Letter - spectral assignments Received: 11 March 2014
Revised: 16 June 2014
Accepted: 19 June 2014
Published online in Wiley Online Library: 10 July 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4108
Four new cembranoids from the soft coral Sarcophyton sp. Zhong-Bin Cheng,a† Qiong Liao,a,b† Ye Chen,a Cheng-Qi Fan,c Zhi-Ying Huang,a,b Xin-Jun Xua* and Sheng Yina* Introduction Soft corals belonging to the genus Sarcophyton have been well recognized as a rich source of secondary metabolites endowed with a range of structural diversity and various biological activities. Members of this genus have been studied widely, resulting in the identification of unprecedented diterpenoids[1–3] and steroids.[4] Some of these metabolites are of considerable interest and merit continuous attention due to their unique structures and significant biological activities, including inhibition of protein tyrosine phosphatase 1B,[2] anti-tumor,[3] antimicrobial,[4] and anti-inflammatory properties.[5] As part of our continuing efforts to discover structurally intriguing biologically significant metabolites from marine invertebrates of the South China Sea,[6,7] we undertook a detailed chemical analysis of Sarcophyton sp., which led to the isolation of four new cembranoids (1–4), together with eight known compounds including five cembranoids (5–9), two carotenoids (10–11), and a tetra-substituted quinone (12) (Fig. 1). Herein, the details of the isolation, structure elucidation, and activity of these compounds are described.
Results and Discussion
Magn. Reson. Chem. 2014, 52, 515–520
* Correspondence to: Xin-Jun Xu and Sheng Yin, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China. E-mail: [email protected]; [email protected] †
Zhong-Bin Cheng and Qiong Liao contributed equally to this work.
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China b Center of Laboratory Animals, Sun Yat-sen University, Guangzhou, Guangdong 510006, China c East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China
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The frozen animal of Sarcophyton sp. (500 g) was extracted with MeOH/CH2Cl2 (1:1) to give a crude extract, which was dispersed in H2O and successively partitioned with petroleum ether and EtOAc. The EtOAc extract was subjected to various column chromatographies to afford compounds 1–12. Compound 1 had a molecular formula of C23H36O4 as established by HRESIMS at m/z 399.2512 [M + Na]+ (calcd 399.2506). The hydrogen-1 nuclear magnetic resonance (1H NMR) spectrum of 1 displayed signals of six methyl singlets at δH 1.32 (3H, s, H3-17), 1.33 (3H, s, H3-16), 1.80 (3H, s, H3-18), 1.28 (3H, s, H3-19), 2.07 (3H, s, 20-OAc), and 3.05 (3H, s, 15-OCH3), three olefinic protons at δH 6.26 (1H, d, J = 10.5 Hz, H-2), 5.95 (1H, d, J = 10.5 Hz, H-3), and 5.45 (1H, t, J = 6.7 Hz, H-11), an oxymethine at δH 2.85 (1H, dd, J = 6.0, 4.2 Hz, H-7), an oxymethylene [δH 4.68 (1H, d, J = 12.1 Hz, H-20a) and 4.59 (1H, d, J = 12.1 Hz, H-20b)], and a series of aliphatic methylene multiplets. The carbon-13 nuclear magnetic resonance (13C NMR) spectrum, in combination with DEPT experiments, resolved 23 carbon resonances attributable to a carbonyl, six methyls, three trisubstituted double bonds, an epoxy moiety,[8,9] seven sp3 methylenes including an oxymethylene carbon, and an oxygenated quaternary carbon. The aforementioned information showed high similarity to that of the co-occurring known analog (1E,3E,7R*,8R*,11E)-1-(2-methoxypropan-2-yl)-4,8,12-trimethyloxabicyclo[12.1.0]-pentadeca-1,3,
11-triene (6)[10] except for the absence of CH3-20 (δH 1.63, δC 15.8 ) in 6 and the presence of an acetylated hydroxymethyl unit (δH 4.68, 4.59, and 2.07; δC 21.0, 61.9, and 171.0) in 1, suggesting that the CH3-20 in 6 was replaced by an acetoxymethyl in 1. This was supported by HMBC correlations (Fig. 2) of H-20 with C-12, C-13, C-14, and the carbonyl (δC 171.0). Detailed 2D NMR (1H 1H COSY and HMBC) analyses of 1 established the gross structure of 1 as depicted (Fig. 2). The relative configuration of 1 was determined to be the same as that of 6 by NOESY experiment and by comparison of their 13C NMR data. In particular, the crucial NOE correlations (Fig. 3) of H-2/H3-18, H-3/H-7, H-11/H2-13, H2-20/H2-10 and the large coupling constant (J2,3 = 10.5 Hz)[10] indicated that the configuration of the conjugated diene Δ1, Δ3, and Δ11 were E, E, and Z, respectively. Moreover, the NOESY correlations of H-7/H-5α, H-9α and H3-19/H2-6 assigned H-7 and CH3-19 to be α and β oriented, respectively. Thus, compound 1 was determined to be (1E,3E,7R*,8R*,11Z)15-methoxy-20-acetoxymethyl-7,8-epoxycembra-1,3,11-triene and named sarcophyton A. Compound 2, a colorless oil, had the molecular formula C20H30O3, as determined by the HRESIMS at m/z 341.2069 [M + Na]+ (calcd 341.2087). The NMR spectroscopic features indicated that 2 had a similar structure as that of sinumaximol E,[5] except for the presence of an extra trisubstituted double bond [(δH 4.99 (1H, t, J = 6.0 Hz, H-11); δC 123.4 (C-11), 134.9 (C-12)] and an additional olefinic methyl [δH 1.65 (3H, s, H3-20); δC 17.9 (C-20)] in 2 instead of the terminal double bond [δH 4.94 (1H, br s), 4.99 (1H, br s); δC 150.0 (C-12), 113.1 (C-20)] and the oxymethine [δH 4.12 (dd, J = 4.5, 7.0 Hz, H-11), δC 75.3 (C-11)] in sinumaximol E. The trisubstituted double bond and the olefin methyl was located by the 1H 1H COSY correlations (Fig. 2) of H-10/H-11, H13/H-14 and by HMBC correlations (Fig. 2) of H-20/C-11, C-12, and C-13. Detailed analyses of the 2D NMR spectra of 2
Z.-B. Cheng et al.
Figure 1. Structures of compounds 1–12.
1
Figure 2. Selected H
1
H COSY (–) and HMBC (→) correlations of 1–4.
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confirmed the gross structure of 2 (Fig. 2). The Z configuration of Δ1,14 was determined to be the same as that of sinumaximol E by comparing the 13C chemical shift values of C-1 and C-14 with those of sinumaximol E (δC 147.0 and 123.4 in 2, δC 146.5, 123.3 in sinumaximol E). The NOESY correlations (Fig. 3) of H-2/H3-18, H-3/H-5α, H3-20/H2-10, and H-11/H-13α assigned both Δ3 and Δ11 as E configuration. The CH3-17 was assigned in α-orientation based on the NOESY correlations of H-2/H-16β, H3-17/H-16α, H-11, and the lack of the correlations between H-2 and H3-17. The H-7 and CH3-19 were assigned to be α and β-oriented, respectively, by comparison of the 13C chemical shift values of C-7 and C-8 of 1 and 2 (ΔδC < 0.3 ppm) and by NOESY correlations of H3-19/H2-6, H-10β and H-7/H-9α, H5α, H-11, H-3 (Fig. 3). Thus, compound 2 was determined to be (1Z,2S*,3E,7S*,8S*,11E,15S*)-15-hydroxy-7,8:2,16-bisepoxycembra1(14),3,11-trien and named sarcophyton B. Compound 3 was isolated as a colorless oil, and its molecular formula was assigned as C20H30O2 based on the sodiated molecular ion peak at m/z 325.2138 [M + Na]+ (calcd 325.2140). Detailed analysis of the 1H NMR spectrum revealed the presence of four
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tertiary methyls including three olefinic methyl singlets [δH 1.28 (3H, s, H3-19), 1.65 (3H, s, H3-17), 1.78 (3H, s, H3-18), and 1.58 (3H, s, H3-20)], three olefinic protons [δH 5.11 (t, J = 6.7 Hz, H-11), 5.17 (1H, d, J = 9.9 Hz, H-3), and 5.68 (1H, dt, J = 15.8, 5.7 Hz, H-6)], an oxymethylene group [δH 4.53 (1H, d, J = 11.6 Hz, H-16α) and 4.46 (1H, d, J = 11.6 Hz, H-16β)], an oxymethine proton [δH 5.42 (1H, J = 9.9 Hz, H-2)], and several aliphatic multiplets. The 13C NMR spectrum displayed signals for four double bonds, four methyls, six methylenes including an oxymethylene carbon, an oxymethine, and an oxygenated quaternary carbon. Aforementioned spectroscopic data resembled those of (+)-7β,8βdihydroxydeepoxysarcophytoxid[11] except for the presence of a disubstituted double bond [δH 5.68 (H-6), 5.57 (H-7); δC 123.9 (C-6), 139.4 (C-7)] in 3 instead of the methylene at C-6 (δC 29.8) and the oxymethine at C-7 (δC 74.4) in (+)-7β,8β-dihydroxydeepoxysarcophytoxid, indicating 3 was the C-6 C-7 dehydrated derivative of (+)-7β,8βdihydroxydeepoxysarcophytoxid and was upheld by the MS data. The postulated gross structure of 3 was confirmed by detailed 2D NMR analyses of 3 (Fig. 2). The configurations of the double bonds Δ1(15), Δ3, and Δ11 were assigned to be Z, E, and E, respectively, by comparison of the 1D NMR data of 3 with those of (+)-7β,8βdihydroxydeepoxysarcophytoxid and by NOESY experiment (Fig. 3). The coupling constant between H-6 and H-7 (J = 15.8 Hz) defined Δ6 to be E configuration. The OH-8 was assigned in α-orientation by analysis of its C-19 chemical shift, as the chemical shift value for the C-19 methyl group is very sensitive to the configuration at C-8,[12] and the value of δC 28.4 for C-19 in 3 is in good agreement with that of the 8α-hydroxyl isomer crassumolide F (δC 27.2), while the 8β-hydroxyl analogs usually resonated at δC 24.8.[13] Thus, compound 3 was determined to be (1Z,2S*,3E,6E,8S*,11E)-8-hydroxy-2,16-epoxycembra-1(15),3,6,11tetraene and named sarcophyton C. Compound 4, a colorless oil, had the molecular formula C20H34O2, as determined by the HRESIMS at m/z 329.2435 [M + Na]+ (calcd 329.2451). The 1D NMR data of 4 were closely comparable with those of (+)-7,8-epoxy-7,8-dihydrocembrene C[14] except that the epoxy functionality [δH 2.83, δC 61.6, 60.1]
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 515–520
Four new cembranoids from the soft coral Sarcophyton sp.
