A new capnosane-type diterpenoid from the South China sea soft coral Lobophytum pauciflorum

Abstract A new rare capnosane-type diterpenoid (1) along with two known compounds sarcophyolides D (2) and arbolide C (3), were isolated from the soft coral Lobophytum pauciflorum collected at Xisha Islands in the South China Sea. The structure of 1 was established based on comprehensive spectroscopic data, literature comparison, and quantum chemical calculations. Compound 1 exhibited moderate anti-inflammatory activities in the zebrafish model at a concentration of 20 μM. GRAPHICAL ABSTRACT


Introduction
Soft coral of the genus Lobophytum is widely distributed in the sea around the world with abundant and structurally specific secondary metabolites such as cembrane diterpenes (Yin et al. 2021), lobane diterpenes (Chang et al. 2020), capnosane diterpenes (Li et al. 2020), and steroids (Huang et al. 2018).The rare capnosane-type diterpenoids produced by the genus Lobophytum were first reported in 2019 and were considered to be formed by transannular cyclisation at C-3 and C-7 of the classical cembrane diterpenoids, which possess a 14-membered carbocyclic backbone (Zhang et al. 2019).
As a continuing project on searching for novel and bioactive marine natural products from soft corals, Lobophytum pauciflorum was collected at Xisha Islands in the South China Sea, leading to the isolation of a new capnosane-type diterpenoid (1) and two known compounds sarcophyolides D (2) (Xi et al. 2013) and arbolide C (3) ( Wang et al., 2014) (Figure 1).Herein, we report the isolation, structure elucidation, and bioactivities of the isolates.
The Z-configuration of C-1/C-2 was confirmed by the NOESY correlation of H-2/CH 3 -16.The J H-2/H-3 (10.8 Hz) value suggested that H-2 and H-3 were trans-axial, which indicated that the double bond was approximately perpendicular to the cyclopentane ring.The NOESY correlation between H-3 and H 3 -18 and the absence of correlation between H-3 and H-7 indicated that H-3 and H 3 -18 were in b-oriented position, while H-7 was in a-oriented position.The NOESY correlations of H-6a/H-9a, H 3 -19 with H-6b/ H-7/H-11, and H-9b with H-3 indicated that the relative configuration of C-8 and C-11 were S Ã and R Ã , respectively (Figure S2).The S Ã -configuration of C-12 was determined by the NOESY correlations of H-11 with H-13/H 3 -20 and H 3 -20 with H-10/H-13 (Figure S2).
Moreover, the NOESY correlations of H-14 with H-3/H-13 suggested the S Ã -configuration of C-14.The R Ã -configuration of C-13 was deduced by the NOESY correlation (Figure S3) of H-13 with H-11/H-14/H-15/H 3 -16/H 3 -20 together with 3 D molecular models and DP4þ probability analysis.S3) were used for DP4þ probability analysis.The results showed that 1a was identified as the correct one by DP4þ analysis with 100% probability (Table S5).Thus, the relative conformation of 1 was determined.Finally, the experimental ECD spectrum of 1 matched reasonably well with the calculated one for 3S4R7R8R11S12R13S14R, which confirmed the absolute configuration of 1 (Figure S4).
All the compounds were evaluated for their anti-inflammatory activities in classic transgenic fluorescent zebrafish models induced by CuSO 4 .The results showed that compound 1 exhibited moderate activity, with an inhibition rate of 37.37% at a concentration of 20 lM (Figure S5).

General experimental procedures
NMR spectra were recorded on a Bruker AVANCE NEO 400 ( 1 H, 400 MHz), an Agilent DD2-500 ( 1 H, 500 MHz; 13 C, 125 MHz), or a JEOL JNM-ECP600 ( 1 H, 600 MHz; 13 C, 150 MHz) spectrometer.Chemical shifts were reported in parts per million (ppm) with coupling constants (J) in Hertz relative to the solvent peaks; d H 7.16 and d C 128.06 for C 6 D 6 ; d H 7.26 and d C 77.16 for CD 3 Cl.HRESIMS data were surveyed on a Micromass Q-Tof Ultima GLOBAL GAA076 LC mass spectrometer.UV and ECD spectra were taken on a Jasco J-810 spectropolarimeter.Optical rotations were recorded on a Jasco P-1020 polarimeter.HPLC was performed on a Waters 2695/2998 instrument with a DAD detector, equipped with an analytic reverse-phase column (ZORBAX 80 A Extend-C18, 250 Â 4.6 mm, 5 mm, 1.0 mL/min).Silica gel (300-400 mesh, Qingdao) was employed for column chromatography (CC), and precoated silica gel plates (GF254, Qingdao) were used for TLC.

Animal materials
The soft coral Lobophytum pauciflorum was collected from Yongle Islands of Xisha Islands of the South China Sea in May 2012.The sample was identified by Pingjyun Sung, National Museum of Marine Biology and Aquarium (NMMBA).The voucher specimen (No.XS-2012-27), frozen at À20 C, was deposited at the School of Medicine and Pharmacy, Ocean University of China, P. R. China.

Zebrafish maintenance
Adult zebrafish were cultivated by the Qilu University of Technology (Jinan, China).Transgenic zebrafish (Tg: zlyz-EGFP) expressing enhanced green fluorescent protein (EGFP) was used in this article.The conditions of the maintenance complied with guidelines of the Organization for Economic Co-operation and Development (OECD).The zebrafish were maintained under a 14/10 h light/dark cycle at the temperature (28 ± 0.5 C) in a closed flow-through system with charcoal-filtered tap water to ensure normal spawning.

CuSo 4 -induced model of zebrafish
Like literature reference (Gui et al. 2020), healthy zebrafish larvae were selected into 24-well plates (n ¼ 10/well) in a 2 mL final volume of embryo medium at 3 dpf and divided into five groups: a control group (fresh fish water), a model group: 40 lM CuSO 4 , a positive drug group: 50 lM indomethacin (Solarbio, China) and drug groups: 20 lM CuSO 4 (Sigma-Aldrich, St. Louis, MO, USA) was added to the drug groups and incubated for 1 h after treatment with different compounds for 2 h.All treatments were performed in triplicate.Each zebrafish larva was photographed by a fluorescence microscope (AXIO, Zoom.V16), and the number of macrophages was counted by using the Image-Pro Plus software.

Quantum chemical calculations
Conformational analyses were carried out in the MM2 force field by the xtb software.The resulting conformers were optimised using DFT at the b3lyp/6-31 þ g Ã level in the methanol by the GAUSSIAN 09 program.The optimised conformations, whose Boltzmann distributions of Gibbs free energies were more than 1.0%, were used for the ECD calculations using the TD-DFT method with the basis set cam-b3lyp/6-311g ÃÃ or the NMR calculations using the GIAO method at the mpw1pw91/6-31 þ g ÃÃ .

Conclusion
In summary, a rare capnosane-type diterpenoid (1) together with two known compounds sarcophyolides D (2) and arbolide C (3) were isolated from the soft coral Lobophytum pauciflorum collected at Yongle Islands of Xisha Islands of the South China Sea.The stereochemistry of 1 was determined by spectroscopic analysis, comparison with those reported in the literature, and quantum chemical calculations.Moreover, compound 1 exhibited a moderate anti-inflammatory effect in zebrafish with an inhibition rate of 37.37% at a concentration of 20 lM.

Disclosure statement
No potential conflict of interest was reported by the authors.