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Mar. Drugs 2013, 11(11), 4318-4327; doi:10.3390/md11114318
Published: 30 October 2013
Abstract: Continuing chemical investigation on the acetone extracts of the soft coral Sarcophyton ehrenbergi collected off the coast of San-hsian-tai, Taitong County, Taiwan led to the isolation of two new diterpenoids, ehrenbergol C and acetyl ehrenberoxide B (1 and 2). The structures of these isolated metabolites were elucidated through extensive spectroscopic analyses. Moreover, in vitro tests show that compounds 1 and 2 displayed antiviral activity towards human cytomegalovirus, with EC50 of 20 and 8.0 µg/mL, respectively.
Marine organisms, which have developed unique metabolic and physiological capabilities to ensure survival in extreme marine habitats, offer the potential to produce novel bioactive secondary metabolites that would not be produced by terrestrial organisms . Soft corals of the genus Sarcophyton have been reported as a rich source of diterpenoids . These constituents, mainly macrocyclic cembranes and their derivatives, represent important chemical defense substances for the animals against their natural predators . Cembranoids have been previously reported to exhibit a range of biological activities including antitumor [3,4,5,6,7,8,9], ichthyotoxic , anti-inflammatory , neuroprotective , antibacterial , antiangiogenic , antimetastatic , anti-osteoporotic , cytotoxic [16,17,18] and antiviral properties [19,20].
Fifteen cembranoids were previously reported from the Taiwanese soft coral Sarcophyton ehrenbergi [19,20,21]. Continuing chemical investigation of the soft coral S. ehrenbergi (Figure 1) collected at San-Hsian-Tai (Taitong County, Taiwan) resulted in the isolation of two new diterpenoids, designated as ehrenbergol C and acetyl ehrenberoxide B (1 and 2) (Figure 2). Herein, we describe the purification, structure elucidation, cytotoxicity and antiviral evaluation of these metabolites.
2. Results and Discussion
Compound 1 was isolated as a colorless oil, [α]25D +95.0 (c 0.2, CHCl3). The IR spectrum of 1 exhibited absorptions due to hydroxyl (3444 cm−1) and conjugated enone (1696 cm−1) functionalities. The presence of the conjugated enone was also confirmed by the UV spectrum [λmax (log ε) 223 nm (3.42)]. HRESIMS exhibited a pseudo molecular ion peak at m/z 401.1939 [M + Na]+, consistent with the molecular formula of C21H30O6.
The structure of 1 was solved by a combination of 1D and 2D NMR methods. The resonances at δC 205.9 (qC), 141.4 (qC), and 168.9 (qC), in the 13C NMR and DEPT spectra suggested the presence of a tetrasubstituted conjugated enone (Table 1). Furthermore, the presence of four oxygenated carbons was inferred from the carbon signals at δC 73.9 (qC), 83.2 (qC), 85.6 (CH), and 75.4 (CH). NMR spectroscopic data [δH 6.76 (1H, t, J = 7.2 Hz) and 3.75 (3H, s); δC 168.2 (C, C-20), 130.7 (C, C-12), 144.7 (CH, C-11), and 51.6 (CH3, COOMe)] revealed the presence of an α,β-unsaturated methyl ester functionality. Six methylene groups were deduced from six triplet signals at δC 36.6, 34.3, 28.5, 24.3, 23.8, and 21.8, a methine signal at δC 60.8, and, finally, three methyl signals at δC 13.4, 28.4, and 24.2.