Figure 3. Key NOE correlations (↔) of 1–4.
Magn. Reson. Chem. 2014, 52, 515–520
Experimental General experimental procedures Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. ESIMS was measured on a Finnigan LCQ Deca instrument, and HRESIMS was performed on a Waters Micromass Q-TOF spectrometer. A Shimadzu LC-20 AT equipped with a SPD-M20A PDA detector was used for HPLC. A YMC-pack ODS-A column (250 × 10 mm, S5 μm, 12 nm) was used for semi-preparative HPLC separation. Silica gel (300–400 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography. All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.).
NMR spectroscopy Nuclear magnetic resonance spectra were recorded on a Bruker Avance-400 spectrometer equipped with a BBFO-5 mm probe in 0.4 mL CDCl3 at 300 K. Solvent signals were used as internal standard (CDCl3: δH = 7.26, δC = 77.0 ppm). The pulse conditions were as follows: For 1H NMR, spectrometer frequency (SF) = 400.13 MHz, spectral width (SW) = 8223.7, pulse 90 width (PW) = 12.9 μs, Fourier transform size (SI) = 32768, acquisition time (AQ) = 3.98, line broadening (LB) = 0.3 Hz, relaxation delay (RD) = 1.0 s, number of dummy scans (DS) = 2 and number of scans (NS) = 32; for 13 C, SF = 100.61 MHz, SW = 24038.5 Hz, PW = 10.3 μs, SI = 32768, AQ = 0.5 s, LB = 3 Hz, RD = 1.5 s, DS = 4, NS = 2400 (1) or 1024 (2) or 6000 (3)
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in (+)-7,8-epoxy-7,8-dihydrocembrene C had been replaced by a vicinal diol[15] [δH 3.53 (1H, d, J = 8.7 Hz), δC 71.5, 75.3] in 4. Detailed 2D NMR analyses of 4 confirmed the gross structure (Fig. 2). All three double bonds (Δ1, Δ3, and Δ11) were assigned as E configuration by comparison of its 1D NMR data with those of (+)-7,8-epoxy-7,8-dihydrocembrene C and were supported by the NOESY correlations of H-2/H3-17, H-2/H3-18, H-3/H2-5, H-11/H2-13, and H2-10/H3-20 (Fig. 3). The H-7 and CH3-19 were both assigned in α-orientation by the apparent NOESY correlations of H-7/H3-19, H-3, H-5α, H-10α, H3-20 and H3-19/H-6α, H-10α. Thus, compound 4 was determined to be (1E,3E,7S*,8S*,11E)7,8-dihydroxycembra-1,3,11-triene. The structure of 4 was very similar to that of (EEE)-7β,8α-dihydroxy-1-isopropyl-4,8,12trimethylcyclotetradeca-1,3,11-triene[16] with the only difference due to the cis-vicinal diol moiety in 4 instead of the transvicinal diol moiety in (EEE)-7β,8α-dihydroxy-1-isopropyl-4,8,12trimethylcyclotetradeca-1,3,11-triene. Compound 4 was named sarcophyton D. The structures of the known compounds were determined as 2-[(E,E,E)-7′,8′-epoxy-4′,8′,12′-trimethylcyclotetradeca-1′,3′,11′-trienyl] propan-2-ol (5),[17] (1E,3E,7R*,8R*,11E)-1-(2-methoxy-propan-2-yl)4,8,12-trimethyloxabicyclo[12.1.0]-pentadeca-1,3,11-triene (6),[10] crassumol C (7),[18] (+)-sarcophytoxide (8),[19] laevigatol A (9),[20] all-trans-(3S,5R,6R,3′S,5′R,6′S)-peridinin (10),[21] all-trans-(9′Z,11′Z)(3R,3′S,5′R,6′R)-pyrrhoxanthin (11),[22] and sarcophytonone (12)[23] by extensive spectroscopic analysis combined with careful comparison with the reported data. All isolated compounds were evaluated for cytotoxicities against the MCF-7 human breast cancer cell line. Only 5, 7, 9, and 12 exhibited very weak growth inhibitory effects with the inhibition greater than 10% at the concentration of 20 μM (11%, 11%, 13%, and 12%, respectively).
Z.-B. Cheng et al. or 3600 (4); for HSQC, SF = 400.13 MHz, SW 1H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4) Hz, SW 13C = 20124.6 Hz, AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.46 (1), or 1.6 (2), or 1.49 (3, 4) s, DS = 16, NS = 10 (1) or 12 (2) or 8 (3) or 6 (4); for HMBC, SF = 400.13 MHz, SW 1H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4) Hz, SW 13C = 20124.6 Hz, AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.46 (1), or 1.6 (2), or 1.49 (3, 4) s, DS = 16, NS = 20 (1) or 18 (2, 3) or 16 (4); for COSY, SF = 400.13 MHz , SW 1 H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4), AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.45 (1) or 1.55 (2) or 1.44 (3), or 1.47 (4) s, DS = 16, NS = 8 (1, 2, 3) or 6 (4); for NOESY, SF = 400.13 MHz, SW 1H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4), AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.96 (1) or 2.06 (2) or 1.99 (3, 4) s, DS = 16, NS = 12 (1), 6 (2), 10 (3), 8 (4).
Animal material The soft coral Sarcophyton sp. were collected from Dongshan Island, Guangdong Province, PR China, in October 2012, at a depth of 4–5 m water. The biological material was frozen immediately until used and was identified by Associate Prof. Cheng-Qi Fan from East China Sea Fisheries Research Institute. A voucher specimen (accession number: Sarcosp201210) has been deposited
Table 1.
1
H NMR (400 MHz) and
No.
13
20-OAc
518
15-OCH3
Extraction and isolation The frozen sample (500 g, wet weight) was chopped and exhaustively extracted with MeOH/CH2Cl2 (1:1) (1 × 3 L) at room temperature. After removal of solvent in vacuo, the residue (10 g) was suspended in H2O (200 mL) and partitioned sequentially to give dried petroleum ether (2 g) and EtOAc (4 g) extracts. The EtOAc extract was subjected to MCI gel column chromatography (CC) eluted with MeOH/H2O (3:7 → 10:0, 6 mL/min) in a gradient to afford four fractions (I IV). Fraction II (240 mg) was separated on reversed-phase C-18 (RP-18) silica gel CC to give three subfractions (sfr. IIa–IIc). Sfr. IIb (35 mg) was purified on semi-preparative RPHPLC equipped with a YMC column (MeOH/H2O, 75:25, 3 mL/min) to yield 10 (6 mg, tR 10.15 min), 4 (4 mg, tR 11.32 min), and 11 (2.8 mg, tR 13.58 min). Fraction III (220 mg) was subjected to silica gel CC (PE/acetone, 20:1 → 0:1, 4 mL/min) to give four subfractions (sfr. IIIa–IIId). Sfr. IIIb (16 mg) was further separated by RP-HPLC (YMC column, MeOH/H2O, 80:20, 3 mL/min) to yield 9 (6 mg, tR 17.86 min). Separation of sfr. IIIc (35 mg) by RP-HPLC (YMC column, MeOH/H2O, 80:20, 3 mL/min) yielded 2 (3 mg, tR 10.27 min), 7 (7.0 mg, tR 14.75 min), and 12 (4 mg, tR 15.70 min). Fraction IV
C NMR (100 MHz) data of compounds 1–4 (in CDCl3, δ in ppm, J in Hz)
1 δH
1 2 3 4 5α 5β 6α 6β 7 8 9α 9β 10α 10β 11 12 13α 13β 14α 14β 15 16α 16β 17 18 19 20
at the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, PR China.
