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Cembrane-type diterpenoids from the South China Sea soft coral Sarcophyton mililatensis

Eight cembrane-type diterpenoids, namely, (+)-(6R)-6-hydroxyisosarcophytoxide (1), (+)-(6R)-6-acetoxyisosarcophytoxide (2), (+)-17-hydroxyisosarcophytoxide (3), sarcomililatins A–D (4–7), and sarcomililatol (8), were isolated from the soft coral Sarcophyton mililatensis collected from Weizhou Island, Guangxi Autonomous Region, together with 2 known related analogues, (+)-isosarcophytoxide (9) and (+)-isosarcophine (10). The structures of these compounds were elucidated by a combination of detailed spectroscopic analyses, chemical methods, and comparison with reported data. The absolute configuration of compound 1 was established by the modified Mosher׳s method, while the absolute configurations of compounds 4 and 5 were assigned by electronic circular dichroism (ECD) spectroscopy and that of compound 8 was established by time-dependent density functional theory electronic circular dichroism (TD-DFT ECD) calculation. In in vitro bioassays, compound 9 displayed significant cytotoxicity against the human cancer cell lines human promyelocytic leukemia cells (HL-60) and human lung adenocarcinoma cells (A-549) with IC50 values of 0.78±0.21 and 1.26±0.80 μmol/L, respectively. Compounds 4 and 9 also showed moderate inhibitory effects on the TNFα-induced Nuclear factor kappa B (NF-κB, a therapeutical target in cancer) activation, showing IC50 values of 35.23±12.42 and 22.52±4.44 μmol/L, respectively.


Introduction
Literature reports concerning the natural products chemistry of soft corals of the cosmopolitan genus Sarcophyton (Alcyonacea, Alcyoniidae) indicate that they are well-known to be a rich source of specialised metabolites, particularly diterpenoids of the cembrane-type 1,2 . To date, more than 220 cembranes have been discovered, besides undefined species, from approximately 18 species of this genus. Moreover, some of them have been reported to be responsible for a diverse range of significant bioactivities, especially cytotoxic and anti-inflammatory effects [2][3][4] . Their excellent bioactivities have for a long time attracted great interest from synthetic organic chemists as challenging targets for total synthesis 5,6 .
Cancer is a group of diseases characterized by uncontrolled cell growth, which has become the major public health concern over the last several decades [12][13][14][15] . NF-κB, as a family of inducible transcription factors in all cells discovered by Sen and Baltimore 16 in 1986, has become one of the major targets for drug development 17 . In particular, the aberrant activation of NF-κB has been frequently observed in various types of human cancers, and suppression of NF-κB can limit the proliferation of cancer cells 18 . Hence, we focus on testing cytotoxic activity and the NF-κB inhibitory effects. According to the results, compound 9 exhibited moderate cytotoxic activity, both compounds 4 and 9 showed moderate NF-κB inhibitory effects on the TNFα-induced NF-κB. Reported herein are the isolation and structural elucidation of these compounds as well as their biological properties.

Results and discussion
Compound 1 was isolated as a colorless oil, and had a molecular formula of C 20  H NMR spectrum. The 13 C NMR spectrum indicated the presence of 20 signals which were attributed by DEPT and HSQC experiments to 4 methyls, 6 methylenes, 5 methines, and 5 quaternary carbons. Of these carbons, 5 were bonded to oxygen and 6 were olefinic (2 were trisubstituted). These data suggested that 1 was a cembrane-type diterpenoid.
A comparison of the NMR data of 1 with those of the cooccurring known cembrane diterpenoid, (þ)-isosarcophytoxide (9) 19,20 , revealed that they were structural analogues, with the only difference being the presence of an additional hydroxyl group at C-6 in 1, in agreement with the mass data. The hydroxyl group Figure 1 Chemical structures of compounds 1-10. The absolute configuration at C-6 of 1 was assigned via a modified Mosher's method. Esterification of 1 with (R)-and (S)-MTPA chloride occurred at the C-6 hydroxyl group to give the (S)-and (R)-MTPA ester derivatives, 1s and 1r, respectively. The observed Δδ H(S-R) value distribution pattern (Fig. 4) established the 6R-configuration for 1. Therefore, the structure of 1 was elucidated as (þ)-(6 R)-6-hydroxyisosarcophytoxide.
