 from the same organism, it is reasonable on biogenetic grounds to assume that 2 possessed the same absolute configuration as that of 1. Therefore, the configurations of the stereogenic carbons of 2 should be assigned as 1S,2S,7S,8S,9S,10S,11S,12R,14S, and 17R (Supplementary Materials, Figures S15-S29). The effects of briaranes 1 and 2 on the release of iNOS and COX-2 from lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells were assessed (Table 3 and Figure 5). It is interesting to note that 2 at 10 μM enhanced the release of iNOS and COX-2 to 122.87% and 113.65%, respectively, as compared to results of the cells stimulated with LPS only.        which confirmed that these two protons were β-oriented at C-7 and C-17, respectively. As briarane 2 was isolated along with 1 from the same organism, it is reasonable on biogenetic grounds to assume that 2 possessed the same absolute configuration as that of 1. Therefore, the configurations of the stereogenic carbons of 2 should be assigned as 1S,2S,7S,8S,9S,10S,11S,12R,14S, and 17R (Supplementary Materials, Figures S15-S29).
The effects of briaranes 1 and 2 on the release of iNOS and COX-2 from lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells were assessed (Table 3 and Figure 5). It is interesting to note that 2 at 10 µM enhanced the release of iNOS and COX-2 to 122.87% and 113.65%, respectively, as compared to results of the cells stimulated with LPS only.

General Experimental Procedures
NMR spectra were recorded on a 400 MHz Jeol NMR (model ECZ 400S, Tokyo, Japan) spectrometer using the residual CHCl3 signal (δH 7.26 ppm) and CDCl3 (δC 77.1 ppm) as internal references for 1 H and 13 C NMR, respectively. ESIMS and HRESIMS were obtained from a Bruker mass spectrometer with 7 Tesla magnets (model: SolariX FTMS system, Bremen, Germany). Column chromatography, HPLC, IR spectra, and optical rotation values were performed according to our earlier research [19].

Animal Material
The specimens coral J. fragilis were collected in July 2019 by hand, using self-contained underwater breathing apparatus (SCUBA) off the coast of Orchid Island (Lanyu Island), Taiwan. The samples were stored in a −20 °C freezer until extraction. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium (NMMBA) (voucher no.: NMMBA-TW-GC-2019-017). This organism was identified by comparison with previous descriptions [1 −3].

General Experimental Procedures
NMR spectra were recorded on a 400 MHz Jeol NMR (model ECZ 400S, Tokyo, Japan) spectrometer using the residual CHCl 3 signal (δ H 7.26 ppm) and CDCl 3 (δ C 77.1 ppm) as internal references for 1 H and 13 C NMR, respectively. ESIMS and HRESIMS were obtained from a Bruker mass spectrometer with 7 Tesla magnets (model: SolariX FTMS system, Bremen, Germany). Column chromatography, HPLC, IR spectra, and optical rotation values were performed according to our earlier research [19].

Animal Material
The specimens coral J. fragilis were collected in July 2019 by hand, using self-contained underwater breathing apparatus (SCUBA) off the coast of Orchid Island (Lanyu Island), Taiwan. The samples were stored in a −20 • C freezer until extraction. A voucher specimen was deposited in the National Museum of Marine Biology and Aquarium (NMMBA) (voucher no.: NMMBA-TW-GC-2019-017). This organism was identified by comparison with previous descriptions [1][2][3].

Extraction and Isolation
Sliced bodies (wet/dry weight = 1125 g/588 g) of the coral specimen were prepared and extracted with a mixture of methanol (MeOH) and dichloromethane (CH 2 Cl 2 ) (1:1) to give a crude extract (29.0 g) which was partitioned between ethyl acetate (EtOAc) and H 2 O. The EtOAc extract (17.0 g) was then applied to a silica gel column chromatograph (C.C.) (500 g) and eluted with gradients of hexanes/acetone