Lobophytones O–T, New Biscembranoids and Cembranoid from Soft Coral Lobophytum pauciflorum

Chemical examination of a Chinese soft coral Lobophytum pauciflorum resulted in the isolation of five new biscembranoids named lobophytones O–S (1–5) and a new “monomeric” cembrane lobophytone T (6). The structures of the new compounds were elucidated by interpretation of 1D and 2D NMR (COSY, HSQC, HMBC, and NOESY) spectroscopic data in association with MS and IR data. Lobophytone Q showed significant inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) release in mouse peritoneal macrophages, while lobophytones Q and T showed inhibitory activities against the bacteria S. aureus and S. pneumoniae.


Introduction
Biscembranes are a class of marine natural products with unusual structure patterns, which are mainly found in the marine soft coral genus of Sarcophyton (S. tortuosum, S. glaucum, S. latum, and OPEN ACCESS S. elegans). They are supposed to be biogenetically derived by a Diels-Alder cycloaddition of cembranoid-diene and cembranoid-dienophile [1][2][3][4][5][6][7][8]. Recently, we found the soft coral Lobophytum pauciflorum also contains a variety of biscembrane analogues, but they presented the structure pattern with antipodal Diels-Alder cycloaddition and were named isobiscembranoids [9]. This finding provided a new marine organism to enrich the chemical diversity of biscembranoids. Our continuing interest in the chemical diversity of this specimen led to the isolation of five new biscembranoids (1)(2)(3)(4)(5) and a new monomeric cembrane (6) (Figure 1). This paper reports the structural elucidation of these new compounds and their bioactivity.

