Four new cembranoids from the South China Sea soft coral Sarcophyton trocheliophorum

Abstract Four new cembrane diterpenes, 1,13-di-epi-13-acetoxy launine P (1), 13-oxo-thunbergol (2), isocrassumol B (3) and 7α, 8α-sarcophine (4), together with two known launine P (5) and sarcophytonin B (6), were isolated from the extract of the South China Sea soft coral Sarcophyton trocheliophorum. The structures were elucidated by spectroscopic analysis, ECD analysis and comparison with literature data. All compounds were tested for their antimicrobial activity and antiviral activity. Compounds 1, 2 and 5 were found to exhibit weak antibacterial activity, while 6 showed strong inhibition activity against Staphylococcus aureus and Bacillus subtilis (MIC＜0.5 µg/mL). Compounds 2 showed moderate inhibitory effect against influenza A viruses H1N1 with IC50 of 17.8 μM. Graphical Abstract

In the course of a continuous search for bioactive secondary metabolites from the South China Sea soft corals Liang et al. 2017), the soft coral Sarcophyton trocheliophorum was collected from Xisha Island (10 m), the South China Sea. Our chemical investigation of the specimen resulted in the isolation of six cembrane-based diterpenes, including four new cembranoids (1-4) and two known related compounds (5 and 6) ( Figure 1). Herein, we report the isolation, structural elucidation, and biological evaluation of these compounds.

Results and discussion
1,13-Di-epi-13-acetoxy launine P (1) was isolated as a colorless oil. [  carbon (d C 76.9), three sp 3 methines, five sp 3 methylenes and six methyls were included ( Figures S3-S7). Comparison of the 13 C NMR data of 1 with those of known compound launine P (5) (Yang et al. 2017) revealed that they should possess the same carbon skeleton except for the acetylation of 13-OH, which was further established by the HMBC correlations from CH-13 to 13-OAc ( Figures S4-S8). The E stereochemistry of the double bond between C-2 and C-3 was determined by the value of the coupling constant J 2, 3 ¼ 15.8 Hz. The geometry of the C-7 À C-8 and C-11 À C-12 double bonds were determined to be E on the basis of the absence NOE ( Figures  S2-S9) correlations of CH-7/CH 3 -19 and CH-11/CH 3 -20. The relative stereochemistry was elucidated from the NOE correlations. The crucial NOE correlations between CH-1/ CH-13, 4-OCH 3 /CH 3 -16 and 4-OCH 3 /CH 3 -17 demonstrated the relative configuration of 1 as 1 R Ã , 4 R Ã , 13S Ã . According to the relative configuration established for 1, 1 R, 4 R, 13S configuration (1a) and 1S, 4S, 13 R configuration (1 b) were used as the model compounds for ECD calculations ( Figure S10). The experimental ECD spectrum of 1 was in good agreement with the calculated ECD spectrum for the 1 R, 4 R, 13S configuration (1a). Hence, the configuration of 1 was unambiguously assigned as shown. A small amount of the soft coral were extracted with chromatogram class methanol. We analyzed it with LC-MS, and extracted the ion peak [M þ Na] þ of 1 ( Figure S48), proving that compound 1 was not an artefact.
13-Oxo-thunbergol (2) was also a colorless oil. , 5.08 (1H, t, J ¼ 6.9 Hz) and 6.56 (1H, t, J ¼ 6.9 Hz] were observed ( Figure S13). The 13 C NMR and DEPT data of 2 revealed that 20 carbon signals attributed to four quaternary carbons (two olefinic carbons at dc 132.2 and 136.8, one carbonyl carbon at dc 202.9), six methines (four olefinic carbons at dc 128.2, 137.2, 128.4 and 143.6), five methylenes and five methyls (Table S3, Figure  S14 and S15). Detailed analysis of 1 H-1 H COSY spectrum of 2 revealed the protons connectivity of the following four structural units: from CH 2 -5 to CH-7 through CH 2 -6, from CH 2 -10 to CH-11, from CH 2 -14 to CH-2 through CH-1, and from CH 3 -16 to CH 3 -17 through CH-15 ( Figure S2 and S16). These fragments connection were proved by HMBC correlations ( Figure S2 and S18). In addition, the ketone group was assigned to C-13 according to HMBC correlations from CH-11 to C-13, from CH 2 -14 to C-13 and from CH 3 -20 to C-13. As a result, the planar structure of 2 was established as a derivative of thunbergol (Carmely et al. 1981) . The E stereochemistry of the double bond C-2 À C-3 was also determined by the value of the coupling constant (J 2, 3 ¼ 15.8 Hz). The geometry of the C-7 À C-8 and C-11 À C-12 double bonds were determined to be E as compound 1. The relative stereochemistry of 2 was established also on the basis of NOESY data. In the NOESY spectrum, CH 3 -18 correlated to CH 3 -16 and CH 3 -17, indicating that CH 3 -18 had an orientation different from CH-1 ( Figure S19). ECD calculations were performed ( Figure S21) to determine the absolute configuration of compound 2 as 1S, 4 R.
Isocrassumol B (3) was obtained as a colorless oil.
Two known compounds were also isolated from the Sarcophyton trocheliophorum extract and identified as launine P (5) (Yang et al. 2017) and sarcophytonin B (6) (Kobayashi and Hirase 1990), by comparison of their 1 H and 13 C NMR and MS spectroscopic data with those reported in the literature.
Compounds 1-6 were tested for their antibacterial activities, against five pathogenic bacteria, Staphylococcus aureus CMCC (B) 26003, methicillin-resistant Staphylococcus aureus (MRSA) ATCC43300, Bacillus subtilis CMCC (B) 63501, Pseudomonas aeruginosa CMCC (B) 10104 and Salmonella paratyphi CMCC (B) 50071. (Table S1). Compounds 6 showed a strong inhibition against Staphylococcus aureus and Bacillus subtilis. While compounds 1, 2 and 5 showed a weak antimicrobial activity, penicillin G as a positive control. For all compounds, no activity was observed in the other three tested bacteria. Moreover, all the isolated compounds were evaluated in vitro antiviral activity against influenza A virus H1N1 (Table S1). Compounds 2 and 5 showed anti-H1N1 virus activity with 96.2% and 89.4% inhibition respectively at 30 mM (acyclovir, 97.0% inhibition). Compounds 2 showed moderate inhibitory effect with IC 50 of 17.8 lM.