6.26 (d, 10.5) 5.95 (d, 10.5) 2.28 (m) 2.34 (m) 1.66 (m) 1.91 (m) 2.85 (dd, 6.0, 4.2) 1.27 (m) 2.10 (m) 2.25 (m) 5.45 (t, 6.7) 2.18 (m) 2.38 (m) 2.31 (m)
1.33 (s) 1.32 (s) 1.80 (s) 1.28 (s) a 4.68 (d, 12.1) b 4.59 (d, 12.1) 2.07 (s) 3.05 (s)
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2 δC 144.8 121.7 120.2 137.9 35.4 25.9 62.4 59.6 38.8 23.3 131.0 135.0 37.5 25.2 77.9 25.5 26.1 17.9 16.7 61.9
δH
5.23 (d, 8.3) 5.40 (d, 8.3) 2.32 (t, 12.5) 2.20 (m) 1.79 (m) 1.54 (m) 2.93 (dd, 6.4, 1.9) 1.18 (m) 2.06 (m) 2.22 (m) 2.04 (m) 4.99 (t, 6.0) 3.35 (dd, 16.7, 12.2) 2.52 (d, 16.7) 5.22 (m)
3.84 (d, 8.9) 3.64 (d, 8.9) 1.44 (s) 1.78 (s) 1.22 (s) 1.65 (s)
3 δC 147.0 79.7 128.6 135.8 36.9 26.9 62.4 59.9 38.3 23.3 123.4 134.9 34.4 123.0 77.3 80.3 26.1 16.9 16.9 17.9
δH
5.42 (d, 9.9) 5.17 (d, 9.9) 2.80 (dd, 14.8, 5.7) 2.70 (dd, 14.8, 5.7) 5.68 (dt, 15.8, 5.7) 5.57 (d, 15.8) 1.72 (m) 2.05 (m) 2.20 (m) 5.11 (t, 6.7) 1.87 (m) 2.07 (m) 2.14 (m) 1.94 (m) 4.53 (d, 11.6) 4.46 (d, 11.6) 1.65 (s) 1.78 (s) 1.28 (s) 1.58 (s)
4 δC 133.7 84.9 126.3 138.5 42.1 123.6 139.4 73.0 43.1 23.1 125.9 134.1 37.8 24.4
δH
6.07 (d, 10.7) 5.98 (d, 10.7) 2.25 (m) 1.49 (m) 2.06 (m) 3.53 (d, 8.7) 1.65 (m) 1.92 (m) 2.18 (m) 5.30 (t, 6.7) 2.19 (m) 2.12 (m) 2.29 (m)
δC 147.2 119.1 121.2 135.2 34.3 27.3 71.5 75.3 38.4 23.0 125.6 138.5 39.0 30.6
128.0 78.4
2.36 (q, 6.8) 1.07 (d, 6.8)
33.8 22.3
10.3 16.5 28.4 15.0
1.08 (d, 6.8) 1.74 (s) 1.14 (s) 1.59 (s)
22.3 17.4 22.7 17.7
21.0 171.0 50.3
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Magn. Reson. Chem. 2014, 52, 515–520
Four new cembranoids from the soft coral Sarcophyton sp. (300 mg) was separated on RP-18 silica gel CC (MeOH/H2O, 6:4 → 10:0, 2 mL/min) to give three subfractions (sfr. IVa–IVc). Sfr. IVa (120 mg) was subjected to further purification using Sephadex LH-20 eluted with ethanol (0.5 mL/min) to yield three subfractions (Sfr. IVa1–IVa3). Sfr. IVa1 (30 mg) was purified on RP-HPLC (YMC column, MeOH/H2O, 80:20, 3 mL/min) to afford 3 (3 mg, tR 10.99 min) and 5 (8 mg, tR 13.25 min). Sfr. IVb (27 mg) was separated by RPHPLC (YMC column, MeOH/H2O, 90:10, 3 mL/min) to yield 1 (5 mg, tR 12.47 min) and 6 (9 mg, tR 18.22 min). Purification of sfr. IVc (38 mg) over Sephadex LH-20 column using MeOH/CH2Cl2 (1:1, 0.5 mL/min) as eluent obtained 8 (23 mg). Sarcophyton A (1): colorless oil; [α]25 6.0 (c 0.41, CHCl3); UV D (MeOH) λmax (log ε) 247 (3.77) nm; IR (KBr) νmax 2977, 2929, 2860, 2825, 1740, 1462, 1373, 1231, 1072, and 1024 cm 1; 1H and 13C NMR data, see Table 1; positive ESIMS m/z 399.1 [M + Na]+; HRESIMS m/z 399.2512 [M + Na]+ (calcd for C23H36O4Na, 399.2506). Sarcophyton B (2): colorless oil; [α]25 D +1.7 (c 0.50, CHCl3); IR (KBr) νmax 3461, 2964, 2934, 2873, 1727, 1662, 1395, 1240, and 1082 cm 1; 1H and 13C NMR data (Table 1); positive ESIMS m/z 319.2 [M + H]+, 301.2 [M H2O + H]+; HRESIMS m/z 341.2069 [M + Na]+ (calcd for C20H30O3Na, 341.2087). Sarcophyton C (3): colorless oil; [α]25 D +20.8 (c 0.34, CHCl3); IR (KBr) νmax 3536, 2961, 2927, 2858, 1739, 1665, 1377, 1260, 1098, and 1034 cm 1; 1H and 13C NMR data (Table 1); positive ESIMS m/z 303.2 [M + H]+, 285.2 [M H2O + H]+; HRESIMS m/z 325.2138 [M + Na]+ (calcd for C20H30O2Na, 325.2140). Sarcophyton D (4): colorless oil; [α]25 11.4 (c 1.51, CHCl3); UV D (MeOH) λmax (log ε) 242 (3.38) nm; IR (KBr) νmax 3447, 2962, 2931, 2874, 1716, 1670, 1457, 1376, 1262, and 1073 cm 1; 1H and 13C NMR data (Table 1); positive ESIMS m/z 307.3 [M + H]+; HRESIMS m/z 329.2435 [M + Na]+ (calcd for C20H34O2Na, 329.2451) Compounds 5-9: 13C NMR data, see Table 2.
Table 2. ppm) carbon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 15-OCH3 7-OAc
13
C NMR (100 MHz) data of compounds 5–9 (in CDCl3, δ in 5
6
7
8
9
148.3 118.3 120.4 137.2 35.5 26.3 62.3 59.9 38.6 23.2 125.0 136.0 41.7 26.0 73.9 29.7 29.7 17.7 17.0 16.0
145.2 121.6 120.3 137.6 35.3 26.2 62.6 59.9 38.9 23.4 124.4 136.4 41.6 25.4 78.0 26.0 25.5 18.0 16.7 15.8 50.3
134.1 83.8 127.7 137.9 35.6 24.8 77.2 75.3 37.4 23.8 123.3 137.1 36.6 24.2 127.3 78.5 10.3 15.8 24.8 15.2
133.1 83.6 126.2 139.2 37.5 25.2 61.8 59.8 39.7 23.4 123.5 136.7 36.6 26.0 127.7 78.3 10.1 15.5 16.8 15.0
70.6 77.4 124.6 140.0 37.9 25.4 61.9 59.7 40.1 23.7 123.9 136.0 35.3 27.3 68.3 98.5 11.1 15.5 16.7 14.9
Cytotoxicity assay Compounds 1–12 were evaluated for cytotoxicities against MCF-7 human breast cancer cell line using the MTT[24] method in 96-well microplates. Cisplatin was used as a positive control. Acknowledgements The authors thank the National Natural Science Foundation of China (No. 81102339) and Guangdong Natural Science Foundation (No. S2011040002429) for providing financial support to this work.
