Metabolites from the Paracel Islands Soft Coral Sinularia cf. molesta

Five new oxygenated sesquiterpenes, molestins A–D (1, 3–5) and epi-gibberodione (2), three new cyclopentenone derivatives, ent-sinulolides C, D, and F ((+)-9–(+)-11), one new butenolide derivative, ent-sinulolide H ((+)-13), and one new cembranolide, molestin E (14), together with 14 known related metabolites (6–8, (–)-9–(–)-11, (±)-12, (–)-13, 15–19) were isolated from the Paracel Islands soft coral Sinularia cf. molesta. The structures and absolute configurations were elucidated based on comprehensive spectroscopic analyses, quantum chemical calculations, and comparison with the literature data. Compound 5 is the first example of a norsesquiterpene with a de-isopropyl guaiane skeleton isolated from the genus Sinularia. Molestin E (14) exhibited cytotoxicities against HeLa and HCT-116 cell lines with IC50 values of 5.26 and 8.37 μM, respectively. Compounds 4, 5, and 8 showed significant inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 218, 344, and 1.24 μM, respectively.

Considering the reported cytotoxic activities of some sesquiterpenes [30], cyclopentenone derivatives [28], and cembranoid diterpenes [36], all the compounds were evaluated for their cytotoxic activities against HeLa (human cervical carcinoma), HCT-116 (human colon carcinoma), BEL-7402 (human hepatocellular carcinoma), K562 (human leukemia), and Jurkat (human acute leukemia T) tumor cell lines. Only three cembranoid diterpenes showed valuable cytotoxicities against the selected cell lines (IC 50 < 10 µM). Molestin E (14) exhibited cytotoxicities against HeLa and HCT-116 cell lines with IC 50 values of 5.26 and 8.37 µM, respectively. Compounds 17 and 18 showed cytotoxicities against HeLa cell lines with IC 50 values of 6.66 and 6.10 µM, respectively. Considering some guaiane-type sesquiterpenes and furanoterpenoids previously reported showed inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) [37][38][39], a major negative regulator in insulin signaling pathways [40]. Inhibiting PTP1B activity could increase insulin sensitivity and is expected to be a potential promising therapeutic for type 2 diabetes and obesity [41]. These isolated sesquiterpenes were also assessed for their inhibitory activities against PTP1B. In addition, the results revealed that two guaiane-type sesquiterpenes (4 and 5) and a furanosesquiterpene (8) displayed strong inhibitory activities against PTP1B with IC 50 values of 218, 344, and 1.24 µM, respectively, lower than the positive control (the IC 50 value of sodium orthovanadate was 881 µM).

General Methods
Optical rotations were measured on a JASCO P-1020 digital polarimeter. Ultra-violet (UV) spectra were measured on a Beckman DU640 spectrophotometer. ECD spectra were obtained on a JASCO J-810 spectropolarimeter. IR spectra were recorded on a Nicolet Nexus 470 FT-IR spectrophotometer in KBr discs. 1D and 2D NMR spectra were recorded on a JEOL JNMECP 500 spectrometer (500 MHz for 1 H and 125 MHz for 13 C) using tetramethylsilane as an internal standard. Chemical shifts are expressed in δ (ppm) referring to the solvent peaks at δ H 7.26 and δ C 77.16 for CDCl 3 , and δ H 3.31 and δ C 49.50 for methanol-d 4 and coupling constant J in Hz. HRESIMS data were acquired on a Thermo Scientific LTQ Orbitrap XL mass spectrometer or a Micromass Q-Tof Ultima GLOBAL GAA076 mass spectrometer, equipped with an electrospray ionization (ESI) source, and the ionization mode was positive or negative. Semipreparative HPLC separations were carried out using an Agilent 1100 series instrument with a diode array detector (DAD) detector, equipped with a reversed-phase column (YMC-Pack ODS-A, 5 µm, 250 × 10 mm). Chiral HPLC analysis and resolution were conducted on a chiral analytical column (Daicel Chiralpack IC, 5 µm, 250 × 4.6 mm). Silica gel (200-300 mesh, 300-400 mesh; Qingdao Marine Chemical Co. Ltd., Qingdao, China), ODS silica gel (50 µm, Merck, Darmstadt, Germany) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used to perform column chromatography (CC). Precoated silica gel plates (GF254, Qingdao Marine Chemical Co. Ltd., Qingdao, China) were used for thin layer chromatography (TLC) analyses, and spots were visualized under UV light and by spraying with 10% H 2 SO 4 in EtOH.

Animal Material
The soft coral S. cf. molesta was collected from the Paracel Islands of the South China Sea in October 2012, at a depth of about 10 m, and was frozen immediately until it was examined. The specimen was identified by Dr. Leen van Ofwegen, a co-author of this paper.. A voucher specimen (NO. XS-2012-22) was deposited in the school of Medicine and Pharmacy, Ocean University of China, China.

ECD Calculations of Compounds 1-5
The quantum chemical calculations were performed by using the density functional theory (DFT) as implemented in Gaussian 09 [42]. The initial structures of compounds 1-5 were built with Spartan 10 software and all trial structures were first minimized based on molecular mechanics calculations. Conformational searches were performed by Spartan 10 software using Merck Molecular Force Field (MMFF), 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/6-31G (d,p) level. The stable conformations of 1-5 were calculated for ECD spectra using TD-DFT method with the basis set RB3LYP/ DGDZVP [23]. Solvent effects of MeOH were evaluated at the same DFT level by using the SCRF/PCM method. The ECD spectra were combined after Boltzmann weighting according to their population contribution ( Figure S84-S88, Supplementary Materials).

PTP1B Inhibitory Assay
The PTP1B inhibitory activities of the isolates were evaluated by the method of pNPP [45], using sodium orthovanadate as a positive control.