Heterodimeric Diterpenoids Isolated from Euphorbia ebracteolata

Nov 17, 2017 - Column chromatography was performed using silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China...
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Heterodimeric Diterpenoids Isolated from Euphorbia ebracteolata Roots and Their Inhibitory Effects on α‑Glucosidase Yunlong Wei,†,‡,# Chao Wang,†,# Zhongbin Cheng,§,# Xiangge Tian,† Jingming Jia,‡ Yonglei Cui,† Lei Feng,*,† Chengpeng Sun,† Baojing Zhang,† and Xiaochi Ma*,† †

College of Pharmacy, Academy of Integrative Medicine, and Liaoning Engineering Technology Centre of Target-based Nature Products for Prevention and Treatment of Ageing-related Neurodegeneration, Dalian Medical University, Dalian 116044, People’s Republic of China ‡ School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China § College of Pharmacy, Henan University, Kaifeng 475004, People’s Republic of China S Supporting Information *

ABSTRACT: Two heterodimeric diterpenoids (1 and 2) comprising abietane lactone and nor-rosane constituent units were isolated from Euphorbia ebracteolata roots. Compound 1 exhibited a moderate inhibitory effect on α-glucosidase (IC50 = 7.94 μM), with a Ki value of 10.8 μM. In silico molecular docking has been performed to investigate the inhibition mechanism. Compound 2 inhibited the acetyl transfer activity of Mycobacterium tuberculosis GlmU (IC50 = 41.85 μM), which is a novel tuberculosis treatment target.

T

he perennial herbaceous plant Euphorbia ebracteolata Hayata (Euphorbiaceae) is distributed throughout East Asia. The roots of this plant, known as “LangDu” in traditional Chinese medicine, exhibit various biological activities and have been used to treat edema, indigestion, cough, asthma, and chronic bronchitis.1 Chemical investigations revealed that these roots contain diterpenoids, triterpenoids, flavonols, sesquiterpenoids, and acetophenones.2−6 Interestingly, various types of diterpenoids, including ent-abietanes, rosanes, isopimaranes, ingenanes, casbanes, and kauranes, were obtained from the roots of E. ebracteolata and were found to exhibit antibacterial, anti-inflammatory, and cytotoxic effects and inhibit B lymphocytes.7−13 In this study, two dimeric diterpenoids, 1 and 2, were isolated from E. ebracteolata and rigorously characterized. The compounds were determined to be heterodimeric diterpenoids consisting of ent-abietane and rosane-type monomeric constituent units. They represent a rare class of natural products, although a dimeric rosane was previously reported.14 The inhibitory effect of compounds 1 and 2 on α-glucosidase was evaluated in vitro, and the αglucosidase inhibition kinetics of compound 1 were also studied. The interactions between 1 and α-glucosidase were investigated by a docking study. The inhibitory effects of both compounds on M.tb. GlmU and their cytotoxicities were also evaluated in vitro.

UV spectrum suggested the presence of an aromatic ring. The H NMR spectrum of 1 exhibited proton signals corresponding to seven methyls (δH 0.68, 0.85, 0.92, 0.94, 1.00, 1.83, 2.08; each 3H; s), two oxygenated methines (δH 3.96 d; J = 7.8, 3.0 Hz; 3.82 s), one aromatic proton (δH 6.98 s), and one terminal olefinic moiety (δH 5.86 dd; J = 18.0, 10.8 Hz; 4.95 dd; J = 18.0, 1.8 Hz; 4.88 dd; J = 10.8, 1.8 Hz) (Table 1). The 13C NMR spectrum revealed the presence of 39 carbons, including two oxygenated methines (δC 73.3, 57.7), an oxygenated tertiary carbon (δC 67.1), a dioxygenated secondary carbon (δC 105.9), an α,β-unsaturated γ-lactone (δC 169.2, 149.4, 131.9, 105.9), a terminal olefinic bond (δC 108.8, 151.2), and an aromatic ring (δC 144.6, 139.3, 135.5, 132.6, 123.5, 117.2) (Table 1). The spectroscopic data indicated that compound 1 is a dimeric diterpenoid consisting of an ent-abietane and an 18-nor-rosane constituent unit with an aromatic ring.13,14 1H−1H COSY, HMBC, and HSQC spectra were collected to determine the 1

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RESULTS AND DISCUSSION Compound 1 had a molecular formula of C39H52O6 established by HRESIMS ([M − H]− m/z 615.3694, calcd for C39H51O6 615.3686), indicating 14 indices of hydrogen deficiency. The λmax values of 201.4, 221.3, 243.5, and 276.0 nm observed in the © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 14, 2017 Published: November 17, 2017 3218

DOI: 10.1021/acs.jnatprod.7b00595 J. Nat. Prod. 2017, 80, 3218−3223

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Table 1. 1H (600 MHz, CDCl3) and 13C (150 MHz, CDCl3) NMR Data for Compounds 1 and 2 (δ in ppm, J in Hz) 1 no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′

17′ 18′ 19′ OH

2

δH

δC

δH

1.96 m, 1.32 m 1.53 m, 1.35 m 1.45 m, 1.21 m

38.8

2.22 m, 1.23 m 1.58 m, 1.37 m 1.43 m, 1.23 m

1.12 dd (12.6, 2.4) 1.81 m, 1.48 m 1.64 m, 2.13 m 2.02 m 3.96 dd (7.8, 3.0)