References [1] K.-H. Lin, Y.-J. Tseng, B.-W. Chen, T.-L. Hwang, H.-Y. Chen, C.-F. Dai, J.-H. Sheu. Org. Lett. 2014, 16, 1314–1317. [2] L.-F. Liang, T. Kurtan, A. Mandi, L.-G. Yao, J. Li, W. Zhang, Y.-W. Guo. Org. Lett. 2013, 15, 274–277. [3] Z. Xi, W. Bie, W. Chen, D. Liu, L. van Ofwegen, P. Proksch, W. Lin. Mar. Drugs 2013, 11, 3186–3196. [4] Z. Wang, H. Tang, P. Wang, W. Gong, M. Xue, H. Zhang, T. Liu, B. Liu, Y. Yi, W. Zhang. Mar. Drugs 2013, 11, 775–787. [5] N. P. Thao, N. H. Nam, N. X. Cuong, T. H. Quang, P. T. Tung, B. H. Tai, B. T. T. Luyen, D. Chae, S. Kim, Y.-S. Koh, P. V. Kiem, C. V. Minh, Y. H. Kim. Chem. Pharm. Bull. 2012, 60, 1581–1589. [6] Z.-H. Sun, Y.-H. Cai, C.-Q. Fan, G.-H. Tang, H.-B. Luo, S. Yin. Mar. Drugs 2014, 12, 672–681. [7] Z.-B. Cheng, H. Xiao, C.-Q. Fan, Y.-N. Lu, G. Zhang, S. Yin. Steroids 2013, 78, 1353–1358. [8] Z.-B. Cheng, W.-W. Shao, Y.-N. Liu, Q. Liao, T.-T. Lin, X.-Y. Shen, S. Yin. J. Nat. Prod. 2013, 76, 664–671. [9] D.-Z. Liu, B.-W. Liang. Magn. Reson. Chem. 2014, 52, 57–59. [10] T. Grkovic, E. L. Whitson, D. C. Rabe, R. S. Gardella, D. P. Bottaro, W. M. Linehan, J. B. McMahon, K. R. Gustafson, T. C. McKee. Bioorg. Med. Chem. Lett. 2011, 21, 2113–2115. [11] N. X. Cuong, T. A. Tuan, P. V. Kiem, C. V. Minh, E. M. Choi, Y. H. Kim. Chem. Pharm. Bull. 2008, 56, 988–992.
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20.9 170.4
All-trans-(3S, 5R, 6R, 3′S, 5′R, 6′S)-peridinin (10): 13C NMR (MeOH-d4, 100 MHz) δC 36.8 (C, C-1), 46.7 (CH2, C-2), 69.4 (CH, C3), 46.6 (CH2, C-4), 72.9 (C, C-5), 118.3 (C, C-6), 204.1 (C, C-7), 104.0 (CH, C-8), 135.2 (C, C-9), 129.3 (CH, C-12), 133.0 (CH, C-13), 134.4 (CH, C-14), 139.1 (CH, C-15), 32.8 (CH3, C-16), 31.2 (CH3, C-17), 29.5 (CH3, C-18), 14.3 (CH3, C-19), 36.2 (C, C-1′), 47.9 (CH2, C-2′), 64.5 (CH, C-3′), 41.6 (CH2, C-4′), 68.9 (C, C-5′), 71.7 (C, C-6′), 134.7 (C, C-7′), 123.0 (CH, C-8′), 125.5 (C, C-9′), 138.3 (CH, C-10′), 148.2 (CH, C-11′), 120.9 (CH, C-12′), 135.2 (CH, C-13), 140.0 (CH, C-14′), 139.1 (CH, C-15′), 30.1 (CH3, C-16′), 25.3 (CH3, C-17′), 21.3 (CH3, C-18′), 170.5 (C, C-19′), 15.5 (CH3, C-20′), 3-OAc (172.3, 20.2) All-trans-(9′Z,11′Z)-(3R,3′S,5’R,6’R)-pyrrhoxanthin (11): 13C NMR (CDCl3, 100 MHz) δC 36.1 (C, C-1), 42.3 (CH2, C-2), 67.9 (CH, C-3), 37.5 (CH2, C-4), 137.2 (C, C-5), 124.3 (C, C-6), 90.1 (C, C-7), 98.6 (CH, C-8), 121.0 (C, C-9), 134.6 (CH, C-12), 130.6 (CH, C-13), 133.8 (CH, C-14), 136.9 (CH, C-15), 28.7 (CH3, C-16), 30.2 (CH3, C-17), 22.4 (CH3, C-18), 18.1 (CH3, C-19), 35.31 (C, C-1′), 47.1 (CH2, C-2′), 64.2 (CH, C-3′), 40.9 (CH2, C-4′), 67.5 (C, C-5′), 70.5 (C, C-6′), 133.7 (C, C-7′), 121.8 (CH, C-8′), 125.0 (C, C-9′), 136.3 (CH, C-10′), 147.0 (CH, C-11′), 119.1 (CH, C-12′), 134.4 (CH, C-13), 137.8 (CH, C-14′), 129.8 (CH, C-15′), 29.5 (CH3, C-16′), 24.9 (CH3, C-17′), 19.9 (CH3, C-18′), 168.7 (C, C-19′), 15.4 (CH3, C-20′), 3-OAc (170.7, 21.3). Sarcophytonone (12): 13C NMR (CDCl3, 100 MHz) δC 177.2 (C, C-1), 39.4 (CH, C-2), 34.2 (CH, C-3), 21.6 (CH2, C-4), 41.7 (CH2, C-5), 72.5 (C, C-6), 40.2 (CH2, C-7), 21.3 (CH2, C-8), 17.1 (CH3, C-9), 26.5 (CH3, C-10), 144.3 (C, C-1′), 140.6 (C, C-2′), 187.6 (C, C-3′), 140.4 (C, C-4′), 140.2 (C, C-5′), 187.2 (C, C-6′), 12.4 (CH3, C-7′), 12.3 (CH3, C-8′), 12.0 (CH3, C-9′).
Z.-B. Cheng et al. [12] C.-H. Chao, Z.-H. Wen, Y.-C. Wu, H.-C. Yeh, J.-H. Sheu. J. Nat. Prod. 2008, 71, 1819–1824. [13] G.-A. Zou, G. Ding, Z.-H. Su, J.-S. Yang, H.-W. Zhang, C.-Z. Peng, H. A. Aisa, Z.-M. Zou. J. Nat. Prod. 2010, 73, 792–795. [14] K. H. Shaker, M. Mueller, M. A. Ghani, H.-M. Dahse, K. Seifert. Chem. Biodivers. 2010, 7, 2007–2015. [15] P. V. Kiem, C. V. Minh, N. X. Nhiem, N. T. Cuc, N. V. Quang, H. L. T. Anh, B. H. Tai, P. H. Yen, N. T. Hoai, K. Y. Ho, N. Kim, S. Park, S. H. Kim. Magn. Reson. Chem. 2014, 52, 51–56. [16] M. R. Rao, U. Venkatesham, K. V. Sridevi, P. S. Reddy, K. Jamil, Y. Venkateswarlu. Indian J. Chem. B Org. 2002, 41B, 1472–1476. [17] J. C. Coll, G. B. Hawes, N. Liyanage, W. Oberhansli, R. J. Wells. Aust. J. Chem. 1977, 30, 1305–1309. [18] S.-T. Lin, S.-K. Wang, C.-Y. Duh. Mar. Drugs 2011, 9, 2705–2716. [19] B. F. Bowden, J. C. Coll, A. Heaton, G. König, M. A. Bruck, R. E. Cramer, D. M. Klein, P. J. Scheuer. J. Nat. Prod. 1987, 50, 650–659.
