Cembranoids with 3,14-Ether Linkage and a Secocembrane with Bistetrahydrofuran from the Dongsha Atoll Soft Coral Lobophytum sp.

Four new cembranoids, lobophylins A–D (1–4), and one novel secocembrane, lobophylin E (5) were isolated from a soft coral Lobophytum sp. The structures of new metabolites were elucidated on the basis of extensive spectroscopic methods. Among these metabolites, 1–4 are rarely found cembranoids possessing a tetrahydrofuran moiety with a 3,14-ether linkage. In addition, 5 is the first secocembrane possessing two tetrahydrofuran moieties with 3,14- and 4,7-ether linkages.


Results and Discussion
The new metabolite lobophylin A (1) exhibited a protonated molecule peak in the HRESIMS at m/z 343.2251 [M + Na] + , establishing the molecular formula C 20 H 32 O 3 and five degrees of unsaturation. The IR spectrum suggested the presence of hydroxy group (ν max 3460 cm −1 ) in 1. The 13 C NMR spectrum of 1 measured in CDCl 3 (Table 1) showed the presence of twenty carbon signals, which were assigned by the assistance of DEPT spectrum to four methyls, six sp 3 methylenes, one sp 2 methylene, four sp 3 methines (including three oxymethines), one sp 2 methine, and two sp 3 quaternary and two sp 2 quaternary carbons. From the 1 H NMR spectroscopic data of 1 (Table 2), the presence of two hydroxy protons resonating at δ 3.98 (dd, J = 9.6, 4.4 Hz) and 4.37 (ddd, J = 12.0, 3.6, 3.6 Hz) were observed. Moreover, the 1 H NMR spectrum revealed the presence of two olefinic methylene protons at δ 4.87 (d, J = 1.6 Hz) and 4.81 (s) and one olefinic methine proton at δ 5.09 (t, J = 6.8 Hz). A proton signal appearing at δ 3.27 ( 1 H, d, J = 6.8 Hz) and correlating with a carbon signal at δ 64.7 in the HMQC spectrum was due to the proton of the trisubstituted epoxide. The planar structure and all of the assignments of 1 H and 13 C NMR data of 1 were determined by the assistance of 2D NMR studies, including 1 H-1 H COSY and HMBC experiments ( Figure 1). 1 Key HMBC correlations of H-3 to C-4; H-7 to C-8; H 2 -13 to C-11 and C-12; H 2 -16 to C-1  and C-15; H 3 -17 to C-1, C-15 and C-16; H 3 -18 to C-3, C-4 and C-5; H 3 -19 to C-7, C-8 and C-9; and H 3 -20 to C-11, C-12 and C-13, permitted the connection of the carbon skeleton. Furthermore, the HMBC cross-peak from H-14 to C-3 suggested that C-3 and C-14 were linked through an oxygen to form a tetrahydrofuran ring. Thus, 1 was revealed as a cembranoid possessing a 3,14-ether linked tetrahydrofuran ring, on the basis of the above analysis.     The relative configuration of 1 elucidated mainly by NOESY spectrum was compatible with that of 1 offered by using the MM2 force field calculations which suggested the most stable conformations as shown in Figure 2. In the NOESY spectrum, it was found that H-1 (δ 2.77, dt, J = 8. Also, H 3 -19 was found to interact with H 2 -6, but not with H-7, revealing the trans geometry of the trisubstituted epoxide. Furthermore, the NOE correlations observed H 3 -20 and H-10 (δ 2.21), but not with H-11, reflected the E geometry of double bond at C-11. On the basis of the above findings and other detailed NOE correlations (Figure 2), the relative structure of 1 was determined.  (Tables 1  and 2), except for the replacement of the two carbon signals of the epoxide moiety in 1 by the signals of a trisubstituted double bond in 2 (δ 126.6, CH, C-7 and 132.8, C, C-8). This double bond was positioned at C-7/C-8 due to the 1 H-1 H COSY correlation found between the H-6 and H-7, the HMBC correlations observed from the olefinic methyl protons at δ 1.65 (3H, s) to C-7, C-8 and C-9. Furthermore, the E geometry of the 7,8-double bond was deduced from the NOE correlation of H 3 -19 with H 2 -6 and not with H-7. Thus, the structure of 2 was determined unambiguously. Literature review revealed a known compound similar to compound 2 but possessing a rare 3,13-bridged tetrahydropyran ring [20].  (Tables 1  and 2) of 3 showed the structural unit of a 3,14-oxa-bridged tetrahydrofuran, too. 1 H-1 H COSY and HMBC (Figure 1) further revealed that 3 possesses a 1,2-disubstituted double bond (δ 118.9 and 142.7, each CH) at C-6 and C-7 and a quaternary oxycarbon at C-8 (δ 73.6, C). On the basis of the above observations, and by the assistance of additional 2D NMR ( 1 H-1 H COSY and HMBC) correlations, it was possible to establish the planar structure of 3 as illustrated in Figure 1. The relative configurations of the five chiral centers at C-1, C-3, C-4, C-8 and C-14 in 3 were thus determined on the basis of NOE correlations (Figure 3). By careful inspection on the NOESY spectrum of 3, it was found that one proton (δ 2.40) of H 2 -5 showed NOE interaction with both H 3 -18 and H-7, and H-7 was NOE correlated with H 3 -19. Therefore, H 3 -18 and H 3 -19 are situated on the same β-face. Furthermore, NOESY spectrum showed correlation of H 3 -20 with one proton (δ 2.19) of CH 2 -10, but not with H-11, revealing the E-configurations of the 11,12-trisubstituted double bond. The above finding, together with J values for both H-6 (15.2 Hz) and H-7 (15.6 Hz), confirmed the E-configuration of the 6,7-double bond. Further NOE analysis revealed that 3 possessed the same configurations at C-1, C-3, C-4 and C-14, as in compound 1 (Figure 3). Based on the above results, the structure of 3 was established.
The HRESIMS spectrum of lobophylin D (4) showed a molecular formula of C 20 H 32 O 3 , the same as that of 3. By analysis 2D NMR spectra, including 1 H-1 H COSY, HMQC and HMBC, 4 was shown to possess the same molecular framework as that of 3. Furthermore, it was found that the NMR data of 4 were very similar to those of 3 (Tables 1 and 2), revealing that 4 might be an isomer of 3. However, the significant downfield shift at C-6 (Δδ C +2.9 ppm) and the upfield shift at C-7 (Δδ C −1.  Lobophylin E (5) was assigned a molecular formula of C 21 H 34 O 4 , according to the HRESIMS and NMR spectroscopic data (Tables 1 and 2). The IR absorption band at 3444 cm −1 revealed the presence of hydroxy group. By the analysis of 13 C and DEPT spectroscopic data, the carbons signals were assigned into five methyls (including one methoxy methyl resonating at δ C 54.3), six sp 3 methylenes, one sp 2 methylene, four sp 3 methines (including two monooxygenated carbons resonating at δ C 82.2 and 80.3 and an acetal carbon resonating at δ C 105.6), one sp 2 methine, one sp 3 quaternary carbons and three sp 2 quaternary carbons (including a normal ketone resonating at δ C 208.9). From the 1 H-1 H COSY spectrum of 5, it was possible to identify three different structure units, which were assembled with the assistance of an HMBC experiment. Key HMBC correlations between H-3 to C-4; H 2 -9 and H 2 -10 to C-8 (carbonyl carbon); H-11 to C-13; H 2 -16 to C-1 and H 3 -17 to C-1, C-15 and C-16; H 3 -18 to C-3, C-4 and C-5; H 3 -19 to C-8 and C-9; and H 3 -20 to C-11, C-12 and C-13 permitted the connection of the carbon skeleton ( Figure 1). Furthermore, the HMBC correlation observed from the methoxy protons (δ 3.34, 3H, s) to the carbon resonating at δ 105.6 positioned a methoxy group at C-7. In considering the degrees of unsaturation and molecular formula, two oxa-bridged ether linkages were placed between C-3/C-14 and C-4/C-7 by HMBC correlations from H-14 to C-3 and H-7 to C-4. The relative configuration of 5 was determined by the interpretation of the NOESY correlations ( Figure 4). It was found that H 3 -18 showed NOE interactions with H-1, H-3 and methoxy protons (H 3 -21). Thus, by considering a molecular model as shown in Figure 4 and assuming the β-orientation of H 3 -18, all of H-1, H-3 and methoxy group should be positioned on the β face. The NOE correlation observed between H-1 and H-14 also reflected the β-orientation of H-14. Furthermore, NOESY spectrum showed NOE interaction of H 3 -20 with H-10, but not with H-11, revealing the E geometry of the C-11/C-12 double bond. From the above evidence and the other NOE correlations ( Figure 4) the relative configurations at chiral centers of 5 was assumed to be 1R*, 3R*, 4R*, 7R* and 14S*. On the basis of the above analysis, the structure of 5 was established.  It is worth noting that metabolites 1-4 are rare cembranoids possessing a tetrahydrofuran moiety with a 3,14-ether linkage, which has been discovered previously in the soft coral Sinularia gibberosa [5,21]. In addition, 5 is the first secocembrane possessing two tetrahydrofuran moieties with 3,14-and 4,7-ether linkages. Our study thus adds the structure diversity of cembranoidal natural compounds.
The cytotoxicity of compounds 1-5 against the proliferation of a limited panel of cancer cell lines, including K562 (human chronic myelogenous leukemia), DLD-1 (human colon adenocarcinoma) and HepG2 and Hep3B (human liver carcinoma), was studied. The results showed that 1-5 are not cytotoxic toward the above cancer cells (IC 50 > 20 µg/mL).

