Further New Highly Oxidative Cembranoids from the Hainan Soft Coral Sarcophyton trocheliophorum

Abstract Three new highly oxidative cembranoids, sarcophytrols D–F (1–3), were obtained from the South China Sea soft coral Sarcophyton trocheliophorum, along with two known related ones (4 and 5). Their structures were elucidated by extensive spectroscopic analyses and by comparison with literature data. The discovery of these new secondary metabolites enriched the family of cembranoids deduced from the title animal. Graphical Abstract

Sarcophyton species are prolific in the South China Sea. In the course of our search for bioactive substances from Chinese marine organisms, Sarcophyton trocheliophorum of the coast of Yalong Bay, Hainan Province, has been collected and chemically investigated, which were found to encompass numerous cembranoids with a variety of oxidation and cyclization patterns, including two unprecedented structures, methyl sarcotroates A and B [7-9, 12, 17]. In addition, many of them exhibited significant inhibitory activities against human PTP1B enzyme [7,8,17]. Recently, in order to find more chemically appealing and biologically active cembrane-based metabolites, S. trocheliophorum was re-collected from the same location while in a different growing period, and a further chemical investigation yielded three new highly oxidative compounds named sarcophytrols D-F (1-3), along with two known ones (4 and 5). Details of the isolation, structure elucidation and biological study of these secondary metabolites are reported herein.

Results and discussion
Samples of S. trocheliophorum (400 g, dry weight) were extracted exhaustively with acetone, and the extract was partitioned between H 2 O and Et 2 O. The Et 2 O soluble fraction was subjected to silica gel chromatography (light petroleum ether/acetone gradient). The lower polar fractions were subsequently purified on repeated column chromatography (silica gel, Sephadex LH-20, reversed phase-C 18 silica gel and semi preparative-HPLC) to afford five pure metabolites, compounds 1-5. A preliminary NMR analysis revealed that all the new molecules shared the same cembrane skeleton. Among them, two known compounds were readily identified as 11,12-epoxy-1(E),3(E), 7(E)-cembratrien-15-ol (4) [18] and sinugibberol (5)  . Based on these NMR and MS variations, the hydroxylation at C-8 accompanying the double-bond migration from D 7 (8) to D 6(7) in the structure of 1 were supported. Assignments of 1 H and 13 C NMR signals of 1 were made by the application of detailed 2D NMR analysis.
As for the relative configuration of compound  (Fig. 1), the olefinic geometries were assigned to 1E and 3E. The 13 C NMR chemical shift of Me-20 (d C \ 20 ppm) and the absence of the ROESY correlations of H-11 (d H 3.08)/Me-20 (d H 1.25) indicated the trans-configuration of the epoxy group at C-11/C-12 in 1, which was the same as that in 4. Additional ROESY interactions of H-11/H-7, H-7/H-9b (d H 1.70), H-9b/H-11 suggested these three protons were co-facial, assigned tentatively as b-orientation. Thus, Me-19 was accordingly b-oriented due to the diagnostic cross-peak of Me-19 (d H 1.40) with H-7, allowing the determination of the structure of 1 as showed in Fig. 2, which was the D 6(7) -8a-hydroxyl derivative of 4.
Sarcophytrol E (2) was also obtained as colorless oil. The molecular formula, C 20 H 30 O 3 , established by HRESIMS (m/ z 343.2243 [M ? Na] ? ), was identical to that of 1. A detailed 2D NMR analysis of 2 and careful comparison with the NMR data of 1 (Tables 1 and 2) revealed that their structures were almost the same. In fact, the only differences between 2 and 1 were the C-19 signal downfield shifted in the 13 C NMR spetrum (d C 28.8 in 1 and d C 31.3 in 2), while the C-6 and C-9 signals upfield shifted (d C 125.0, 40.1 in 1 and d C 123.6, 38.9 in 2, respectively). The observed differences can be rationalized when the two compounds are C-8 epimers, which was confirmed by the ROESY interaction of H-6 (d H 5.81)/Me-19 (d H 1.36) (Fig. 3). Since the hydroxyl group at C-8 of 1 was a-oriented, the opposite configuration at this center is therefore tentatively suggested for 2.
The HRESIMS of sarcophytrol F (3) established the molecular formula C 20 H 32 O 4 (m/z 359.2188 [M ? Na] ? ), 16 mass units more than that of sinugibberol (5) [19]. The 1 H and 13 C NMR data of 3 showed great similarity as those of 5 (Tables 1 and 2), some minor differences were observed in relation to the functional group. The presence of a secondary hydroxyl group in the molecule was readily recognized by a signal resonating at d H 3.70 (1H, ddd, J = 8.7, 7.7, 4.0 Hz) in its 1 H NMR spectrum, and by a carbon signal at d C 69.1 (CH) in the 13 C NMR and DEPT spectra. The oxygenated methine proton was secured at C-10 by a COSY cross-peak between H-9 (d H 2.44, 2.30) and H-11 (d H 3.57), and the HMBC correlations with C-8, C-9 and C-11 (Fig. 4). Due to the presence of the 10-OH, 13 C NMR chemical shifts of C-9 to C-12 were all reasonably downfield shifted with respect to those of 5.
Finally, the relative configuration of 3 was determined by ROESY experiment and by comparison of the NMR data with 5 (Fig. 4). The 13  , indicated the trans-configurations of these two epoxy groups at C-3/C-4 and C-11/C-12 in 3, which were the same as those in 5. The similar 13 C NMR data of C-3 and C-4 in 3 and 5 further confirmed the same stereochemistry of this epoxy group. Furthermore, the ROESY correlations of H-11/H-13a (d H 1.44), H-3/H-13a, suggested that H-11 and H-3 were co-facial, leading to the determination of the relative configurations at C-11 and C-12 where the other epoxy group resided on. In addition, the ROESY correlations of H-10/Me-20 indicated that 10-OH and H-11 were co-facial. Due to the scarcity of material, the modified Mosher's method could not be able to apply for the determination of the absolute configuration in C-10 position in 3 at this moment. Thus the structure of sarcophytrol F (3) was tentatively determined as 10-hydroxyl derivative of 5.
All the compounds were tested for the cytotoxic activities and inhibitory activities against human protein tyrosine phosphatase 1B (PTP1B), a key target for the treatment of type-II diabetes and obesity [20]. Unfortunately, none of them showed inhibitory effects toward the above bioassays. Further study should be conducted to understand the real biological/ecological role of these metabolites in the life cycle of the animal, as well as to carry out other biological evaluations such as antioxidant, anti-inflammatory, anti-fouling activities, etc.

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. HRESIMS spectra were recorded on a Waters-Micromass Q-TOF Ultima Global electrospray mass spectrometer. NMR spectra were measured on a Bruker-DRX-500 spectrometer with the residual CHCl 3 (d H 7.26 ppm, d C 77.0 ppm) as internal standard. Chemical shifts are expressed in d (ppm) and coupling constants (J) in Hz. 1 H and 13 C NMR assignments were supported by 1 H-1 H COSY, HSQC, and HMBC experiments. Commercial silica gel (Qing Dao Hai Yang Chemical Group Co., 200-300 and 400-600 mesh), C 18 reversed-phase silica gel

Animal Material
The soft corals S. trocheliophorum were collected by scuba at Yalong Bay, Hainan Province, China, in February 26,