[20] T. H. Quang, T. T. Ha, C. V. Minh, P. V. Kiem, H. T. Huong, N. T. T. Ngan, N. X. Nhiem, N. H. Tung, B. H. Tai, D. T. T. Thuy, S. B. Song, H.-K. Kang, Y. H. Kim. Bioorg. Med. Chem. 2011, 19, 2625–2632. [21] J. Krane, T. Aakermann, S. Liaaen-Jensen. Magn. Reson. Chem. 1992, 30, 1169–1177. [22] G. Englert, T. Aakermann, S. Liaaen-Jensen. Magn. Reson. Chem. 1993, 31, 910–915. [23] L. Li, C.-Y. Wang, C.-L. Shao, L. Han, X.-P. Sun, J. Zhao, Y.-W. Guo, H. Huang, H.-S. Guan. J. Asian Nat. Prod. Res. 2009, 11, 851–855. [24] T. Mosmann. J. Immunol. Methods 1983, 65, 55–63.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Magn. Reson. Chem. 2014, 52, 515–520
Revised: 16 June 2014
Accepted: 19 June 2014
Published online in Wiley Online Library: 10 July 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4108
Four new cembranoids from the soft coral Sarcophyton sp. Zhong-Bin Cheng,a† Qiong Liao,a,b† Ye Chen,a Cheng-Qi Fan,c Zhi-Ying Huang,a,b Xin-Jun Xua* and Sheng Yina* Introduction Soft corals belonging to the genus Sarcophyton have been well recognized as a rich source of secondary metabolites endowed with a range of structural diversity and various biological activities. Members of this genus have been studied widely, resulting in the identification of unprecedented diterpenoids[1–3] and steroids.[4] Some of these metabolites are of considerable interest and merit continuous attention due to their unique structures and significant biological activities, including inhibition of protein tyrosine phosphatase 1B,[2] anti-tumor,[3] antimicrobial,[4] and anti-inflammatory properties.[5] As part of our continuing efforts to discover structurally intriguing biologically significant metabolites from marine invertebrates of the South China Sea,[6,7] we undertook a detailed chemical analysis of Sarcophyton sp., which led to the isolation of four new cembranoids (1–4), together with eight known compounds including five cembranoids (5–9), two carotenoids (10–11), and a tetra-substituted quinone (12) (Fig. 1). Herein, the details of the isolation, structure elucidation, and activity of these compounds are described.
Results and Discussion
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* Correspondence to: Xin-Jun Xu and Sheng Yin, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China. E-mail: [email protected]; [email protected] †
Zhong-Bin Cheng and Qiong Liao contributed equally to this work.
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China b Center of Laboratory Animals, Sun Yat-sen University, Guangzhou, Guangdong 510006, China c East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China
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The frozen animal of Sarcophyton sp. (500 g) was extracted with MeOH/CH2Cl2 (1:1) to give a crude extract, which was dispersed in H2O and successively partitioned with petroleum ether and EtOAc. The EtOAc extract was subjected to various column chromatographies to afford compounds 1–12. Compound 1 had a molecular formula of C23H36O4 as established by HRESIMS at m/z 399.2512 [M + Na]+ (calcd 399.2506). The hydrogen-1 nuclear magnetic resonance (1H NMR) spectrum of 1 displayed signals of six methyl singlets at δH 1.32 (3H, s, H3-17), 1.33 (3H, s, H3-16), 1.80 (3H, s, H3-18), 1.28 (3H, s, H3-19), 2.07 (3H, s, 20-OAc), and 3.05 (3H, s, 15-OCH3), three olefinic protons at δH 6.26 (1H, d, J = 10.5 Hz, H-2), 5.95 (1H, d, J = 10.5 Hz, H-3), and 5.45 (1H, t, J = 6.7 Hz, H-11), an oxymethine at δH 2.85 (1H, dd, J = 6.0, 4.2 Hz, H-7), an oxymethylene [δH 4.68 (1H, d, J = 12.1 Hz, H-20a) and 4.59 (1H, d, J = 12.1 Hz, H-20b)], and a series of aliphatic methylene multiplets. The carbon-13 nuclear magnetic resonance (13C NMR) spectrum, in combination with DEPT experiments, resolved 23 carbon resonances attributable to a carbonyl, six methyls, three trisubstituted double bonds, an epoxy moiety,[8,9] seven sp3 methylenes including an oxymethylene carbon, and an oxygenated quaternary carbon. The aforementioned information showed high similarity to that of the co-occurring known analog (1E,3E,7R*,8R*,11E)-1-(2-methoxypropan-2-yl)-4,8,12-trimethyloxabicyclo[12.1.0]-pentadeca-1,3,
11-triene (6)[10] except for the absence of CH3-20 (δH 1.63, δC 15.8 ) in 6 and the presence of an acetylated hydroxymethyl unit (δH 4.68, 4.59, and 2.07; δC 21.0, 61.9, and 171.0) in 1, suggesting that the CH3-20 in 6 was replaced by an acetoxymethyl in 1. This was supported by HMBC correlations (Fig. 2) of H-20 with C-12, C-13, C-14, and the carbonyl (δC 171.0). Detailed 2D NMR (1H 1H COSY and HMBC) analyses of 1 established the gross structure of 1 as depicted (Fig. 2). The relative configuration of 1 was determined to be the same as that of 6 by NOESY experiment and by comparison of their 13C NMR data. In particular, the crucial NOE correlations (Fig. 3) of H-2/H3-18, H-3/H-7, H-11/H2-13, H2-20/H2-10 and the large coupling constant (J2,3 = 10.5 Hz)[10] indicated that the configuration of the conjugated diene Δ1, Δ3, and Δ11 were E, E, and Z, respectively. Moreover, the NOESY correlations of H-7/H-5α, H-9α and H3-19/H2-6 assigned H-7 and CH3-19 to be α and β oriented, respectively. Thus, compound 1 was determined to be (1E,3E,7R*,8R*,11Z)15-methoxy-20-acetoxymethyl-7,8-epoxycembra-1,3,11-triene and named sarcophyton A. Compound 2, a colorless oil, had the molecular formula C20H30O3, as determined by the HRESIMS at m/z 341.2069 [M + Na]+ (calcd 341.2087). The NMR spectroscopic features indicated that 2 had a similar structure as that of sinumaximol E,[5] except for the presence of an extra trisubstituted double bond [(δH 4.99 (1H, t, J = 6.0 Hz, H-11); δC 123.4 (C-11), 134.9 (C-12)] and an additional olefinic methyl [δH 1.65 (3H, s, H3-20); δC 17.9 (C-20)] in 2 instead of the terminal double bond [δH 4.94 (1H, br s), 4.99 (1H, br s); δC 150.0 (C-12), 113.1 (C-20)] and the oxymethine [δH 4.12 (dd, J = 4.5, 7.0 Hz, H-11), δC 75.3 (C-11)] in sinumaximol E. The trisubstituted double bond and the olefin methyl was located by the 1H 1H COSY correlations (Fig. 2) of H-10/H-11, H13/H-14 and by HMBC correlations (Fig. 2) of H-20/C-11, C-12, and C-13. Detailed analyses of the 2D NMR spectra of 2
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Figure 1. Structures of compounds 1–12.
1
Figure 2. Selected H
1
H COSY (–) and HMBC (→) correlations of 1–4.
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confirmed the gross structure of 2 (Fig. 2). The Z configuration of Δ1,14 was determined to be the same as that of sinumaximol E by comparing the 13C chemical shift values of C-1 and C-14 with those of sinumaximol E (δC 147.0 and 123.4 in 2, δC 146.5, 123.3 in sinumaximol E). The NOESY correlations (Fig. 3) of H-2/H3-18, H-3/H-5α, H3-20/H2-10, and H-11/H-13α assigned both Δ3 and Δ11 as E configuration. The CH3-17 was assigned in α-orientation based on the NOESY correlations of H-2/H-16β, H3-17/H-16α, H-11, and the lack of the correlations between H-2 and H3-17. The H-7 and CH3-19 were assigned to be α and β-oriented, respectively, by comparison of the 13C chemical shift values of C-7 and C-8 of 1 and 2 (ΔδC < 0.3 ppm) and by NOESY correlations of H3-19/H2-6, H-10β and H-7/H-9α, H5α, H-11, H-3 (Fig. 3). Thus, compound 2 was determined to be (1Z,2S*,3E,7S*,8S*,11E,15S*)-15-hydroxy-7,8:2,16-bisepoxycembra1(14),3,11-trien and named sarcophyton B. Compound 3 was isolated as a colorless oil, and its molecular formula was assigned as C20H30O2 based on the sodiated molecular ion peak at m/z 325.2138 [M + Na]+ (calcd 325.2140). Detailed analysis of the 1H NMR spectrum revealed the presence of four
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tertiary methyls including three olefinic methyl singlets [δH 1.28 (3H, s, H3-19), 1.65 (3H, s, H3-17), 1.78 (3H, s, H3-18), and 1.58 (3H, s, H3-20)], three olefinic protons [δH 5.11 (t, J = 6.7 Hz, H-11), 5.17 (1H, d, J = 9.9 Hz, H-3), and 5.68 (1H, dt, J = 15.8, 5.7 Hz, H-6)], an oxymethylene group [δH 4.53 (1H, d, J = 11.6 Hz, H-16α) and 4.46 (1H, d, J = 11.6 Hz, H-16β)], an oxymethine proton [δH 5.42 (1H, J = 9.9 Hz, H-2)], and several aliphatic multiplets. The 13C NMR spectrum displayed signals for four double bonds, four methyls, six methylenes including an oxymethylene carbon, an oxymethine, and an oxygenated quaternary carbon. Aforementioned spectroscopic data resembled those of (+)-7β,8βdihydroxydeepoxysarcophytoxid[11] except for the presence of a disubstituted double bond [δH 5.68 (H-6), 5.57 (H-7); δC 123.9 (C-6), 139.4 (C-7)] in 3 instead of the methylene at C-6 (δC 29.8) and the oxymethine at C-7 (δC 74.4) in (+)-7β,8β-dihydroxydeepoxysarcophytoxid, indicating 3 was the C-6 C-7 dehydrated derivative of (+)-7β,8βdihydroxydeepoxysarcophytoxid and was upheld by the MS data. The postulated gross structure of 3 was confirmed by detailed 2D NMR analyses of 3 (Fig. 2). The configurations of the double bonds Δ1(15), Δ3, and Δ11 were assigned to be Z, E, and E, respectively, by comparison of the 1D NMR data of 3 with those of (+)-7β,8βdihydroxydeepoxysarcophytoxid and by NOESY experiment (Fig. 3). The coupling constant between H-6 and H-7 (J = 15.8 Hz) defined Δ6 to be E configuration. The OH-8 was assigned in α-orientation by analysis of its C-19 chemical shift, as the chemical shift value for the C-19 methyl group is very sensitive to the configuration at C-8,[12] and the value of δC 28.4 for C-19 in 3 is in good agreement with that of the 8α-hydroxyl isomer crassumolide F (δC 27.2), while the 8β-hydroxyl analogs usually resonated at δC 24.8.[13] Thus, compound 3 was determined to be (1Z,2S*,3E,6E,8S*,11E)-8-hydroxy-2,16-epoxycembra-1(15),3,6,11tetraene and named sarcophyton C. Compound 4, a colorless oil, had the molecular formula C20H34O2, as determined by the HRESIMS at m/z 329.2435 [M + Na]+ (calcd 329.2451). The 1D NMR data of 4 were closely comparable with those of (+)-7,8-epoxy-7,8-dihydrocembrene C[14] except that the epoxy functionality [δH 2.83, δC 61.6, 60.1]
Copyright © 2014 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 515–520
Four new cembranoids from the soft coral Sarcophyton sp.