Compound 2, which was obtained as a colorless oil, gave the molecular formula C 22 H 32 O 4 on the basis of its (þ)-HR-ESI-MS ion peak at m/z 383.2197 [M þ Na] þ (Calcd. for C 22 H 32 O 4 Na, 383.2193), requiring 7 degrees of unsaturation. The IR spectrum displayed a strong absorption at 1732 cm -1 , consistent with the presence of a saturated ester carbonyl group. The 1 H and 13 C NMR spectra ( Table 1) of 2 were virtually identical to those of 1, with the exception of an acetoxy moiety in 2 instead of the C-6 hydroxyl group in 1. This replacement caused the 13 C NMR resonance of C-6 to be shifted downfield (from δ C 65.4 to δ C 67.9). The position of the acetoxy group at C-6 was further secured by the HMBC correlation ( Fig. 2) from H-6 (δ H 5.78) and the ester carbonyl carbon (δ C 170.2). The similar NOESY correlation (Fig. 3) patterns of 2 and 1 indicated that they have the same relative configuration. Finally, the absolute configuration of 2 was assigned as 2S,6R,11R,12R, the same as those of 1. This is because acetylation of 1 yielded 2, which gave optical rotations {½α 25 D þ51 (c 0.09, CH 3 OH); ½α 25 D þ65 (c 0.09, CHCl 3 )}, compared to those {½α 25 D þ53 (c 0.5, CH 3 OH); ½α 25 D þ63 (c 0.24, CHCl 3 )} observed for the natural sample of 2. The structure of 2 was thereby proposed as (þ)-(6 R)-6-acetoxyisosarcophytoxide.
A literature search revealed that the assigned structure of 2 was the same as that of sarcophytonoxide A, a known cembrane diterpenoid isolated previously from the soft coral Sarcophyton ehrenbergi 22 . Furthermore, the 1 H and 13 C NMR data of 2 were also the same as those of sarcophytonoxide A. However, when the optical rotation of 2{dextrorotatory, ½α 25 D þ53 (c 0.5, CH 3 OH)}, recorded in the same conditions, was compared with that reported for sarcophytonoxide A {levorotatory, ½α 25 D -36.8 (c 0.5, CH 3 OH)} 22 , it appeared quite equal in value but opposite in sign. This result indicated that the 2 compounds are enantiomers and that the absolute configuration of sarcophytonoxide A should be 2R,6S,11S,12S.
Compound 3 was isolated as a colorless oil. Its molecular formula of C 20 H 30 O 3 , the same as that of 1, was deduced from the (þ)-HR-ESI-MS ion peak at m/z 341.2077 [M þ Na] þ (Calcd. for C 20 H 30 O 3 Na, 341.2087). A comparison of the 1 H and 13 C NMR data (Table 1) of 3 and 1 indicated similarities between them. In fact, the structure of 3 differed from that of 1 only by the location of the hydroxyl group from C-6 to C-17 in 3. This deduction was based on the chemical shift observed for C-6 (δ C 22.8) and C-17 (δ C 57.0), which was further confirmed by the 1 H-1 H COSY cross-peaks ( Fig. 2) of H 2 -5 (δ H 2.31 and 2.18)/H 2 -6 (δ H 2.42 and 1.09)/H-7 (δ H 5.00) and the HMBC correlations ( Fig. 2) from H 2 -17 to C-1 (δ C 136.8), C-15 (δ C 132.0), and C-16 (δ C 75.9). The relative configurations of all the asymmetric centers were determined to be the same as those of 1 on the basis of the NOESY experiment (Fig. 3). According to the previous findings 2, 19 , the absolute configuration at C-2 of 3 was proposed as S from the large dextrorotatory optical rotation {½α 25 D þ98 (c 0.1, CHCl 3 )}. The absolute configuration of 3 was tentatively assigned as 2S,11R,12R. Hence, the structure of 3 was shown to be (þ)-17hydroxyisosarcophytoxide.