|Table 1. NMR data for compound 1.|
|Position||δH a (J in Hz)||δC b, type||HMBC||COSY||NOESY|
|3||2.13, d (2.0)||60.8, CH||2, 4, 16||16, 18||5a|
|5a||3.11, ddd (12.0, 8.8, 3.2)||34.3, CH2||6b, 18||3|
|6a||1.56, m||28.5, CH2||7|
|6b||1.84, m||7||5a, 7||7|
|7||3.82, dd (10.4, 6.0)||85.6, CH||8, 9||6a, 6b||6b, 19|
|9a||1.59, m||36.6, CH2||8, 10, 11|
|9b||1.45, m||8, 10, 11||10|
|10a||1.98, m||9b, 11||13b|
|10b||1.99, m||23.8, CH2||9, 12||9b, 11||11, 19|
|11||6.76, t (7.2)||144.7, CH||9, 10, 20||10||10b|
|13a||2.46, m||24.3, CH2||11, 12, 14, 20||14|
|13b||2.63, m||1, 11, 12, 14, 20||14||14a|
|14a||2.46, m||21.8, CH2||1, 2, 13, 15||13||13b|
|16||4.92, d (6.0)||75.4, CH||1, 4, 15||3, 17||17, 18|
|17||1.94, s||13.4, CH3||1, 15, 16||16||14b, 16|
|18||1.42, s||28.4, CH3||3, 4, 5||5a||7, 5b, 16|
|19||1.09, s||24.2, CH3||7, 8, 9||6a, 7, 10b|
|OMe||3.75, s||51.6, CH3||20|
a Spectra were measured in CDCl3 (400 MHz); b Spectra were measured in CDCl3 (100 MHz).
The combined use of 1H–1H COSY and HMQC on 1 allowed us to distinguish four spin systems (Figure 3a–d). A HMBC experiment was used to assemble the skeletal fragments through quaternary carbons and heteroatoms. Thus, these substructures were connected through HMBC correlations between the protons H2-14 (δH 2.46) and the carbons C-1 (δC 141.4), C-2 (δC 205.9), C-15 (δC 168.9), and C-12 (δC 130.7), between the methine proton H-3 (δH 2.13) and carbon C-2, between the methyl protons Me-19 and the carbon C-7, C-8 and C-9, between the methyl protons Me-18 (δH 1.42) and carbons C-3 (δC 60.8), C-4 (δC 83.2), and C-5 (δC 34.3), and between H2-13/H-11/OMe-20 and the carbon C-20. These relationships are represented in Figure 3.
All these data allowed us to identify compound 1 having the same planar framework as lobocrasol isolated from soft coral Lobophytum crassum . With the gross structure of 1 in hand, the relative stereochemistry of compound 1 was deduced from NOESY correlations and Chem3D Ultra 9.0 (Figure 4). The Z geometry of the Δ11 double bond was established by the NOESY correlation observed between H-11 and H-10b and between H-10a and H-13b. NOESY correlations between H-7/H3-19, H-7/H3-18, and H3-18/H-3 indicated that these protons are on the same face of the ring system, thereby establishing the relative configuration of 1. The relative stereochemistry of C-7 and C-8 were different from lobocrasol; however, the absolute structure was not determined due to the limited amount of the sample.
Compound 2 analyzed for C22H36O4 from HRESIMS and 13C NMR spectroscopic data (Table 2), corresponding to five degrees of unsaturation. The IR spectrum of 2 at 3445 cm−1 demonstrated a broad absorption band diagnostic of hydroxy group. The presence of one oxygenated methine [δH 4.05 (t, 1H, J = 3.2 Hz) and δC 75.3 (C-7)] and an oxygenated quaternary carbon [δC 80.4 (C-12)] implied that an oxygen bridge is probably present between C-7 and C-12, which was supported by the HMBC correlations from H-7 to C-12. The NMR spectroscopic data (Table 2) indicated that 2 possesses an acetoxy [δH 1.66 (3H, s); δC 169.8, 22.1] and a conjugated diene [δH 6.05 (1H, d, J = 8.4 Hz) and 6.37 (1H, d, J = 8.4 Hz); δC 151.0 (C, C-1), 117.9 (CH, C-2), 123.5 (CH, C-3), and 132.6 (C, C-4)]. The above functionalities suggest that metabolite 2 must consist of a 14-membered ring diterpenoid incorporating an oxepane ring, a hydroxy, an acetoxy and a conjugated diene. Correlations deduced from extensive analyses of the 1H–1H COSY correlations of 2 in C6D6 enabled initially the establishment of five partial structures. The structural fragments were subsequently interconnected by the HMBC correlations (Figure 3). Two oxygen bearing carbons at δC 87.0 (C) and 78.3 (CH) were ascribable to C-8 and C-11 on the basis of the HMBC correlations from Me-19 