Tetrahydrofuran Cembranoids from the Cultured Soft Coral Lobophytum crassum

Three new cembranoids, culobophylins A–C (1–3), along with two known compounds (4 and 5) were isolated from the cultured soft coral Lobophytum crassum. The structures of these compounds were elucidated on the basis of their spectroscopic data and comparison of the NMR data with those of known analogues. Among these metabolites, 2 is rarely found in cembranoids possessing an isopropyl moiety with an epoxide group. Compound 1 exhibited significant cytotoxic activity against HL60 and DLD-1 cancer cell lines.


Results and Discussion
The EtOAc extract of the freeze-dried specimen was fractionated by silica gel column chromatography and the eluted fractions were further separated utilizing normal phase HPLC to yield metabolites 1-5 (Chart 1).The new metabolite culobophylin A (1) had a molecular formula of C 20 H 30 O 3 , which was determined by HRESIMS and NMR spectroscopic data.The IR spectrum of 1 showed absorption bands at 3458 and 1694 cm −1 , suggesting the presence of hydroxy and carbonyl groups.The 13 C NMR data of 1 showed the presence of 20 carbons (Table 1): three methyls, six sp 3 methylenes, one sp 2 methylene, three sp 3 methines (including two oxygenated carbons at δ 76.6 and 75.6), two sp 2 methines, and one sp 3 quaternary carbon.The remaining three signals appearing in the lower field region of the spectrum are due to the quaternary carbons of three olefinic carbons (δ 148.2, 132.9 and 131.8) and one aldehyde carbonyl (δ 194.7).From the 1 H NMR (Table 1) spectrum of 1, the presence of one aldehyde proton resonating as a singlet at δ H 9.56 was observed.Moreover, the 1 H NMR data revealed the presence of two olefinic methylene protons (δ 6.33, J = 1.5 Hz; 6.14, d, J = 1.5 Hz) and two olefinic methine protons (δ 5.18, dd, J = 5.0, 5.0 Hz; 4.84, d, J = 7.5 Hz).Furthermore, two oxygenated methines (δ 4.66, ddd, J = 11.0,6.0, 5.0 Hz; 3.96, dd, J =9.5, 4.0 Hz) and three methyls (δ 1.61, s; 1.56, s; 1.10, s) were also designated from the 1 H NMR signals.The planar structure and all of the 1 H and 13 C chemical shifts of 1 were elucidated by 2D NMR spectroscopic analysis, in particular 1 H-1 H COSY and HMBC experiments (Figure 2).From the 1 H-1 H COSY correlations (Figure 2), it was possible to establish three partial structures of consecutive proton spin systems extending from H 2 -5 to H-7; H-9 to H-11; H 2 -13 to H-3.The following key HMBC correlations permitted 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.The relative configuration of 1 elucidated mainly from the 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 3.In the NOESY spectrum, both H 3 -18 and H-14 showed NOEs with H-1 but not with H-3.Thus, assuming the β-orientation of H-1, H 3 -18 and H-14 should be positioned on the β face.Moreover, H-3 should be positioned on the α face.Also, the NOE correlations of H 3 -19 with H 2 -6 but not with H-7 and H 3 -20, with H-10a (δ 2.33) but not with H-11, indicated the E configuration of the double bonds between C-7/C-8 and C-11/C-12.Furthermore, the relative stereochemistry of 1 was mostly confirmed to be the same as that of 4 by comparison of the proton chemical shifts and coupling constants [17].On the basis of the above findings and other detailed NOE correlations, the structure of 1 was established unambiguously.Culobophylin B (2) was isolated as a colorless oil with the molecular formula C 20 H 32 O 3 , which possesses five degrees of unsaturation, as indicated by HRESIMS (m/z 343.2251, [M + Na] + ) and NMR spectroscopic data (Table 1).In addition, 1 H and 13 C NMR spectroscopic data (Table 1) of 2 showed the structural unit of a 3,14-oxa-bridged tetrahydrofuran.By comparison of the NMR data of 2 with that of 4, it was found that the 1 H and 13 C NMR data of 2 were very similar to those of 4 [17].However, the 1 H and 13 C NMR spectroscopic data revealed that the signals corresponding to one 1,1-disubstituted carbon-carbon double bond in 4 were not present and were replaced by one 1,1-disubstituted epoxide in 2 (δ H 2.51, 1H, d, J = 4.5 Hz and δ H 2.43, 1H, d, J = 5.0 Hz; δ C 54.2, C and δ C 50.8 CH 2 ) (Table 1). 1 H-1 H COSY and HMBC (Figure 2) further revealed that 2 possesses one 1,1-disubstituted epoxide at C-15.On the basis of the above observations, and with the assistance of additional 2D NMR ( 1 H-1 H COSY and HMBC) correlations, it was possible to establish the planar structure of 2, as illustrated in Figure 2. The relative stereochemistries of all stereocenters except C-15 of 2 were confirmed to be the same as those of 1 and 4 by comparison of the proton shifts, coupling constants, and NOE correlations of 2 with those of 1 and 4.
Culobophylin C (3) was obtained as a colorless oil and showed a [M + Na] + ion peak in the HRESIMS spectrum corresponding to the molecular formula C 20 H 30 O 3 , the same as that of 2. IR absorptions were observed at 3425 cm −1 , suggesting the presence of a hydroxy group in 3. The 13 C NMR spectrum of 3 showed twenty signals accounting for three methyls, five sp 3 methylenes, two sp 2 methylenes, five sp 3 methines , one sp 2 methine and four quaternary carbons (including one oxygenated carbon at δ 74.4 and three olefinic carbons with resonances at δ 146.5, 144.4 and 132.5).The 1 H NMR data revealed the presence of four olefinic methylene protons (δ 5.28, 5.12, 4.83 and 4.73, each a singlet).Two proton signals at δ 3.46 ddd (1H, 8.5, 2.5, 2.0) and 3.10 (1H, d, J = 2.0 Hz) correlated with two carbon signals at δ 54.5 and 61.6 and in the HMQC spectrum of 3 were attributed to the proton of one 1,2-disubstituted epoxide.The planar structure and all of the 1 H and 13 C chemical shifts of 3 were elucidated by 2D NMR spectroscopic analysis, in particular 1 H-1 H COSY and HMBC experiments (Figure 2).Thus, 3 was found to possess three double bonds at C-8/C-19, C-11/C-12 and C-15/C-16, one hydroxy group at C-4, one 1,2-disubstituted epoxide at C-6/C-7, and an oxa-bridged ether linkage at C-3/C-14.The relative configurations of the five chiral centers at C-3, C-4, C-6, C-7 and C-14 in 3 were elucidated by detailed NOE analysis, as shown in Figure 4.In these experiments, it was found that H 3 -18 showed NOE interactions with H-14 and H-7.Thus, assuming the β-orientation of H 3 -18, H-7 and H-14 should be positioned on the β face.The NOE correlation observed between H-14 and H-1 also reflected the β-orientation of H-1.Furthermore, the 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.On the basis of these results and other detailed NOE correlations, the structure of 3 was established unambiguously.The cytotoxicities of compounds 1-5 against HL60, MDA-MB-231, DLD-1 and HCT-116 cancer cells are shown in Table 2.The results show that compound 1, the most potent of compounds 1-5, exhibited cytotoxicity against the HL60, MDA-MB-231, DLD-1 and HCT-116 cancer cell lines with IC 50 s of 3.0, 16.8, 4.6 and 16.3 μg/mL, respectively.Furthermore, compound 2 exhibited moderate to weak cytotoxic activity against HL60, DLD-1 and HCT-116 cancer cell lines (the IC 50 values were 6.8, 16.2 and 16.7 μg/mL for HL60, DLD-1 and HCT-116, respectively).The other tested compounds were not cytotoxic (IC 50 > 20 μg/mL) toward the above four cancer cell lines.The in vitro anti-inflammatory effects of 1-5 were also tested.Furthermore, the anti-inflammatory activity of 1-5 against the accumulation of pro-inflammatory iNOS and COX-2 proteins in RAW264.7 macrophage cells stimulated with LPS was evaluated using immunoblot analysis.At a concentration of 10 µM, compounds 1-5 did not inhibit COX-2 and iNOS proteins expression relative to the control cells stimulated with LPS only (Figure 5).

