Sarcoeleganolides C–G, Five New Cembranes from the South China Sea Soft Coral Sarcophyton elegans

Five new cembranes, named sarcoeleganolides C–G (1–5), along with three known analogs (6–8) were isolated from soft coral Sarcophyton elegans collected from the Yagong Island, South China Sea. Their structures and absolute configurations were determined by extensive spectroscopic analysis, QM-NMR, and TDDFT-ECD calculations. In addition, compound 3 exhibited better anti-inflammation activity compared to the indomethacin as a positive control in zebrafish at 20 μM.


Results
Sarcoeleganolide C (1), which was isolated as a colorless oil, gave a mula of C20H28O3 by its HREIMS ion peak at m/z 317.2114 [M + H] + , implying of unsaturation. The 1D NMR data (Table 1) and HSQC spectrum of 1 rev ence of 20 carbons belonging to four methyls (three olefinic, and one sp 3 h methylenes (all sp 3 hybridized), four methines (two olefinic and two oxyge quaternary carbons (four olefinic, one sp 3 hybridize, and one carbonyl). T cate that compound 1 was a cembrane-type diterpenoid.

Results
Sarcoeleganolide C (1), which was isolated as a colorless oil, gave a molecular formula of C 20 H 28 O 3 by its HRESIMS ion peak at m/z 317.2114 [M + H] + , implying seven degrees of unsaturation. The 1D NMR data (Table 1) and HSQC spectrum of 1 revealed the presence of 20 carbons belonging to four methyls (three olefinic, and one sp 3 hybridized), six methylenes (all sp 3 hybridized), four methines (two olefinic and two oxygenated), and six quaternary carbons (four olefinic, one sp 3 hybridize, and one carbonyl). These data indicate that compound 1 was a cembrane-type diterpenoid. The planar framework of 1 was elucidated by 1 H-1 H COSY and HMBC spectra (Figure 2). Four spin systems were established by the 1 H-1 H COSY correlations from H-2 to H-3; H-5 to H-7; H-9 to H-11, and H-13 to H-14. As previously reported, 3, 4-epoxycembranolides [21,22], a trisubstituted epoxide ring located at C-3 and C-4, were deduced by the downfield chemical shift of C-3 (δ C 61.5) and C-4 (δ C 61.5) and HMBC correlations from H 3 -18 to C-3, C-4, and C-5. Based on the above data, together with the key HMBC correlation from H 3 -19 to C-7, C-8, and C-9; H 3 -20 to C-11, C-12, and C-13; H 3 -17 to C-1, C-15, and C-16; H-14a (δ H 2.74) to C-1, C-2, and C-15 the connection of the carbon skeleton was permitted. Thus, compound 1 was deduced as a cembranoid possessing a trisubstituted epoxide. In the NOESY spectrum of 1 (Figure 3), the correlations of H 3 -19/H-6a (δ H 2.20), H 3 -20/H-10a (δ H 2.26) indicate that the ∆ 7 and ∆ 11 double bonds could be of an E-configuration. The NOESY correlation of H-2/H 3 -18 indicates that these protons were on the same side. In addition, considering the geometry of the 3-(E)-olefin in co-isolates, the epoxide of 1 should be in an anti-relationship between H-3 and H 3 -18, which was further confirmed by the 13 C NMR chemical shift calculation for the DP4 + calculations (Supplementary Materials, Figures S1 and S2) [23]. Finally, the absolute configurations of 1 were defined as 2S, 3R, and 4R by TDDFT-ECD calculations ( Figure 4). H3-20/H-10a (δH 2.26) indicate that the Δ 7 and Δ 11 double bonds could be of an E-configuration. The NOESY correlation of H-2/H-18 indicates that these protons were on the same side. In addition, considering the geometry of the 3-(E)-olefin in co-isolates, the epoxide of 1 should be in an anti-relationship between H-3 and H3-18, which was further confirmed by the 13 C NMR chemical shift calculation for the DP4 + calculations (Supplementary Materials, Figures S1 and S2) [23]. Finally, the absolute configurations of 1 were defined as 2S, 3R, and 4R by TDDFT-ECD calculations ( Figure 4).     Sarcoeleganolide D (2), a colorless oil, had a molecular formula of C21H30O4 on the basis of its HREIMS ion peak at m/z 347.2221 [M + H] + , requiring seven degrees of unsaturation. The 1 H and 13 C NMR data of 2 ( Table 1) resemble that of (−)-sartrochine (7), a known cembranoid previously isolated from the soft coral Sarcophyton trochliphroum. In fact, the structure of 2 was truly similar to 7, with the exception of a methoxyl at C-2 in 2 instead of the proton in 7. This deduction was further proven by the HMBC correlation ( Figure 2) from the H3-21 (δH 3.14) to C-2, along with the significant downfield shift observed for C-2 (δC 108.3). Then, the relative configurations of 2 were deduced on the basis of the NOESY experiment (   Table 1) resemble that of (−)-sartrochine (7), a known cembranoid previously isolated from the soft coral Sarcophyton trochliphroum. In fact, the structure of 2 was truly similar to 7, with the exception of a methoxyl at C-2 in 2 instead of the proton in 7. This deduction was further proven by the HMBC correlation ( Figure 2) from the H 3 -21 (δ H 3.14) to C-2, along with the significant downfield shift observed for C-2 (δ C 108.3). Then, the relative configurations of 2 were deduced on the basis of the NOESY experiment (  (Table 1) of 3 and 6 indicate similarities between them. The 2D NMR data of 3 ( Figure S3 and Figure 2) indicate the plane structure was identical to 6, suggesting that 3 should be a stereoisomer of 6. The relative configurations of 3 were deduced by the NOESY spectrum (Figure 3). By the NOESY correlation of H 3 -19/H-7, the geometry of the ∆ 7 double bonds was assigned to be a Z-configuration, which was further confirmed by the downfield chemical shift of C-19 (δ C 22.4), revealing the major difference in configurations between 3 and 6. The  (Table 1) and HSQC spectrum confirm the presence of 21 carbons, five methyls (three olefinic, one oxygenated, and one sp 3 hybridized), five methylenes (all sp 3 hybridized), four methines (three olefinic and one oxygenated), and seven quaternary carbons (five olefinic, one oxygenated, and one carbonyl). By analysis of these data above, compound 4 was speculated to be a cembrane nucleus.