Figure 3. Key NOE correlations (↔) of 1–4.
Magn. Reson. Chem. 2014, 52, 515–520
Experimental General experimental procedures Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a Bruker AM-400 spectrometer at 25 °C. ESIMS was measured on a Finnigan LCQ Deca instrument, and HRESIMS was performed on a Waters Micromass Q-TOF spectrometer. A Shimadzu LC-20 AT equipped with a SPD-M20A PDA detector was used for HPLC. A YMC-pack ODS-A column (250 × 10 mm, S5 μm, 12 nm) was used for semi-preparative HPLC separation. Silica gel (300–400 mesh, Qingdao Haiyang Chemical Co., Ltd.), C18 reversed-phase silica gel (12 nm, S-50 μm, YMC Co., Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography. All solvents were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.).
NMR spectroscopy Nuclear magnetic resonance spectra were recorded on a Bruker Avance-400 spectrometer equipped with a BBFO-5 mm probe in 0.4 mL CDCl3 at 300 K. Solvent signals were used as internal standard (CDCl3: δH = 7.26, δC = 77.0 ppm). The pulse conditions were as follows: For 1H NMR, spectrometer frequency (SF) = 400.13 MHz, spectral width (SW) = 8223.7, pulse 90 width (PW) = 12.9 μs, Fourier transform size (SI) = 32768, acquisition time (AQ) = 3.98, line broadening (LB) = 0.3 Hz, relaxation delay (RD) = 1.0 s, number of dummy scans (DS) = 2 and number of scans (NS) = 32; for 13 C, SF = 100.61 MHz, SW = 24038.5 Hz, PW = 10.3 μs, SI = 32768, AQ = 0.5 s, LB = 3 Hz, RD = 1.5 s, DS = 4, NS = 2400 (1) or 1024 (2) or 6000 (3)
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in (+)-7,8-epoxy-7,8-dihydrocembrene C had been replaced by a vicinal diol[15] [δH 3.53 (1H, d, J = 8.7 Hz), δC 71.5, 75.3] in 4. Detailed 2D NMR analyses of 4 confirmed the gross structure (Fig. 2). All three double bonds (Δ1, Δ3, and Δ11) were assigned as E configuration by comparison of its 1D NMR data with those of (+)-7,8-epoxy-7,8-dihydrocembrene C and were supported by the NOESY correlations of H-2/H3-17, H-2/H3-18, H-3/H2-5, H-11/H2-13, and H2-10/H3-20 (Fig. 3). The H-7 and CH3-19 were both assigned in α-orientation by the apparent NOESY correlations of H-7/H3-19, H-3, H-5α, H-10α, H3-20 and H3-19/H-6α, H-10α. Thus, compound 4 was determined to be (1E,3E,7S*,8S*,11E)7,8-dihydroxycembra-1,3,11-triene. The structure of 4 was very similar to that of (EEE)-7β,8α-dihydroxy-1-isopropyl-4,8,12trimethylcyclotetradeca-1,3,11-triene[16] with the only difference due to the cis-vicinal diol moiety in 4 instead of the transvicinal diol moiety in (EEE)-7β,8α-dihydroxy-1-isopropyl-4,8,12trimethylcyclotetradeca-1,3,11-triene. Compound 4 was named sarcophyton D. The structures of the known compounds were determined as 2-[(E,E,E)-7′,8′-epoxy-4′,8′,12′-trimethylcyclotetradeca-1′,3′,11′-trienyl] propan-2-ol (5),[17] (1E,3E,7R*,8R*,11E)-1-(2-methoxy-propan-2-yl)4,8,12-trimethyloxabicyclo[12.1.0]-pentadeca-1,3,11-triene (6),[10] crassumol C (7),[18] (+)-sarcophytoxide (8),[19] laevigatol A (9),[20] all-trans-(3S,5R,6R,3′S,5′R,6′S)-peridinin (10),[21] all-trans-(9′Z,11′Z)(3R,3′S,5′R,6′R)-pyrrhoxanthin (11),[22] and sarcophytonone (12)[23] by extensive spectroscopic analysis combined with careful comparison with the reported data. All isolated compounds were evaluated for cytotoxicities against the MCF-7 human breast cancer cell line. Only 5, 7, 9, and 12 exhibited very weak growth inhibitory effects with the inhibition greater than 10% at the concentration of 20 μM (11%, 11%, 13%, and 12%, respectively).
Z.-B. Cheng et al. or 3600 (4); for HSQC, SF = 400.13 MHz, SW 1H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4) Hz, SW 13C = 20124.6 Hz, AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.46 (1), or 1.6 (2), or 1.49 (3, 4) s, DS = 16, NS = 10 (1) or 12 (2) or 8 (3) or 6 (4); for HMBC, SF = 400.13 MHz, SW 1H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4) Hz, SW 13C = 20124.6 Hz, AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.46 (1), or 1.6 (2), or 1.49 (3, 4) s, DS = 16, NS = 20 (1) or 18 (2, 3) or 16 (4); for COSY, SF = 400.13 MHz , SW 1 H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4), AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.45 (1) or 1.55 (2) or 1.44 (3), or 1.47 (4) s, DS = 16, NS = 8 (1, 2, 3) or 6 (4); for NOESY, SF = 400.13 MHz, SW 1H = 3472.2 (1) or 3937.0 (2) or 3787.9 (3) or 3816.8 (4), AQ = 0.29 (1) or 0.26 (2) or 0.27 (3) or 0.27 (4) s, RD = 1.96 (1) or 2.06 (2) or 1.99 (3, 4) s, DS = 16, NS = 12 (1), 6 (2), 10 (3), 8 (4).
Animal material The soft coral Sarcophyton sp. were collected from Dongshan Island, Guangdong Province, PR China, in October 2012, at a depth of 4–5 m water. The biological material was frozen immediately until used and was identified by Associate Prof. Cheng-Qi Fan from East China Sea Fisheries Research Institute. A voucher specimen (accession number: Sarcosp201210) has been deposited
Table 1.
1
H NMR (400 MHz) and
No.