General Experimental Procedures
Optical rotation values were measured using a Jasco P-1010 digital polarimeter.IR spectra were recorded on a Varian Digilab FTS 1000 Fourier transform infrared spectrophotometer.The NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR instrument at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR, respectively, in CDCl 3 .ESIMS and HRESIMS data were recorded with a Bruker APEX II mass spectrometer.Gravity column chromatography was performed on silica gel (230-400 mesh, Merck).TLC was carried out on precoated Kieselgel 60 F254 (0.2 mm, Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating.High-performance liquid chromatography was performed using a system comprised of a Hitachi L-7100 pump and a Rheodyne 7725 injection port.A preparative normal phase column (250 × 21.2 mm, 5 μm) was used for HPLC.

Animal Material
Specimens of the soft coral Lobophytum crassum were collected off the coast of Pingtung, southern Taiwan, and transplanted to a 120-ton cultivating tank equipped with a flow-through sea water system in July 2003.The cultured soft coral was harvested in December 2010.A voucher specimen (specimen no.2010CSC-1) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

In Vitro Anti-Inflammatory Assay
Macrophage (RAW264.7)cell line was purchased from ATCC.In vitro anti-inflammatory activities of compounds 1-5 were measured by examining the inhibition of lipopolysaccharide (LPS) induced upregulation of iNOS (inducible nitric oxide synthetase) and COX-2 (cyclooxygenase-2) proteins in macrophages cells using western blotting analysis [20,21].

Molecular Mechanics Calculations
Implementation of the MM2 force filed in Chem3D Pro software [22], was used to calculate the molecular models.

Figure 3 .
Figure 3. Computer-generated model of 1 using MM2 force field calculations and key NOE correlations.

Figure 4 .
Figure 4. Computer-generated model of 3 using MM2 force field calculations and key NOE correlations.

Figure 5 .
Figure 5.Effect of compounds 1-5 at 10 μM on the expression of iNOS and COX-2 proteins of RAW264.7 macrophage cells examined by immunoblot analysis.(A) Immunoblots of iNOS and β-actin; (B) immunoblots of COX-2 and β-actin.Values represent mean ± SEM (n = 6).The relative intensity of the LPS-only-stimulated group was taken as 100%.* Significantly different from the LPS-only-stimulated group (* P < 0.05).a Stimulated with LPS; b stimulated with LPS in the presence of 1-5.

Table 1 .
Cont. 500 MHz in CDCl 3 ; b 125 MHz in CDCl 3 ; c J values (Hz) are given in parentheses; d Numbers of attached protons were deduced by DEPT experiments. a