Terpenoids from the Soft Coral Sinularia sp. Collected in Yongxing Island

Three new sesquiterpenoids (sinuketal (1), sinulins A and B (2 and 3)) and two new cembranoids (sinulins C and D (4 and 5)), as well as eight known sesquiterpenoids (6–13) and eight known cembranoids (14–21), were isolated from the Xisha soft coral Sinularia sp. Their structures were elucidated by extensive spectroscopic analysis. Compound 1 possesses an unprecedented isopropyl-branched bicyclo [6.3.0] undecane carbon skeleton with unique endoperoxide moiety, and a plausible biosynthetic pathway of it was postulated. According to the reported biological properties of endoperoxide, the antimalarial, cytotoxic, antiviral, and target inhibitory activities of 1 were tested. Compound 1 showed mild in vitro antimalarial activity against Plasmodium falciparum 3D7, weak cytotoxic activities toward Jurkat, MDA-MB-231, and U2OS cell lines, inhibitory effects against influenza A viruses H1N1 and PR8, as well as mild target inhibitory activity against acetylcholinesterase. The other compounds were evaluated for cytotoxicities against HeLa, HCT-116, and A549 tumor cell lines and target inhibitory activities against protein tyrosine phosphatase 1B (PTP1B). Compound 20 exhibited cytotoxicities against HeLa and HCT-116, and compounds 5, 11, and 15 showed mild target inhibitory activities against PTP1B.


Results and Discussion
Sinuketal (1) was obtained as a light-yellow oil. The molecular formula was determined as C 15 (Table 1) showed the presence of two methyl doublets (δ H 0.89 (3H, d, J = 6.7 Hz) and 0.95 (3H, d, J = 6.7 Hz)), an AX coupling system (δ H 2.28 (1H, d, J = 13.4 Hz) and 3.14 (1H, d, J = 13.4 Hz)), and two terminal olefinic protons (δ H 4.90 (1H, brs) and 4.93 (1H, brs)). Its 13 C NMR and DEPT spectroscopic data (Table 1) exhibited a total of 15 carbon resonances, including 2 methyls, 6 sp 3 methylenes, 3 sp 3 methines, 2 sp 3 oxygenated unprotonated carbons (including an anomeric carbon δ C 107.4 (C)), and 1 terminal double bond (δ C 113.0 (CH 2 ) and 149.4 (C)), accounting for one degree of unsaturation. The above NMR signals suggested the structure of a tricyclic sesquiterpenoid. All the connections of H and protonated C were assigned by detailed analysis of heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectra.  -14))/H-10/H 2 -9 ( Figure 2). This was further supported by HMBC correlations from H 2 -7 to C-6 and from H 3 -14 to C-13, C-12 and C-11, from H-11 to C-1, C-9 and C-10, and from H 2 -1 to C-2 ( Figure 2). HMBC correlations from H-1a and H 2 -2 to C-3 and from H-9α and H-10 to C-3 indicated the presence of a cyclopentane moiety. HMBC correlations from H 2 -15 to C-9 and C-7, from H 2 -7 to C-8, from H-6α and H 2 -7 to C-5, from H-6β to C-4, from H-4β to C-3, C-5 and C-10, and from H-4α to C-6 implied the cyclooctane moiety. Consequently, a 5/8 fused bicyclic system was established, which was further supported by HMBC correlations from H-4β to C-2 and from H-11 to C-9. Considering the last degree of unsaturation according to molecular formula, an AX coupling methylene, and downfield chemical shifts of oxygenated C-3 (δ 98.0) and anomeric C-5 (δ 107.4), a (C-3)-O-O-(C-5)-OH moiety should be present. Thus, the planar structure of 1 was established as shown in Figure 2.  The relative configuration of 1 was established by nuclear Overhauser effect spectroscopy (NOESY) experiment ( Figure 3) in combination with conformational analysis, and density functional theory-NMR (DFT-NMR). A trans configuration for H-11 and H-10 in the cyclopentane ring was deduced from the NOE correlations of H-10 with H-12 and H 3 -13, which was consistent with the absence NOE effect of H-10 with H-11. The endoperoxide group should be on the same side of the cyclooctane ring, owing to unfavorable distortions on the basis of molecular model analysis. NOE correlations of H-11 with H-9α and of H-9α with H-4α indicated cis-fused bicyclo [6.3.0] undecane. The above evidence suggested two stereochemical candidates of 1 (1a: 3R,5R,10S,11S; 1b: 3S,5S,10R,11R). In order to gain further support for determination of the relative configuration of 1, 13 C NMR chemical shifts were calculated by the Gaussian 09 program package. The stable conformations of all the eight isomers of 1 were calculated by DFT gauge-including atomic orbitals (GIAO) model at RB3LYP/6-31+G(2d,p) level [11]. Tables S3 and S4 show the experimental and calculated 13 C chemical shifts (relative to TMS-resonance calculated at the same level), as well as the calculated data after linear regression scaling. As a result, the calculation data of 1a ( Figure 4a) and its enantiomer 1b gives the most reasonable correlation coefficient R 2 value of 0.997, with mean absolute error 1.5 ppm and maximum absolute error 5.4 ppm (at C-15). Thus, the GIAO-based 13 C NMR chemical shifts calculation data strongly supported the assigned carbon and relative configuration of 1. Therefore, the relative configuration of 1 was unambiguously assigned as 3R*,5R*,10S*,11S*.  The absolute configuration of 1 was determined by time-dependent density functional theory calculation of electronic circular dichroism (TDDFT/ECD) method with the basis set RB3LYP/DGDZVP [12][13][14]. The measured ECD spectrum of 1 in methanol exhibited a negative Cotton effect (CE) at 238 nm, matching well with the calculated ECD spectra for 1a ( Figure 4b). Therefore, the absolute configuration of (−)-1 was established as 3R,5R,10S,11S.