Results and Discussion
Repeated column chromatography of the EtOAc-soluble fraction obtained from the EtOH extract of the soft coral L. pauciflorum led to the isolation of five biscembranoids and a cembranoid. All biscembranoids shared the partial structure related to methyl tortuosoate, a cembranoid-dienophile [6].
Lobophytone O (1) has a molecular formula of C 41 H 64 O 9 as determined by HR-ESIMS m/z 723.4428 [M + Na] + (calcd for C 41 H 64 NO 9 Na, 723.4442) and NMR data, requiring 10 degrees of unsaturation. The 1 H NMR spectrum exhibited the resonances for nine methyls including two olefinic methyl singlets ( H 1.65, 1.66) and an OMe ( H 3.42), while the 13 C NMR spectrum presented 41 carbon resonances including three ketone and one ester carbonyl carbon, four olefinic carbons, and five oxygen-bearing sp 3 carbons. Six degrees of unsaturation, as accounted by the functional groups out of 10 in the molecule, suggested the presence of a tetracyclic nucleus. The 1 H and 13 C NMR data of 1 featured a biscembrane skeleton, closely related to that of ximaolide C as formerly isolated from Sarcophyton tortuosum [3]. COSY in association with HMQC relationships enabled all protons and their corresponding carbons in the molecule to be assigned (Tables 1 and 2). Comparison of NMR data revealed that rings A and B of 1 were the same as in ximaolide C. The difference was found in ring C, where a hydroxy group of 1 replaced a chlorine atom at C-31 of ximaolide C. This was evident from the presence of a hydroxy proton at  H 3.98 (s), and its HMBC correlations with C-30 ( C 89.2) and C-31 ( C 73.6), and by the missing chlorine isotope pattern in the mass spectrum.  The relative configurations of the stereogenic centers in 1 were determined on the basis of NOE relationships ( Figure 2) and J values. The NOE relationship between H-2 ( H 3.47) and OMe suggested cis-conjunction of rings A and B. The exclusive -face of the methyl ester group as found in all known biscembranes led to the biogenetical assignment of the methyl ester at C-1 of 1 to be  Figure 2). In addition, the NOE relationship between H-30/H 3 -40 (δ H 0.97) suggested H 3 -40 to be -oriented.  The 1D and 2D NMR spectroscopic analysis revealed that the structure of 3 was also closely related to that of 1. The presence of two D 2 O exchangeable protons at δ H 4.18 (s) and 4.46 (br) was attributed to two hydroxy groups, which were deduced to be attached at C-31 and C-33, based on the HMBC interactions from OH-31 (δ H 4.18, s) to C-30 (δ C 90.1), C-31 (δ C 73.2) and C-32 (δ C 40.7), and the COSY correlation between OH-33 (δ H 4.46, bs) and H-33 (δ H 4.82). The chlorine atom of 3 was assumed to be substituted at C-26 according to the upfield-shifted C-26 (δ C 65.1), which showed around Δδ 5 less than the same carbon of 1 linked by hydroxy group. The relative configurations of the chiral centers in 3 were in agreement with those of 1 based on the similar NMR and NOE data.
The molecular formula of lobophytone R (4) 87 (3H, s), in addition to four olefinic protons for an AB spin system at δ H 6.02 (d, J = 11.3 Hz) and 6.03 (d, J = 11.3 Hz) and for the exo-methylene at δ H 4.86 (brs) and 4.82 (brs). The COSY correlations established the segments from C-2 to C-3, C-5 to C-7, C-9 to C-11, and C-13 to C-14, while 13 C NMR and HMQC data indicated the methines of C-7 (δ C 85.0), C-11 (δ C 70.5) and C-14 (δ C 69.7) bearing oxygen atom. The HMBC relationships connected each segment to form a backbone that featured a cembrane with 14-membered ring. An isopropene was deduced to position at C-1 based on the HMBC cross-peaks from H 2 -16 (δ H 4.86, 4.82) and H 3 -17 (δ H 1.80) to C-1 (δ C 143.5), while additional HMBC correlations indicated two vinyl groups to be conjugated at C-1/C-2 and C-3/C-4. The HMBC correlations from H 3 -19 (δ H 1.01) to C-7, C-8 (δ C 69.2, Cq), and C-9 (δ C 32.2), and from H 3 -20 (δ H 1.05) to C-11, C-12 (δ C 73.7, Cq), and C-13 (δ C 44.3), in addition to the HMBC relationship from H-7 (δ H 3.26) to C-11 established an ether bridge which linked C-7 and C-11. The acetoxy group was deduced to position at C-14 on the basis of the HMBC correlations from H-14 (δ H 6.01) to the acetyl carbonyl carbon and to C-1, C-2, and also C-15 (δ C 144.2, Cq). Thus, C-8 and C-12 were hydroxylated, and the gross structure of 6 was established as shown in Figure 1 The "monomeric" cembrane lobophytone T (6) is likely a precursor (diene) to form the right part of biscembranes when it reacted with methyl tortuosoate [6]. Thus, it provided new evidence to support the plausible biogenetic pathway to generate biscembranes. It is noted that all of the isolated biscembranoids (1-5) maintained the same partial structure of ring A, which was considered to be derived from methyl tortuosoate as a cembranoid-dienophile. The difference was found in ring C where epoxide rearrangement, oxidation, acetylation, and halogen substitution occurred.
In primary bioassays, compound 3 showed significant inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages with IC 50 = 2.8 µM, whereas the other compounds showed weak activity (IC 50 > 10 µM). In addition, all compounds were weakly cytotoxic toward the mouse peritoneal macrophages (IC 50 > 10 µM). The antibiotic assay indicated that compounds 3 and 6 exhibited strong inhibition against Staphylococcus aureus, S. pneumoniae, and Saccharomyces cerevisiae with the inhibitory rates around 90% at 20 µg/mL, but all compounds possessed weakly inhibitory activity against Pseudomonas aerugonisa, Escherichia coli, Candida albicans, and Aspergillus fumigatus.

General Procedures
Optical rotations were measured on a Perkin-Elmer 243B polarimeter. IR spectra were determined on a Thermo Nicolet Nexus 470 FTIR spectrometer. 1 H and 13 C NMR and 2D NMR spectra were recorded on an Avance-500 FT 500 MHz NMR spectrometer using TMS as an internal standard. δ values are expressed in parts per million (ppm), and J values are reported in Hertz (Hz). HR-ESIMS data were obtained from Bruker APEX IV instrument. Low pressure column chromatography was carried out with silica gel (160-200 and 200-300 mesh), and GF 254 silica gel for TLC was provided by Qingdao Marine Chemistry Co. Ltd. HPLC chromatography was performed on an Alltech instrument (426-HPLC pump, Alltech UV-vis-200 detector) equipped with Kromasil semipreparative column (10 µm, ODS, 10 mm × 250 mm) and YMC-Pack C 8 (5 µm, 10 mm × 250 mm) column.