Compound 4 was obtained as a colorless oil with the molecular formula of C 20 H 28 O 5 on the basis of (þ)-HR-ESI-MS ion peak at m/z 371.1822 [M þ Na] þ (Calcd. for C 20 H 28 O 5 Na, 371.1829) and 13 C NMR data ( Table 1), suggesting that 4 possessed 7 degrees of  unsaturation. Its IR spectrum exhibited a broad absorption at 3356 cm À 1 (OH) and strong absorptions at 1751 and 1678 cm À 1 , consistent with the presence of an α,β-unsaturated γ-lactone moiety. This were supported by the 13 C NMR signals at δ C 174.7 (C-16), 161.3 (C-1), 123.8 (C-15), and 79.1 (C-2) and UV absorption maxima at 246 and 275 nm 23 . The third oxygen atom was determined to be part of a trisubstituted epoxy ring, which was confirmed by the appearance of signals at δ H 2.69 (1 H, dd, J ¼ 7.8, 4.8 Hz, H-11) and δ C 61.7 (C-11) and 61.0 (C-12). The NMR signals of an oxygenated quaternary carbon and an exchangeable proton were observed at δ C 84.3 (C-8) and δ H 7. 35 (1 H, s, 8-OOH), respectively, strongly implying that the remaining 2 oxygen atoms were involved in a hydroperoxy group 24 . This conclusion was also supported by the significant downfield shift of the resonance for C-8 in 4 with respect to that of the corresponding carbon (δ C 72.6) for mayolide B, a known cembranoid with the same 14-membered ring substituted with a hydroxy group at C-8 previously isolated the soft coral Sinularia mayi 25 . Further analysis of the 13 C NMR and DEPT spectra of 4 displayed 20 signals for 4 methyls, 5 methylenes, 5 methines (3 olefinic and 2 oxygenated), 5 quaternary carbons (3 olefinic and 2 oxygenated), and one conjugated ester carbonyl carbon (C-16). The planar structure of 4 was extensively elucidated by 1 H-1 H COSY and HMBC spectra (Fig. 2). The 1 H-1 H COSY cross-peaks readily determined the presence of 4 spin systems from H-2 to H-3; H 2 -5 to H-7; H 2 -9 to H-11; and H 2 -13 to H 2 -14. The significant HMBC correlations from Me-17 to C-1, C-15, and C-16; Me-18 to C-3, C-4, and C-5; Me-19 to C-7, C-8, and C-9; Me-20 to C-11, C-12, and C-13;H-2 to C-1, C-15, and C-16; and H 2 -14 to C-1 and C-15 constructed the cembrane skeleton as depicted in Fig. 1. The hydroperoxy group and the epoxy ring in 4 were placed at C-8 and C-11/C-12, respectively, based on the strong HMBC correlations from Me-19 to C-8 and from Me-20 to C-11 and C-12.
The relative configuration of 4 was deduced from interpretation of the coupling constants and a NOESY experiment (Fig. 3). The large coupling constants (J 6,7 ¼ 15.6 Hz) and the chemical shift of the C-18 methyl group (δ C 17.1) established the E geometries of the Δ 3 and Δ 6 double bonds, and this was further supported by the strong NOESY cross-peaks of H-6 (δ H 5.97)/Me-18 (δ H 1.83) and Me-19 (δ H 1.47) and of H-5a (δ H 2.86)/H-3 (δ H 4.91) and H-7 (δ H 5.62). The observed correlations in the NOESY spectrum as shown in Fig. 3 assigned H-11, Me-19, and Me-20 as β-oriented, while H-2 was the α-orientation. Furthermore, the absolute configuration at C-2 in 4 was defined by an ECD experiment. The ECD spectrum of 4 (Fig. 5) showed a negative Cotton effect at 249 nm (Δɛ ¼ -3.1) and a positive Cotton effect at 221 nm (Δɛ ¼ þ33.4), indicative of a 2Sconfiguration [26][27][28] . Accordingly, the structure of 4 was characterized as shown, and the compound was designated with the trivial name sarcomililatin A.
