New Bicyclic Cembranoids from the South China Sea Soft Coral Sarcophyton trocheliophorum

Nine new bicyclic cembranoids, sarcophytrols M–U(1–9), were isolated from the South China Sea soft coral Sarcophyton trocheliophorum as minor components, along with one known related cembranoid 10. Their structures were elucidated by detailed spectroscopic analysis and chemical conversion. The chemical structures of these metabolites are characterized by the different patterns of the additional cyclization within the 14-member skeleton, which leading to the formation of furan, pyran, oxepane, and peroxyl rings, respectively. Among them, sarcophytrols R and S(6 and 7) share a rare decaryiol skeleton with an unusual C12/C15 cyclization. In addition, the absolute configurations of sarcophytrols M and T(1 and 8) were determined by the modified Mosher’s method. The research of these new secondary metabolites provided a further understanding of the diversity of cyclized cembranoids from the title species.


Results and Discussion
Samples of S. trocheliophorum (dry weight 400 g) were extracted exhaustively with acetone, and the extract was partitioned between water and Et 2 O. The Et 2 O solvable fraction was subjected to repeated chromatography as usual work 3,4,9,16 , to afford ten pure metabolites, compounds 1-10 ( Fig. 1). A preliminary NMR analysis revealed that all the new molecules shared the same cembrane skeleton. Among them, the known compound was readily identified as sarglaucol (10) 19 by comparison of its spectral data and [α] D values with those reported in the literatures.
The HRESIMS of sarcophytrol M (1) established the molecular formula C 20 H 34 O 4 . 1 H and 13 C NMR spectra of 1 (Tables 1 and 2) were reminiscent of a known cembranoid, (2E,7E)-4,11-dihydroxy-1,12-oxidocembra-2,7-diene (11), previously isolated from Sinularia ovispiculata 20 . The distinct difference between them was the presence of a hydroxyl at C-15 (δ C 72.4) in 1, which was further confirmed by the observation of HMBC correlations from H 3 -16 (δ H 1.12)/H 3 -17 (δ H 1.06) to C-15 (Fig. 2). Similar to 11, a trans-disubstituted olefin at C-2/C-3 was recognized by the doublet coupling constant (15.4 Hz) of H-2 (δ H 5.62) and H-3 (δ H 5.92), while the chemical shift of Me-19 (δ C < 20 ppm) indicated the 7E assignment in 1 20,21 . The relative configurations of the stereogenic centers were determined by NOE relationships (Fig. 2), which exhibited similar key cross-peaks to those in the cembranoids sinulariols D and F 7 . The NOE correlation between H-3 and H-11 (δ H 3.54) implied that the trans-disubstituted olefin and the hydroxyl group at C-11 were hind the same face toward the five-membered ring. Thus, Me-20 was oriented to the same face with the isopropyl group, and tentatively assigned to be α-oriented. In addition, the significant NOE correlations of H-2/H 3 -16 and H-2/H-14b (δ H 1.67) suggested that H-2 and the isopropyl group to be on the same side of 1. The trans-disubstituted olefin group favoring 'up' or 'down'-orientation toward the 14-membered ring, and thus H-3 was oriented to 'up' face. The configuration of H-11 was assigned as β mainly on the basis of the aforementioned strong NOE correlation between H-11 and H-3 and the very weak NOE correlation between H-11 and H 3 -20 (δ H 1.11). According to the NOE correlation from H-3 to H 3 -18 (δ H 1.27) and the lack of correlation from H-2 to H 3 -18, we tentatively assigned the configuration of H 3 -18 to β face. To obtain its absolute configuration, two aliquots of compound 1 were treated with (R)-and (S)-α-methoxy-α-trifluoromethylphenyl acetyl (MTPA) chlorides to obtain the (S)-and (R)-esters, respectively. Analysis of Δ δ SR values (δ S -δ R ) observed for the signals of the protons close to 11-OH (Fig. 3) indicated the S configuration at this carbon. Consequently, the absolute configuration of compound 1 was determined as (1S, 4S, 11S, 12R).