General Experimental Procedures
The melting points were determined using a Fisher-Johns melting point apparatus. Optical rotation values were measured with a JASCO P-1010 digital polarimeter. IR spectra were recorded on a VARIAN DIGLAB FTS 1000 Fourier transform infrared spectrophotometer. The NMR spectra were recorded on a VARIAN MERCURY PLUS 400 FT-NMR (or Varian Unity INOVA 500 FT-NMR) instrument at 400 MHz (or 500 MHz) for 1 H NMR and 100 MHz (or 125 MHz) for 13 C NMR, respectively, in CDCl 3 . ESIMS were recorded on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, 230-400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254S plates (Merck, 1.05560) were used for TLC analysis. High-performance liquid chromatography (HPLC) was performed on a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 210 nm. A semipreparative reversed-phase column (250 × 10 mm, 5 µm) and a preparative normal phase column (250 × 21.2 mm, 5 µm) was used for HPLC.

Animal Material
The soft coral Lobophytum sp. was collected by hand using SCUBA off the coast of Dongsha Atoll, in April, 2007, at a depth of 10 m, and stored in a freezer until extraction. A voucher specimen (Specimen No. DA2007-04-20) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

Molecular Mechanics Calculations
Implementation of the MM2 force filed in Chem3D Pro software from Cambridge Soft Corporation, Cambridge, MA, USA (ver. 9.0, 2005), was used to calculate molecular models.