Compound 5 was isolated as a colorless oil, possessing the same molecular formula of C 20 (Table 2) of 6 also revealed similarities to 4, except for the location of one of the double bond from the Δ 3 to Δ 2 accompanied by hydroxylation occurring at C-3 in 6. These observations were supported by the HMBC correlations (Fig. 2) from the olefinic proton H-3 resonating at δ H 5.31 to C-1 (δ C 151.0), C-2 (δ C 147.8), C-4 (δ C 73.7), C-5 (δ C 47.0), and C-18 (δ C 29.6) and from the methyl proton singlet H 3 -18 (δ H 1.55) to C-3 (δ C 115.6), C-4, and C-5. Its relative configurations at C-8, C-11, and C-12 were proven the same as those of 4 on the basis of a NOESY experiment (Fig. 3). The diagnostic NOESY cross-peaks of H 3 -19/H-7 and of H 3 -18/one of the H 2 -5 protons (δ H 2.53) and H-6 (δ H 5.81) suggested that Me-18 was α-oriented. Finally, from a biogenetic point of view, the   absolute configurations at C-8, C-11, and C-12 of 6 were suggested to be the same as those of 4. The absolute configuration of 6 was tentatively defined as 4S,8R,11R,12R. Accordingly, the structure of 6 (the trivial name sarcomililatin C) was characterized as depicted.  (Table 2) of 7 showed similarities to those of 6. However, the signal resonating at δ H 7.40 (1 H, s) for the hydroperoxy group at C-8 in 6 was absent. Also, the 2 mutually coupled olefinic proton signals at δ The relative configurations of all the asymmetric centers in 7 remained intact, with respect to those of 6, which was supported by a NOESY experiment. Analogously, the absolute configurations at C-11 and C-12 of 7 were suggested to be the same as those of 4. The absolute configuration of 7 was tentatively defined as 4S,11R,12R. Therefore, the structure of 7 (the trivial name sarcomililatin D) was proposed as depicted.
Compound 8, assigned the trivial name sarcomililatol, was isolated as a colorless oil and exhibited the molecular formula C 20 H 32 O 2 as determined by its HR-EI-MS peak at m/z 304.2409 [M] þ (Calcd. for C 20 H 32 O 2 , 304.2402), suggestive of 5 degrees of unsaturation, of which one was accounted for by a 14-membered macrocyclic ring, 3 by 2 olefinic bonds, and one by an ether linkage. The intense IR absorption at 3362 cm À 1 was indicative of the presence of a hydroxyl group. The 1 H NMR spectrum (  3 -19). The 13 C NMR spectrum (Table 2) showed the presence of 20 carbon signals which were classified with the aid of DEPT and HSQC experiments as 5 methyl, 5 methylene, 6 methine (3 sp 2 hybridized and 2 oxygenated), and 4 quaternary (2 sp 2 hybridized and one oxygenated) carbons. The aforementioned data of 8 revealed that it should be a stereoisomer of (þ)-11,12epoxysarcophytol A, which was first isolated from the soft coral Lobophytum sp. 30 . When comparing their 13 CNMR spectra, the signals of C-11and C-12 were shown to be markedly different [δ C 62.4 and 61.6 for 8; δ C 58.4 and 59.8 for (þ)-11,12epoxysarcophytol A, respectively], indicating that the structural difference between them resided in the different configuration of the 11,12-ether linkage. Detailed analysis of its 1 H-1 H COSY, HSQC, and HMBC spectra (Fig. 2) allowed the assignment for all proton and carbon resonances of 8.
The relative configuration of 8 was deduced from interpretation of a NOESY experiment (Fig. 3) Thus, the relative configuration of 8 was determined as 1Z,3E,7E,11R*,12R*,14S*. However, the absolute configuration of the chiral center at C-14 in 8 was not determined by a modified Mosher's method due to the limited quantity of sample.
In order to determine the absolute configuration of 8, the TD-DFT ECD calculation method was applied, which has proven to be a powerful and reliable method for the elucidation of the absolute configurations of natural products 9 . As shown in Fig. 6, the ECD spectrum (CH 3 CN) of 8 displayed a positive π Àπ* Cotton effect at 242 nm. The conformational searches of (11R,12R,14S)-8 were carried out using the torsional sampling (MCMM) method and OPLS_2005 force field. Conformers above 1% population were re-optimized at the B3LYP/6-311 G(d,p) level with IEFPCM (Polarizable Continuum Model using the Integral Equation Formalism variant) solvent model for acetonitrile ( Supplementary Information Fig. S1 and Table S1). For the resulting geometries, ECD spectra were obtained by TD-DFT calculations performed with the same functional basis set and solvent model as the energy optimization. Finally, the Boltzmannaveraged ECD spectrum of (11 R,12 R,14 S)-8 highly matched to the experimental ECD spectrum of 8, while the enantiomer showed completely opposite curve. Consequently, the absolute configuration of 8 was determined to be 11R,12R,14S.