Animal Material
All collections of the soft coral Sinularia sp. were carried out in Yongxing Island (16 •

Computational Section
The quantum chemical calculations were carried out by Gaussian 09 [35] software (Gaussian Inc. Wallingford, CT, USA) using the density functional theory (DFT). The initial stereochemical structures were built with Spartan 10 software and first minimized based on molecular mechanics calculations. Then, conformational search was performed by Spartan 10 software using MMFF force filed, and conformers occurring within a 10 kcal/mol energy window from the global minimum were chosen for geometry optimization in the gas phase with the DFT method at the B3LYP/DGDZVP level. The B3LYP/DGDZVP harmonic vibrational frequencies were further calculated to confirm their stability. The stable conformations were calculated by DFT GIAO model at RB3LYP/6-31+G(2d,p) level to calculate 13 C NMR chemical shifts. In the ECD calculations, the spin-allowed excitation energies and rotatory (Rn) and oscillator strengths (f n) of the lowest excited states of stable conformers were calculated using TD-DFT method with the basis set RB3LYP/DGDZVP. Solvent effects of methanol solution were evaluated at the same DFT level by using the SCRF/PCM method in agreement with the experiment condition. Electronic transitions were expanded as Gaussian curves with a full width at half maximum (FWHM) for each peak of 0.32 eV. The ECD spectra were combined after Boltzmann weighting according to their population contribution.

Immunosuppressive Activity
The determination of immunosuppressive activity of compound 1 on interleukin 2 (IL-2) secretion in Jurkat T-cells was performed as the literature reported [37], using DMSO and FK506 (Tacrolimus) as negative and positive controls, respectively.

Cytotoxic Activities
The cytotoxicities against Jurkat, MDA-MB-231, U2OS, HeLa, HCT-116, and A549 cell lines were assayed by MTT method [38] with Adriamycin as a positive control. All the cell lines were purchased from Shanghai Institute of Cell Biology (Shanghai, China).

Antiviral Activities
The antiviral activities against influenza A virus (H1N1) and (PR8) were evaluated by the CPE inhibition assay as the literature reported [39]. Ribavirin was used as positive control, and compounds with an inhibition rate of >70%, >50%, and <30% at 50 µg/mL were respectively regarded having strong, moderate, and weak activities.

Target Inhibitory Activities
Evaluation of the inhibitory effects on acetylcholinesterase was performed by the spectrophotometric method developed by Ellman with slight modifications as reported [40], and on c-Met and PTP1B by using the enzyme-linked immunosorbent assay (ELISA) methodology [41].