13
20-OAc
518
15-OCH3
Extraction and isolation The frozen sample (500 g, wet weight) was chopped and exhaustively extracted with MeOH/CH2Cl2 (1:1) (1 × 3 L) at room temperature. After removal of solvent in vacuo, the residue (10 g) was suspended in H2O (200 mL) and partitioned sequentially to give dried petroleum ether (2 g) and EtOAc (4 g) extracts. The EtOAc extract was subjected to MCI gel column chromatography (CC) eluted with MeOH/H2O (3:7 → 10:0, 6 mL/min) in a gradient to afford four fractions (I IV). Fraction II (240 mg) was separated on reversed-phase C-18 (RP-18) silica gel CC to give three subfractions (sfr. IIa–IIc). Sfr. IIb (35 mg) was purified on semi-preparative RPHPLC equipped with a YMC column (MeOH/H2O, 75:25, 3 mL/min) to yield 10 (6 mg, tR 10.15 min), 4 (4 mg, tR 11.32 min), and 11 (2.8 mg, tR 13.58 min). Fraction III (220 mg) was subjected to silica gel CC (PE/acetone, 20:1 → 0:1, 4 mL/min) to give four subfractions (sfr. IIIa–IIId). Sfr. IIIb (16 mg) was further separated by RP-HPLC (YMC column, MeOH/H2O, 80:20, 3 mL/min) to yield 9 (6 mg, tR 17.86 min). Separation of sfr. IIIc (35 mg) by RP-HPLC (YMC column, MeOH/H2O, 80:20, 3 mL/min) yielded 2 (3 mg, tR 10.27 min), 7 (7.0 mg, tR 14.75 min), and 12 (4 mg, tR 15.70 min). Fraction IV
C NMR (100 MHz) data of compounds 1–4 (in CDCl3, δ in ppm, J in Hz)
1 δH
1 2 3 4 5α 5β 6α 6β 7 8 9α 9β 10α 10β 11 12 13α 13β 14α 14β 15 16α 16β 17 18 19 20
at the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, PR China.
6.26 (d, 10.5) 5.95 (d, 10.5) 2.28 (m) 2.34 (m) 1.66 (m) 1.91 (m) 2.85 (dd, 6.0, 4.2) 1.27 (m) 2.10 (m) 2.25 (m) 5.45 (t, 6.7) 2.18 (m) 2.38 (m) 2.31 (m)
1.33 (s) 1.32 (s) 1.80 (s) 1.28 (s) a 4.68 (d, 12.1) b 4.59 (d, 12.1) 2.07 (s) 3.05 (s)
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2 δC 144.8 121.7 120.2 137.9 35.4 25.9 62.4 59.6 38.8 23.3 131.0 135.0 37.5 25.2 77.9 25.5 26.1 17.9 16.7 61.9
δH
5.23 (d, 8.3) 5.40 (d, 8.3) 2.32 (t, 12.5) 2.20 (m) 1.79 (m) 1.54 (m) 2.93 (dd, 6.4, 1.9) 1.18 (m) 2.06 (m) 2.22 (m) 2.04 (m) 4.99 (t, 6.0) 3.35 (dd, 16.7, 12.2) 2.52 (d, 16.7) 5.22 (m)
3.84 (d, 8.9) 3.64 (d, 8.9) 1.44 (s) 1.78 (s) 1.22 (s) 1.65 (s)
3 δC 147.0 79.7 128.6 135.8 36.9 26.9 62.4 59.9 38.3 23.3 123.4 134.9 34.4 123.0 77.3 80.3 26.1 16.9 16.9 17.9
δH
5.42 (d, 9.9) 5.17 (d, 9.9) 2.80 (dd, 14.8, 5.7) 2.70 (dd, 14.8, 5.7) 5.68 (dt, 15.8, 5.7) 5.57 (d, 15.8) 1.72 (m) 2.05 (m) 2.20 (m) 5.11 (t, 6.7) 1.87 (m) 2.07 (m) 2.14 (m) 1.94 (m) 4.53 (d, 11.6) 4.46 (d, 11.6) 1.65 (s) 1.78 (s) 1.28 (s) 1.58 (s)
4 δC 133.7 84.9 126.3 138.5 42.1 123.6 139.4 73.0 43.1 23.1 125.9 134.1 37.8 24.4
δH
6.07 (d, 10.7) 5.98 (d, 10.7) 2.25 (m) 1.49 (m) 2.06 (m) 3.53 (d, 8.7) 1.65 (m) 1.92 (m) 2.18 (m) 5.30 (t, 6.7) 2.19 (m) 2.12 (m) 2.29 (m)
δC 147.2 119.1 121.2 135.2 34.3 27.3 71.5 75.3 38.4 23.0 125.6 138.5 39.0 30.6
128.0 78.4
2.36 (q, 6.8) 1.07 (d, 6.8)
33.8 22.3
10.3 16.5 28.4 15.0
1.08 (d, 6.8) 1.74 (s) 1.14 (s) 1.59 (s)
22.3 17.4 22.7 17.7
21.0 171.0 50.3
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Magn. Reson. Chem. 2014, 52, 515–520
Four new cembranoids from the soft coral Sarcophyton sp. (300 mg) was separated on RP-18 silica gel CC (MeOH/H2O, 6:4 → 10:0, 2 mL/min) to give three subfractions (sfr. IVa–IVc). Sfr. IVa (120 mg) was subjected to further purification using Sephadex LH-20 eluted with ethanol (0.5 mL/min) to yield three subfractions (Sfr. IVa1–IVa3). Sfr. IVa1 (30 mg) was purified on RP-HPLC (YMC column, MeOH/H2O, 80:20, 3 mL/min) to afford 3 (3 mg, tR 10.99 min) and 5 (8 mg, tR 13.25 min). Sfr. IVb (27 mg) was separated by RPHPLC (YMC column, MeOH/H2O, 90:10, 3 mL/min) to yield 1 (5 mg, tR 12.47 min) and 6 (9 mg, tR 18.22 min). Purification of sfr. IVc (38 mg) over Sephadex LH-20 column using MeOH/CH2Cl2 (1:1, 0.5 mL/min) as eluent obtained 8 (23 mg). Sarcophyton A (1): colorless oil; [α]25 6.0 (c 0.41, CHCl3); UV D (MeOH) λmax (log ε) 247 (3.77) nm; IR (KBr) νmax 2977, 2929, 2860, 2825, 1740, 1462, 1373, 1231, 1072, and 1024 cm 1; 1H and 13C NMR data, see Table 1; positive ESIMS m/z 399.1 [M + Na]+; HRESIMS m/z 399.2512 [M + Na]+ (calcd for C23H36O4Na, 399.2506). Sarcophyton B (2): colorless oil; [α]25 D +1.7 (c 0.50, CHCl3); IR (KBr) νmax 3461, 2964, 2934, 2873, 1727, 1662, 1395, 1240, and 1082 cm 1; 1H and 13C NMR data (Table 1); positive ESIMS m/z 319.2 [M + H]+, 301.2 [M H2O + H]+; HRESIMS m/z 341.2069 [M + Na]+ (calcd for C20H30O3Na, 341.2087). Sarcophyton C (3): colorless oil; [α]25 D +20.8 (c 0.34, CHCl3); IR (KBr) νmax 3536, 2961, 2927, 2858, 1739, 1665, 1377, 1260, 1098, and 1034 cm 1; 1H and 13C NMR data (Table 1); positive ESIMS m/z 303.2 [M + H]+, 285.2 [M H2O + H]+; HRESIMS m/z 325.2138 [M + Na]+ (calcd for C20H30O2Na, 325.2140). Sarcophyton D (4): colorless oil; [α]25 11.4 (c 1.51, CHCl3); UV D (MeOH) λmax (log ε) 242 (3.38) nm; IR (KBr) νmax 3447, 2962, 2931, 2874, 1716, 1670, 1457, 1376, 1262, and 1073 cm 1; 1H and 13C NMR data (Table 1); positive ESIMS m/z 307.3 [M + H]+; HRESIMS m/z 329.2435 [M + Na]+ (calcd for C20H34O2Na, 329.2451) Compounds 5-9: 13C NMR data, see Table 2.
Table 2. ppm) carbon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 15-OCH3 7-OAc
13
C NMR (100 MHz) data of compounds 5–9 (in CDCl3, δ in 5
6
7
8
9
148.3 118.3 120.4 137.2 35.5 26.3 62.3 59.9 38.6 23.2 125.0 136.0 41.7 26.0 73.9 29.7 29.7 17.7 17.0 16.0
145.2 121.6 120.3 137.6 35.3 26.2 62.6 59.9 38.9 23.4 124.4 136.4 41.6 25.4 78.0 26.0 25.5 18.0 16.7 15.8 50.3
134.1 83.8 127.7 137.9 35.6 24.8 77.2 75.3 37.4 23.8 123.3 137.1 36.6 24.2 127.3 78.5 10.3 15.8 24.8 15.2
133.1 83.6 126.2 139.2 37.5 25.2 61.8 59.8 39.7 23.4 123.5 136.7 36.6 26.0 127.7 78.3 10.1 15.5 16.8 15.0
70.6 77.4 124.6 140.0 37.9 25.4 61.9 59.7 40.1 23.7 123.9 136.0 35.3 27.3 68.3 98.5 11.1 15.5 16.7 14.9
Cytotoxicity assay Compounds 1–12 were evaluated for cytotoxicities against MCF-7 human breast cancer cell line using the MTT[24] method in 96-well microplates. Cisplatin was used as a positive control. Acknowledgements The authors thank the National Natural Science Foundation of China (No. 81102339) and Guangdong Natural Science Foundation (No. S2011040002429) for providing financial support to this work.