Sarcophytrol N (2) has a molecular formula of C 21 H 36 O 4 , as established by HRESIMS and NMR data, 14 mass units more than that of 1. The 1 H NMR data of 2 (Tables 1 and 2) were closely reminiscent to those of 1, except for the newly appeared signal at δ H 3.23 (3H, s), which suggested the presence of an additional methoxyl group in 2, consistent with one carbon resonance at δ C 50.6 (q) in its 13 C NMR spectrum. The introduction of the methoxyl group in 2 resulted in the significant downfield shift of C-15 from δ C 72.4 (s) in 1 to 78.4 (s) in 2 and the upfield shifts of C-16/C-17. Furthermore, the methoxyl group was secured at C-15 by a diagnostic HMBC correlation from methoxyl group to C-15. Furthermore, ROESY correlations of compound 2 were similar with those of 1. On the basis of above evidences, compound 2 was identified as 15-methoxyl derivative of 1.  Sarcophytrol P (4) was isolated as an optically active colorless oil with molecular formula C 20 H 34 O 4 , the same as 1. The NMR spectra of 4 (Tables 1 and 2) were strongly reminiscent of those of 1 and a careful 2D NMR analysis suggested that they share the same gross structure. In fact, the only difference was found in the segment from C-2 to C-5, where 13 C NMR chemical shifts of C-3, C-4 and C-5 in 4 were all upfield shifted while that of γ-carbon C-2 was downfield shifted with respect of 1. This evidence clearly suggested that the relative configuration of the hydroxyl group at C-4 of 4 was different from that of 1. The NOE interactions between H-2 (δ H 5.55) and H 3 -18 (δ H 1.33) in 1 rather than H-3 (δ H 6.07) and H 3 -18 in 4 confirmed this assignment. Herein, 4 was 4-epimer of 1.
Sarcophytrol Q (5) possesses the same molecular formula C 22 H 36 O 5 as 4, established by HRESIMS, whereas its NMR data (Tables 1 and 2) were almost identical to those of sinulariol Z (12) 22 . The distinction was attributed to the NMR data of 5 presenting an extra hydroxyl group at C-15, which was further confirmed by the observation of HMBC correlations from H 3 -16 (δ H 1.12)/H 3 -17 (δ H 1.07) to C-15 (δ C 74.6). Due to the presence of the 15-OH, 13 C NMR chemical shifts of C-15, C-16, C-17, and C-1 were all downfield shifted reasonably while those of γ -carbons 2 and 14 were upfield shifted with respect of those of 12. Furthermore, the significant NOE interactions between H-3 (δ H 5.98) and H-11 (δ H 3.34) and the lack of NOE correlations of H-11/H 3     The remaining two degrees of unsaturation strongly indicated that 6 has a bicyclic structure with a carbocyclic ring bridged by oxygen. The 13 C NMR spectrum of 6 contained five oxygenated carbon signals at δ C 74.04 (CH), 76.80 (C), 69.49 (CH), 75.32 (C) and 74.44 (C), which were assigned by 2D NMR spectrum to C-3, C-4, C-11, C-12 and C-15, respectively. In order to define the carbon atoms linked to these functionalities, 2D NMR experiments (in DMSO-d 6 ) of 6 were measured. The formation of an ether bridge across C-12 and C-15 was deduced from the observation of OH-3 (δ H 4.31), OH-4 (δ H 3.95) and OH-11 (δ H 4.10). The location of these three -OH groups are determined by the 1 H-1 H COSY correlations of H-3 (δ H 3.90) with OH-3 and H-11 (δ H 3.40) with OH-11, and the HMBC correlations from OH-4 to C-3 (δ C 72.9), C-4 (δ C 75.1), C-5 (δ C 38.1).