The growth inhibitory potential towards various cancer cell lines of numerous cembrane-type diterpenoids has been documented widely. Accordingly, the cytotoxicities of all the isolates were evaluated in vitro against HL-60 and A-549 by using the MTT and SRB methods, respectively. However, all of the tested compound, except for the known compound (þ)-isosarcophytoxide (9), were inactive (IC 50 410 μmol/L). Compound 9 exhibited strong cytotoxic activities, with IC 50 values of 0.7870.21 and 1.2670.80 μmol/L against HL-60 and A-549 cells, respectively, comparable to the positive control (adriamycin, IC 50 0.07 μmol/L for HL-60 and 0.01 μmol/L for A-549). In addition, all of the isolated compounds were also subjected to testing in vitro for their inhibitory activities against the tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB, a transcription factor that plays a critically important role in regulation of cell cycle as well as influencing cell death pathways and has been used as a key target for the treatment of inflammatory diseases and cancer 32,33 . The results indicated that sarcomilitatin A (4) and (þ)-isosarcophytoxide (9)

General experimental procedures
Optical rotations were measured on a Perkin-Elmer 241MC polarimeter (PerkinElmer, Fremont, CA, USA). UV spectroscopic spectra were recorded in chromatographic grade CH 3 OH on a Varian Cary 300 UVÀVis spectrophotometer (Varian, Palo Alto, CA, USA); peak wavelengths are reported in nm. ECD spectra were recorded on a Jasco J-810 spectropolarimeter (JASCO, Japan) at ambient temperature using chromatographic grade CH 3 OH and acetonitrile as solvent. IR spectra were recorded on a Nicolet 6700 spectrometer (Thermo Scientific, Waltham, MA, USA); peaks are reported in cm -1 . The NMR spectra were measured at 300 K on Bruker DRX 400 and Avance 600 MHz NMR spectrometers (Bruker Biospin AG, Fallanden, Germany). Chemical shifts are Scheme 1 Putative biosynthetic pathways toward the formation of compounds 4-6.
reported in parts per million (δ) in CDCl 3 (δ H reported referred to CHCl 3 at 7.26 ppm; δ C reported referred to CDCl 3 at 77.16 ppm) and coupling constants (J) in Hz; assignments were supported by 1 H-1 H COSY, HSQC, HMBC, and NOESY experiments. EIMS and HREIMS spectra (70 eV) were recorded on a Finnigan-MAT-95 mass spectrometer (ThermoFisher Scientific, Waltham, USA). ESI-MS and HR-ESI-MS were carried out on a Bruker Daltonics Esquire 3000 plus instrument (Bruker Daltonics K. K., Kanagawa, Japan) and a Waters Q-TOF Ultima mass spectrometer (Waters, MA, USA), respectively. Semi-preparative HPLC was performed on an Agilent-1260 system equipped with a DAD G1315D detector using ODS-HG-5 (250 mm Â 9.4 mm, 5 mm) by eluting with CH 3 OH-H 2 O or CH 3 CN-H 2 O system at 3 mL/min. Commercial silica gel (200À300 and 400À500 mesh; Qingdao, China) was used for column chromatography (CC). Precoated SiO 2 plates (HSGF-254; Yantai, China) were used for analytical TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde H 2 SO 4 reagent. Sephadex LH-20 (Amersham Biosciences) was also used for CC. All solvents used for extraction and isolation were of analytical grade.

Extraction and isolation
The frozen animals (170 g, dry weight) were cut into pieces and ultrasonically extracted with acetone at room temperature (1 L Â 3). The combined acetone extracts were filtered and concentrated in vacuo, affording a brown residue, which was suspended in H 2 O (4 L) and then partitioned with Et 2 O (3 times with 2 L each). The Et 2 O-soluble portion (5.0 g) was concentrated in vacuo, and then fractionated by silica gel CC (column: 40 cm Â 8 cm) eluting with petroleum ether (PE, 60-90 1C)-acetone (98:2-0:100) to afford nine fractions (A-I), which were combined on the basis of the analysis of TLC.

(þ)-(6R)-6-
where k is the Hill coefficient. Bortezomib was used as a positive control with an IC 50 value of 0.03 μmol/L.