References [1] K.-H. Lin, Y.-J. Tseng, B.-W. Chen, T.-L. Hwang, H.-Y. Chen, C.-F. Dai, J.-H. Sheu. Org. Lett. 2014, 16, 1314–1317. [2] L.-F. Liang, T. Kurtan, A. Mandi, L.-G. Yao, J. Li, W. Zhang, Y.-W. Guo. Org. Lett. 2013, 15, 274–277. [3] Z. Xi, W. Bie, W. Chen, D. Liu, L. van Ofwegen, P. Proksch, W. Lin. Mar. Drugs 2013, 11, 3186–3196. [4] Z. Wang, H. Tang, P. Wang, W. Gong, M. Xue, H. Zhang, T. Liu, B. Liu, Y. Yi, W. Zhang. Mar. Drugs 2013, 11, 775–787. [5] N. P. Thao, N. H. Nam, N. X. Cuong, T. H. Quang, P. T. Tung, B. H. Tai, B. T. T. Luyen, D. Chae, S. Kim, Y.-S. Koh, P. V. Kiem, C. V. Minh, Y. H. Kim. Chem. Pharm. Bull. 2012, 60, 1581–1589. [6] Z.-H. Sun, Y.-H. Cai, C.-Q. Fan, G.-H. Tang, H.-B. Luo, S. Yin. Mar. Drugs 2014, 12, 672–681. [7] Z.-B. Cheng, H. Xiao, C.-Q. Fan, Y.-N. Lu, G. Zhang, S. Yin. Steroids 2013, 78, 1353–1358. [8] Z.-B. Cheng, W.-W. Shao, Y.-N. Liu, Q. Liao, T.-T. Lin, X.-Y. Shen, S. Yin. J. Nat. Prod. 2013, 76, 664–671. [9] D.-Z. Liu, B.-W. Liang. Magn. Reson. Chem. 2014, 52, 57–59. [10] T. Grkovic, E. L. Whitson, D. C. Rabe, R. S. Gardella, D. P. Bottaro, W. M. Linehan, J. B. McMahon, K. R. Gustafson, T. C. McKee. Bioorg. Med. Chem. Lett. 2011, 21, 2113–2115. [11] N. X. Cuong, T. A. Tuan, P. V. Kiem, C. V. Minh, E. M. Choi, Y. H. Kim. Chem. Pharm. Bull. 2008, 56, 988–992.
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20.9 170.4
All-trans-(3S, 5R, 6R, 3′S, 5′R, 6′S)-peridinin (10): 13C NMR (MeOH-d4, 100 MHz) δC 36.8 (C, C-1), 46.7 (CH2, C-2), 69.4 (CH, C3), 46.6 (CH2, C-4), 72.9 (C, C-5), 118.3 (C, C-6), 204.1 (C, C-7), 104.0 (CH, C-8), 135.2 (C, C-9), 129.3 (CH, C-12), 133.0 (CH, C-13), 134.4 (CH, C-14), 139.1 (CH, C-15), 32.8 (CH3, C-16), 31.2 (CH3, C-17), 29.5 (CH3, C-18), 14.3 (CH3, C-19), 36.2 (C, C-1′), 47.9 (CH2, C-2′), 64.5 (CH, C-3′), 41.6 (CH2, C-4′), 68.9 (C, C-5′), 71.7 (C, C-6′), 134.7 (C, C-7′), 123.0 (CH, C-8′), 125.5 (C, C-9′), 138.3 (CH, C-10′), 148.2 (CH, C-11′), 120.9 (CH, C-12′), 135.2 (CH, C-13), 140.0 (CH, C-14′), 139.1 (CH, C-15′), 30.1 (CH3, C-16′), 25.3 (CH3, C-17′), 21.3 (CH3, C-18′), 170.5 (C, C-19′), 15.5 (CH3, C-20′), 3-OAc (172.3, 20.2) All-trans-(9′Z,11′Z)-(3R,3′S,5’R,6’R)-pyrrhoxanthin (11): 13C NMR (CDCl3, 100 MHz) δC 36.1 (C, C-1), 42.3 (CH2, C-2), 67.9 (CH, C-3), 37.5 (CH2, C-4), 137.2 (C, C-5), 124.3 (C, C-6), 90.1 (C, C-7), 98.6 (CH, C-8), 121.0 (C, C-9), 134.6 (CH, C-12), 130.6 (CH, C-13), 133.8 (CH, C-14), 136.9 (CH, C-15), 28.7 (CH3, C-16), 30.2 (CH3, C-17), 22.4 (CH3, C-18), 18.1 (CH3, C-19), 35.31 (C, C-1′), 47.1 (CH2, C-2′), 64.2 (CH, C-3′), 40.9 (CH2, C-4′), 67.5 (C, C-5′), 70.5 (C, C-6′), 133.7 (C, C-7′), 121.8 (CH, C-8′), 125.0 (C, C-9′), 136.3 (CH, C-10′), 147.0 (CH, C-11′), 119.1 (CH, C-12′), 134.4 (CH, C-13), 137.8 (CH, C-14′), 129.8 (CH, C-15′), 29.5 (CH3, C-16′), 24.9 (CH3, C-17′), 19.9 (CH3, C-18′), 168.7 (C, C-19′), 15.4 (CH3, C-20′), 3-OAc (170.7, 21.3). Sarcophytonone (12): 13C NMR (CDCl3, 100 MHz) δC 177.2 (C, C-1), 39.4 (CH, C-2), 34.2 (CH, C-3), 21.6 (CH2, C-4), 41.7 (CH2, C-5), 72.5 (C, C-6), 40.2 (CH2, C-7), 21.3 (CH2, C-8), 17.1 (CH3, C-9), 26.5 (CH3, C-10), 144.3 (C, C-1′), 140.6 (C, C-2′), 187.6 (C, C-3′), 140.4 (C, C-4′), 140.2 (C, C-5′), 187.2 (C, C-6′), 12.4 (CH3, C-7′), 12.3 (CH3, C-8′), 12.0 (CH3, C-9′).
Z.-B. Cheng et al. [12] C.-H. Chao, Z.-H. Wen, Y.-C. Wu, H.-C. Yeh, J.-H. Sheu. J. Nat. Prod. 2008, 71, 1819–1824. [13] G.-A. Zou, G. Ding, Z.-H. Su, J.-S. Yang, H.-W. Zhang, C.-Z. Peng, H. A. Aisa, Z.-M. Zou. J. Nat. Prod. 2010, 73, 792–795. [14] K. H. Shaker, M. Mueller, M. A. Ghani, H.-M. Dahse, K. Seifert. Chem. Biodivers. 2010, 7, 2007–2015. [15] P. V. Kiem, C. V. Minh, N. X. Nhiem, N. T. Cuc, N. V. Quang, H. L. T. Anh, B. H. Tai, P. H. Yen, N. T. Hoai, K. Y. Ho, N. Kim, S. Park, S. H. Kim. Magn. Reson. Chem. 2014, 52, 51–56. [16] M. R. Rao, U. Venkatesham, K. V. Sridevi, P. S. Reddy, K. Jamil, Y. Venkateswarlu. Indian J. Chem. B Org. 2002, 41B, 1472–1476. [17] J. C. Coll, G. B. Hawes, N. Liyanage, W. Oberhansli, R. J. Wells. Aust. J. Chem. 1977, 30, 1305–1309. [18] S.-T. Lin, S.-K. Wang, C.-Y. Duh. Mar. Drugs 2011, 9, 2705–2716. [19] B. F. Bowden, J. C. Coll, A. Heaton, G. König, M. A. Bruck, R. E. Cramer, D. M. Klein, P. J. Scheuer. J. Nat. Prod. 1987, 50, 650–659.
[20] T. H. Quang, T. T. Ha, C. V. Minh, P. V. Kiem, H. T. Huong, N. T. T. Ngan, N. X. Nhiem, N. H. Tung, B. H. Tai, D. T. T. Thuy, S. B. Song, H.-K. Kang, Y. H. Kim. Bioorg. Med. Chem. 2011, 19, 2625–2632. [21] J. Krane, T. Aakermann, S. Liaaen-Jensen. Magn. Reson. Chem. 1992, 30, 1169–1177. [22] G. Englert, T. Aakermann, S. Liaaen-Jensen. Magn. Reson. Chem. 1993, 31, 910–915. [23] L. Li, C.-Y. Wang, C.-L. Shao, L. Han, X.-P. Sun, J. Zhao, Y.-W. Guo, H. Huang, H.-S. Guan. J. Asian Nat. Prod. Res. 2009, 11, 851–855. [24] T. Mosmann. J. Immunol. Methods 1983, 65, 55–63.
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