Animal Material
Soft coral Lobophytum pauciflorum was collected from the inner coral reef at a depth of 10 m in Sanya Bay, Hainan Island of China, in 2008. Fresh samples were frozen immediately. The specimen was identified by Leen van Ofwegen (National Museum of National History Naturalis). The coral specimen (HSF-6) was deposited at State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China.

Extraction and Isolation
The frozen soft coral L. pauciflorum (2.3 kg) was homogenized and then extracted with EtOH (twice, 5 L/each time). The concentrated extract was desalted by dissolving in MeOH to yield a MeOH soluble fraction which was concentrated to an oily residue (92.7 g). This residue was partitioned between H 2 O and EtOAc, and then the water phase was extracted with n-BuOH. The EtOAc fraction (12.1 g) was subjected to silica gel (200-300 mesh) column chromatography, eluting with a gradient (petroleum ether-acetone, 20:1, 10:1, 3:1, 1:1) to obtain seven fractions (F1-F7).Fractions F1-F4 mainly contained lipids and steroids, as detected by 1 H NMR, while F5 and F6 showed the 1 H NMR spectral features of terpenoids. Thus, F5 and F6 were combined (1.69 g) and subsequently subjected to Sephadex LH-20 column eluting with CH 2 Cl 2 -MeOH (1:2) to afford 6 portions (P1-P6). P2 (180 mg) showing blue-green spots after spraying with anisaldehyde reagent was further separated on reversed-phase semi-preparative HPLC with CH 3 CN-H 2 O (61%) as a mobile phase to obtain 1 (12.5 mg). P3 (49.2 mg) was separated on ODS HPLC with MeOH-H 2 O (82%) as a mobile phase to afford 4 (7.6 mg), while 2 (8.1 mg) was obtained from P4 (40.5 mg) by the same separation process as that for P3. Followed by the same process as for P3 and P4, 5 (7.1 mg), 3 (17.7 mg), 3 (14.5 mg), and 6 (5.4 mg) were separated and purified from P5 (112.7 mg) by reversed-phase semi-preparative HPLC (ODS) with CH 3  Absorbance was measured at 548 nm in incubated media with Griess reagent for 10 min. Viable adherent cells were stained with MTT (2 µg/mL) for 4 h. The medium was then removed and the produced formazan crystals were dissolved in DMSO (200 µL). Absorbance was measured at 540 nm.
One-way analysis of variance was applied for all statistical analyses by independent experiments, and data were represented as means ± standard error of the measurement. Individual values were compared by t-test and a P-value < 0.01 to evaluate the significant.

Antimicrobial and Antifungal Assays
Antimicrobial and antifungal bioassays were conducted in triplicate following the method as recommended by the National Center for Clinical Laboratory Standards (NCCLS) [10]. The bacterial strains Staphylococcus aureus, S. pneumoniae, Escherichia coli, and Pseudomonas aeruginosa were grown on Mueller-Hinton agar. The yeasts, Candida albicans and Saccharomyces cerevisiae, were grown on Sabouraud dextrose agar, and the fungus, Aspergillus fumigatus, was grown on potato dextrose agar. Targeted microbes (3-4 colonies) were prepared from broth culture (bacteria: 37 °C for 24 h; fungus: 28 °C for 48 h), and the final spore suspensions of bacteria (in MHB medium), yeasts (in SDB medium), and fungus (in PDB medium) were 10 6 and 10 5 cells/mL and 10 4 mycelial fragments/mL, respectively. Testing compounds (10 mg/mL as stock solution in DMSO and serial dilutions) were transferred to a 96-well clear plate in triplicate, and the suspension of the test microorganisms were added to each well (200 µL) (antimicrobial peptide AMP, streptomycin, and fluconazole were used as positive controls). After incubation, the absorbance at 595 nm was measured with a microplate reader (TECAN), and the inhibition rate was calculated and plotted versus test concentrations.