The E geometry of the Δ 1 and Δ 9 double bonds was deduced by the ROESY correlations of H-2 (δ H 5.36)/H 3 -17 (δ H 1.31) and of H-7 (δ H 5.30)/H 2 -9 (δ H 2.12) (Fig. 4). Moreover, H-14a (δ H 2.85) correlated with H-3 (δ H 4. 19) and H-11 (δ H 3.57), suggesting H-3 and H-11 to be on the same side of 6. When H-3 was assigned tentatively as β-orientation, H-11 was accordingly oriented in β-face. The presence of the cross-peak between H-2 and H 3 -18 (δ H 1.27) in combination with the absence of the correlations of H-3/H-2 and H-3/H 3 -18 in the ROESY spectrum, clarified H 3 -18 and H-2 to be oriented in opposite to H-3. Finally, the α-oriented H 3 -20 was tentatively deduced by the absence of correlations between H 3 -20 and H-11. Sarcophytrol S (7) was isolated as an optically active, colorless oil {[α] D 20 − 39.3 (c 0.07, MeOH)}. Its molecular formula, C 20 H 32 O 3 , was established by HRESIMS, 18 units less than that of 6. Careful comparison of NMR data of 7 and 6 (Tables 1 and 2) revealed that the former differs from the latter only by the presence of a terminal methylene group (δ H 5.13, 4.93; δ C 111.37, 147.92) in 7 instead of a methyl (δ H 1.27; δ C 22.91) and a oxygenated quarternary carbon (δ C 76.8) in 6, in agreement with 18 mass units difference between them, while the rest of the molecules was the same. Base on the HMBC correlations between H 2 -18 (δ H 5.13, 4.93) and C-3 (δ C 75.24) and C-5 (δ C 28.32), the terminal methylene group was located at C-4. Moreover, 1 H-1 H COSY, HSQC, and HMBC experiments allowed the unambiguous definition of the structure of 7. Analogously to 6, the relative stereochemistry of three chiral centers at C-3, C-11 and C-12 was elucidated to be the same as those of 6 by the ROESY experiments (Fig. 4).
The relative stereochemistry of the chiral centers at C-4, C-7, C-11 and C-12 was established by a ROESY experiment running on 9. In MM2 energy-minimized conformation (Fig. 5), H-7 was suggested to be axial position toward the hexatomic ring (β-orientation). The diagnostic correlations between H-7 (δ H 4.71) and H-5a (δ H 1.87) as well as H 3 -18 (δ H 1.28) and H-5a suggested H-5a and H 3 -18 were determined as axial and equatorial position, respectively (β-orientation). The ROESY correlation of H-11 (δ H 3.44) with H 3 -20 (δ H 1.28) suggested the H-11 and H 3 -20 are in the same face, which was compatible with that of the co-exist cembranoid sarglaucol (10) 19 . However, the relationship of H-7 and H-11 cannot be determined from the ROESY spectrum.
In conclusion, nine new cembranoids, sarcophytrols M-U (1-9), were isolated from the South China Sea soft coral Sarcophyton trocheliophorum, along with one known related cembranoid 10. Among them, the characteristic chemical features of them are the diverse types of cyclized rings: furan rings possessed by sarcophytrols M-P (1-4), pyran rings formed in sarcophytrols Q-S (5-7), while oxepane and peroxyl rings appeared in sarcophytrols T (8) and U (9), respectively. In addition, sarcophytrols R and S (6 and 7) share a rare bicyclic skeleton of the decaryiol-type. These group of diterpenes were first reported from the same genus of soft coral S. decaryi 11 , and described so far only from the species of soft corals Lobophytum sp. 12 . The co-isolation of these diterpenes is an example of the productivity of the title animal.
In light of a wide range of biological activities and pharmacological properties of cembranoids 13 , we performed in vitro investigation of inhibitory activity against human protein tyrosine phosphatase 1B (PTP1B) enzyme, a promising drug target for the treatment of type 2 diabetes and obesity 17 , for compounds 1-10, since the previously reported cembranoids from the title animals displayed significant PTP1B inhibitory activity 3,4,16 . Unfortunately, the bioassay result showed that none of the tested compounds exhibited interesting PTP1B inhibitory activities. In addition, the antitumor and antibacterial activities were also tested for compounds 1-10. However, they exhibited neither cytotoxicities against the human tumor cell lines HL-60 and K-562, nor antibacterial activity against Pseudomonas aeruginosa.

Methods
General experimental procedures. Optical rotations were measured on a Perkin-Elmer 341 polarimeter.