to C-7, C-8, and C-9 and from Me-20 to C-11, C-12, and C-13. The attachment of isopropyl to C-1 was established on the grounds of HMBC correlations from Me-16/Me-17 to C-15 and C-1. The positions of the conjugated double bonds at C-1/C-2 and C-3/C-4 were confirmed by the HMBC cross-peaks from Me-18 to C-3, C-4, and C-5, as well as a COSY correlation between H-2 and H-3. The planar structure of compound 2 was thus elucidated. The relative configuration and the detailed 1H NMR spectroscopic data assignments of 2 were determined mainly by the assistance of the NOESY experiment (Figure 5). The crucial NOE correlations between H-2/H-3, H-2/Me-16, H-2/Me-18, H-2/H-15, H-3/H-7 (δH 4.05), and H-3/H-14a (δH 2.64) indicated that the geometries of the two olefins at C-1/C-2 and C-3/C-4 were assigned as both E. The coupling constant between H-2 and H-3 (J2,3 = 8.4 Hz)  further suggested the s-trans geometry of the conjugated double bonds. Furthermore, the crucial NOE correlations between H-7/H-9a (δH 2.13), H-11/H-9a, H-11/H-10a (δH 1.74), H-11/H-13a (δH 1.97), Me-19/H-6b (δH 1.84), Me-19/H-10b (δH 1.78), Me-20/H-14b, Me-20/H-10b, and H-3/H-14a (δH 2.64) demonstrated the 7R*, 8S*, 11S*, and 12R* configurations as depicted in Figure 5. Accordingly, the structure of 2 was determined as (7R*,8S*,11S*,12R*,1Z,3E)-8-acetoxy-11-hydroxy-7,12-epoxycembra-1(2),3-diene.
|Table 2. NMR data for compound 2.|
|Position||δH a (J in Hz)||δC b, type||HMBC||COSY||NOESY|
|2||6.05, d (8.4)||117.9, CH||14, 15||3, 15, 16, 18|
|3||6.37, d (8.4)||123.5, CH||5, 18||18||2, 7, 13a, 14a|
|5a||2.10, m||37.9, CH2||2||6a||6b|
|6a||2.04, m||30.8, CH2||5a, 7|
|6b||1.84, m||7||5a, 19|
|7||4.05, t (3.2)||75.3, CH||6a, 6b||3, 9a, 13a|
|9a||2.13, m||36.6, CH2||7, 11|
|10a||1.74 m||29.2, CH2||9b|
|10b||1.08, m||11||19, 20|
|11||3.16, dd (10.0, 2.8)||78.3, CH||10b||9a, 10a, 13a|
|13a||1.97, m||37.4, CH2||12||14a||3, 7, 11|
|14a||2.64, m||24.3, CH2||1||13a||3|
|15||2.32, m||36.9, CH||1, 16, 17||2, 16, 17|
|16||1.06, d (6.8)||21.5, CH3||1, 15, 17||15||2, 15|
|17||1.04, d (6.8)||21.8, CH3||1, 15, 16||15||15|
|18||1.76, s||17.9, CH3||3, 4, 5||3||2|
|19||1.48, s||18.9, CH3||7, 8, 9||6b, 10b|
|20||1.11, s||18.1, CH3||11, 12, 13||10b, 14b|
|OAc||1.66, s||22.1, CH3||21|
a Spectra were measured in C6D6 (400 MHz); b Spectra were measured in C6D6 (100 MHz).
The cytotoxicities of compounds 1 and 2 against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells are shown in Table 3. Compounds 1 and 2 were also examined for antiviral activity against human cytomegalovirus (HCMV) using a human embryonic lung (HEL) and displayed antiviral activity against human cytomegalovirus, with EC50s of 20 and 8.0 µg/mL, respectively.
|Table 3. Cytotoxicity and anti-HCMV activity of 1 and 2.|
3. Experimental Section
3.1. General Experimental Procedures
Optical rotations were determined with a JASCO P1020 digital polarimeter. UV and IR spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. NMR spectra were recorded on a Varian MR 400 NMR spectrometer at 400 MHz for 1H and 100 MHz for 13C. 1H NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δH 7.27 for CHCl3 or δH 7.15 for C6D6, and coupling constants are expressed in Hertz (Hz). 13C NMR chemical shifts are expressed in δ (ppm) referring to the solvent peak δC 77.0 for CDCl3 or δC 128.0 for C6D6. MS were recorded by a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, Germany, 230–400 mesh) and LiChroprep RP-18 (Merck, 40–63 µm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for thin-layer chromatography (TLC) analysis. High-performance liquid chromatography (HPLC) was carried out using a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 220 nm together with a semi-preparative reversed-phased column (Merck, Hibar LiChrospher RP-18e, 5 µm, 250 × 25 mm).