Sarcoeleganolide G (5) was isolated as a colorless oil with a molecular formula of C 20 H 28 O 3 , established by the HRESIMS ion peak at m/z 339.1928 [M + Na] + . A survey of the literature revealed that the 1D NMR data of compound 5 (Table 1) were similar to those of compound 8, a known cembrane diterpenoid isolated from the Red Sea soft coral Sarcophyton glaucum. In fact, compound 5 had the same functional groups as 8, except for the migration of the ∆ 8 double bonds in 8 to the ∆ 12 double bonds in 5, and the hydroxy group at the C-7 position in 8 to C-11 in 5. These variations of the functional groups were further proven by the HMBC correlations from H 3 -20 to C-11, C-12, and C-13, and from H 3 -19 to C-7, C-8 and C-9. Furthermore, other detailed HMBC correlations and 1 H-1 H COSY correlations helped complete the planar framework of 5 ( Figure 2). In the NOESY spectrum of 5 (Figure 3), the correlations of H-3/H-5a (δ H 2.20), H-2/H 3 -18, and H-7/H 2 -9 (δ H 2.03) indicate that the geometries of ∆ 3 and ∆ 7 double bonds were of an E-configuration. By the NOESY correlations of H-2/H-14a (δ H 2.26) and H-11/H-14a (δ H 2.26), the relative configurations were defined as 2S* and 11S*. Finally, the absolute configurations of 5 were defined by TDDFT-ECD calculations (Figure 4).
Although the anti-inflammatory activity of cembranoids in zebrafish models has been reported previously [24], it is still not very common. Hence, we aimed to seek newer cembranoids with anti-inflammatory activity in zebrafish models. These new compounds (1)(2)(3)(4)(5) were evaluated for anti-inflammatory activity in CuSO4-induced transgenic fluorescent zebrafish. CuSO 4 can produce an intense acute inflammatory response in the neuromast and mechanosensorial cells in the lateral line of zebrafish, stimulating the infiltration of macrophages [25][26][27]. Then the number of macrophages surrounding the neuromast in the zebrafish was observed and imaged under a fluorescence microscope (Supplementary Materials, Section 3). The results are shown in Figure 5. In CuSO 4 -induced transgenic fluorescent zebrafish, compound 3 could alleviate migration and decreased the number of macrophages surrounding the neuromast in the zebrafish, showing stronger anti-inflammatory activity than the indomethacin, which was used as the positive control at 20 µM, while other compounds showed no anti-inflammatory activity, as shown in Figure 5.
of macrophages [25][26][27]. Then the number of macrophages surrounding the neuromast in the zebrafish was observed and imaged under a fluorescence microscope ( Supplementary  Materials, Section 3). The results are shown in Figure 5. In CuSO4-induced transgenic fluorescent zebrafish, compound 3 could alleviate migration and decreased the number of macrophages surrounding the neuromast in the zebrafish, showing stronger anti-inflammatory activity than the indomethacin, which was used as the positive control at 20 μM, while other compounds showed no anti-inflammatory activity, as shown in Figure 5.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). The UV spectra were recorded on a Beckman DU640 spectrophotometer (Beckman Ltd., Shanghai, China). The CD spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan). The NMR spectra were measured by Agilent 500 MHz (Agilent, Beijing, China), JEOL JNMECP 600 spectrometers (JEOL, Beijing, China). The 7.26 ppm and 77.16 ppm resonances of CDCl3 were used as internal references for the 1 H and 13 C NMR spectra, respectively. The 7.16 ppm and 128.06 ppm resonances of C6D6 were used as internal references for the 1 H and 13 C NMR spectra, respectively. The HRESIMS spectra were measured on Micromass Q-Tof Ultima GLOBAL GAA076LC mass spectrometers (Autospec-Ultima-TOF, Waters, Shanghai, China). Semi-preparative HPLC was performed using a Waters 1525 pump (Waters, Singapore) equipped with a 2998 photodiode array detector and a YMC C18 column (YMC, 10 × 250 mm, 5 μm). Silica gel (200-300 mesh, 300-400 mesh, and silica gel H, Qingdao Marine Chemical Factory, Qingdao, China) was used for column chromatography.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). The UV spectra were recorded on a Beckman DU640 spectrophotometer (Beckman Ltd., Shanghai, China). The CD spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan). The NMR spectra were measured by Agilent 500 MHz (Agilent, Beijing, China), JEOL JNMECP 600 spectrometers (JEOL, Beijing, China). The 7.26 ppm and 77.16 ppm resonances of CDCl 3 were used as internal references for the 1 H and 13 C NMR spectra, respectively. The 7.16 ppm and 128.06 ppm resonances of C 6 D 6 were used as internal references for the 1 H and 13 C NMR spectra, respectively. The HRESIMS spectra were measured on Micromass Q-Tof Ultima GLOBAL GAA076LC mass spectrometers (Autospec-Ultima-TOF, Waters, Shanghai, China). Semi-preparative HPLC was performed using a Waters 1525 pump (Waters, Singapore) equipped with a 2998 photodiode array detector and a YMC C18 column (YMC, 10 × 250 mm, 5 µm). Silica gel (200-300 mesh, 300-400 mesh, and silica gel H, Qingdao Marine Chemical Factory, Qingdao, China) was used for column chromatography.