General experimental procedures
Optical rotations acquired with a Jasco P-1010 digital polarimeter. IR and UV spectra were obtained with a Thermo Scientific Nicolet iS5 FT-IR spectrometer and a Thermo Scientific Evolution 201 spectrophotometer, respectively. The 1 H and 13 C NMR spectra were recorded in CDCl 3 with a Bruker AV-600 spectrometer. HRESIMS data were conducted with an Agilent 6230 TOF LC/MS spectrometer. Medium-pressure liquid chromatography (MPLC) was performed using a Bonna-Agela FLEXA purification instrument. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd.) and ODS (50 mm, YMC, Tokyo, Japan) were used for column chromatography. TLC analysis was carried out using silica gel GF 254 (Qingdao Marine Chemistry Co. Ltd., Qingdao, China). Reversed-phase (RP) HPLC was performed on a Waters 1525 binary HPLC pump equipped with a Waters 2996 photodiode detector and a semipreparative ODS-MS-II column(250 Â 10 mm, 5 lm).

Coral material
Soft coral Sarcophyton trocheliophorum (No.XSSC201908) were collected from the coast of Xisha Island in the South China Sea by scuba diving, at the depth of 10 m, and were frozen immediately after collection.

Antiviral activities
The antiviral activities against influenza A virus (H1N1) was evaluated as the literature reported (Grassauer et al. 2008). Acyclovir was used as positive control.

Conclusion
The present study on the soft coral S. trocheliophorum led to the isolation of four new cembranoids (1-4) and two related known cembranoids (5, 6). The structure was elucidated by NMR spectra, ECD analysis and compared with the literature data. All isolated compounds were evaluated for their antibacterial and antiviral activities. Compounds 1, 2 and 5 were found to exhibit weak antibacterial activity, while 6 showed strong inhibition activity against Staphylococcus aureus and Bacillus subtilis (MIC＜0.5 lg/mL). Compounds 2 showed moderate inhibitory effect against influenza A viruses H1N1 with IC 50 of 17.8 lM.