3.2. Biological Material
The soft coral S. ehrenbergi was collected by SCUBA at San-Hsian-Tai, Taitong County, Taiwan, in July 2009 at a depth of 6 m and stored in a freezer until extraction. The voucher specimen (ST-13) was identified by Professor Chang-Feng Dai, National Taiwan University and deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.
3.3. Extraction and Isolation
A specimen of soft coral S. ehrenbergi (2.0 kg) was minced and extracted with acetone (4 × 2 L) at room temperature. The combined acetone extracts were then partitioned between H2O and EtOAc. The resulting EtOAc extract (23.8 g) was subjected to gravity silica gel 60 column chromatography (Si 60 CC) using n-hexane and n-hexane/EtOAc of increasing polarity, to give 20 fractions. Fraction 14 (2.0 g), eluted with n-hexane/EtOAc (10:1), was further subjected to Sephadex LH-20 (acetone) to give 7 subfractions. The fraction 14-2-2 (0.108 g), was further subjected to RP-18 flash column (MeOH/H2O, 60:40 to 100% MeOH) to give 5 fractions. A subfraction 14-2-2-4 (12.9 mg), was purified by RP-18 HPLC (MeOH/H2O, 85:15) to afford 2 (2.3 mg, 0.0012%).The fraction 19 (0.2 g), eluted with n-hexane/EtOAc (1:8), was further subjected to RP-18 flash column (MeOH/H2O, 50:50 to 100% MeOH) to give 6 fractions. The subfraction 19-1, eluted with MeOH/H2O (50:50), was purified by RP-18 HPLC (MeOH/H2O, 50:50) to afford 1 (2.1 mg, 0.001%).
Ehrenbergol C (1): White amorphous powder; +95.0 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 223 (3.42) nm; IR (neat) νmax 3444, 2975, 2929, 1696, 1652, 1439, 1385, 1284, 1192, 1087, 1038, 754 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data in Table 1; HRESIMS m/z 401.1939 [M + Na]+ (calcd for C21H30O6Na, 401.1940).
Acetyl ehrenberoxide B (2): White amorphous powder; +25.0 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 242 (3.2) nm; IR (neat) νmax 3445, 2957, 2871, 1733, 1456, 1377, 1258, 1088, 1034, 772 cm−1; 1H NMR (C6D6, 400 MHz) and 13C NMR (C6D6, 100 MHz) data in Table 1; HRESIMS m/z 387.2512 [M + Na]+ (calcd for C22H36O4Na, 387.2511).
3.4. Cytotoxicity Assay
Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [24,25,26]. The provision of the P-388 cell line was supported by J.M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection. To measure the cytotoxic activities of tested compounds, five concentrations with three replications were performed on each cell line. Mithramycin was used as a positive control.
3.5. Anti-HCMV Assay
To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products with three replications. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of a 24-well dish. Antiviral activity was expressed as IC50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [26,27,28].
This investigation of Taiwanese soft coral S. ehrenbergi collected has led to the isolation of two new ehrenbergol C and acetyl ehrenberoxide B (1 and 2). The carbon framework of 1 was identical to a cytotoxic diterpene, lobocrasol isolated from soft coral Lobophytum crassum. However, the stereochemistry of C-7 and C-8 of 1 were different from lobocrasol. Compounds 1 and 2 were not cytotoxic towards P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma) tumor cells, and human embryonic lung (HEL) cells. However, compounds 1 and 2 displayed antiviral activity towards human cytomegalovirus, with IC50 of 20 and 8.0 μg/mL, respectively.
This research was financially supported by grants from the National Science Council (NSC102-2320-B-110-003-MY3) and NSYSUKMU Joint Project (NSYSUKMU 2013-P018) awarded to Chang-Yih Duh.
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