Animal Material
The soft coral Sarcophyton elegans was collected from Xisha Island (YaGong Island) in the South China Sea in 2018 and frozen immediately after collection.

Extraction and Isolation
A frozen specimen of Sarcophyton elegans (7.2 kg, wet weight) was homogenized and then exhaustively extracted with CH 3 OH six times (3 days each time) at room temperature. The combined solutions were concentrated in vacuo and were then subsequently desalted by redissolving with CH 3 OH to yield a residue (178.0 g). The crude extract was subjected to silica gel vacuum column chromatography eluted with a gradient of petroleum/acetone (400:1-1:1, v/v) and subsequently eluted with a gradient of CH 2 Cl 2 /MeOH (20:1-1:1, v/v) to obtain fourteen fractions (Frs.1-Frs.14). Each fraction was detected by TLC. Frs.5 was subjected to a silica gel vacuum column chromatography (petroleum/acetone, from 100:1 to 1:1, v/v) to give three subfractions Frs.5.1-Frs. 5

Anti-Inflammatory Activity Assay
Healthy macrophage fluorescent transgenic zebrafish (Tg: zlyz-EGFP) was provided by the Biology Institute of the Shandong Academy of Science (Jinan, China). Zebrafish maintenance and the anti-inflammation assay were carried out as previously described [26]. Each zebrafish larva was photographed by a fluorescence microscope (AXIO, Zom.V16), and the number of macrophages around the nerve mound was calculated using Image-Pro Plus 6.0 software (Rockville, MD, USA) [28]. One-way analysis of variance was performed using GraphPad Prism 7.00 software (San Diego, CA, USA) [29]. Sarcoeleganolides C-G (1-5) were tested for anti-inflammatory activities with zebrafish models. Three days postfertilization (dpf) healthy macrophage fluorescent transgenic zebrafish were used as animal models to evaluate the anti-inflammatory effects of 1-5.
Author Contributions: X.T., G.L., and P.L. designed the experiments. C.W. performed the experiments, isolated the compounds, and analyzed spectral data. J.Z., X.S., and K.L. prepared the Supplementary Materials. F.L. performed the anti-inflammatory assay. C.W. wrote the paper. All authors have read and agreed to the published version of the manuscript.