3.82 s

1.83 0.85 0.94 0.68 6.98

s s s s s

2.62 m 1.60 m, 1.52 m 1.71 m

2.01 m, 1.50 m 1.66 m, 1.27 m 1.42 m, 1.18 m 5.86 dd (18.0, 10.8) 4.95 dd (18.0, 1.8) 4.88 dd (10.8, 1.8) 1.00 s 2.08 s 0.92 s 7.10 s

correlations between C-11 and H-9; C-12 and H-9 and H-14; C-8 and H-6α (δH 1.81), H-7β (δH 2.13), and H3-14; and C-16 and H-17 (Figure 1), suggesting that the abietane substructure is similar to jolkinolide B, an ent-abietane diterpenoid lactone isolated from Euphorbia f ischeriana,15 except for the seco-11,12epoxide moiety. The HMBC correlations between H-1′ and C2′, C-3′, and C-5′ and between H-18′ and C-3′, C-4′, and C-5′ revealed the presence of the aromatic A ring in the nor-rosane moiety. Similarly, the presence of the Δ15′(16′) olefinic bond was confirmed by the HMBC data. Based on these spectroscopic data, the nor-rosane constituent unit was determined to be ebractenoid F isolated from E. ebracteolata.10 Additionally, the long-range correlation observed in the HMBC spectrum between H-1′ and C-12 indicated the presence of the ether linkage between C-12 (abietane) and C-2′ (nor-rosane) in the dimeric diterpenoid. Thus, compound 1 was determined to be a dimeric diterpenoid with a 2D structure, as shown by the X-ray diffraction analysis (Figure 2). The relative configuration of the abietane structure was established based on the NOESY effects (Figure 1) between H3-20 and H-11; H-9 and H-5; H-5 and H7β (δH 2.13); and H-7α (δH 1.64) and H-14. Furthermore, H317′ was correlated with H-8′ in the NOESY experiment. The Xray diffraction analysis not only confirmed the 2D structure and relative configuration as deduced from the NOESY experiment but also defined the (5R, 8S, 9R, 10R, 11R, 12R, 14R, 8′S, 9′S, 13′S) absolute configuration of 1 based on the Flack/Hooft parameter of −0.00(5)/0.03(5) obtained using Cu Kα radiation (CCDC 1554072) (Figure 2). Therefore, the structure of compound 1 was defined as a heterodimeric diterpenoid comprising abietane and nor-rosane constituent units and named eupractenoid A. Compound 2 had the same molecular formula (C39H52O6) as compound 1, as determined by the HRESIMS and 13C NMR data. The proton signals in the 1H NMR spectrum of 2 indicated the presence of seven methyls (δH 0.94, 0.95, 0.98, 1.00, 1.04, 1.75, 2.08; each 3H; s), two oxygenated methines (δH 3.70 t, J = 9.6 Hz, 4.68 s), one aromatic proton (δH 6.93 s), and one terminal olefinic moiety (δH 5.85 dd; J = 18.0, 10.8 Hz; 4.95 dd; J = 18.0, 1.2 Hz; 4.88 dd; J = 10.8, 1.8 Hz) (Table 1). The 13C NMR spectrum of 2 exhibited 39 carbon signals, including those indicating the presence of two oxygenated methines (δC 76.6, 80.3), an oxygenated tertiary carbon (δC 77.5), a dioxygenated secondary carbon (δC 107.4), an α,βunsaturated γ-lactone moiety (δC 170.2, 148.0, 133.4, 107.4), a terminal olefinic unit (δC 109.0, 150.9), and an aromatic ring (δC 115.5, 144.3, 142.0, 129.5, 132.5, 145.2). Similar 1, compound 2 was also determined to be a dimeric diterpenoid consisting of abietane and nor-rosane constituent units based on the NMR data. The abietane substructure was revealed to be similar to jolkinolide B, and the nor-rosane substructure was identified as ebractenoid F based on the 1D and 2D NMR data analysis. However, the C-8, C-11, C-14, and C-2′ resonances were deshielded in the 13C NMR spectrum of 2. In the 1H NMR spectrum, a hydroxy proton was observed at δH 3.73, which was linked to C-8 based on the HMBC correlations between the OH proton (δH 3.73) and C-8 and C-7 and was indicative of the ring-opening of the 8,14-epoxide moiety in jolkinolide B. Furthermore, the long-range correlation in the HMBC spectrum from H-14 (δH 4.68) to C-3′ (δC 144.3) and the NOE association between H-14 and H3-18′ (Figure S1, Supporting Information) revealed the presence of an ether linkage between C-14 and C-3′. Therefore, the structure of compound 2 was similar to that of compound 1, except for the

18.2 41.5 33.4 53.2 20.8 34.8 67.1 59.0 39.6 73.3 105.9 149.4 57.7 131.9 169.2 8.8 22.0 33.5 14.9 117.2 135.5 144.6 123.5 132.6 27.2 25.5 36.3 36.5 139.3 33.9 32.8 36.4 39.6 151.2 108.8

22.7 11.5 21.3

2.55 m 1.89 m, 1.47 m 1.59 m, 1.29 m 1.97 m 3.70 t (9.6)

4.68 s

1.75 0.99 0.95 1.04 6.93

s s s s s

2.62 m 1.63 m, 1.55 m 1.67 m

1.97 m, 1.48 m 1.66 m, 1.38 m 1.43 m, 1.19 m 5.85 dd (18.0, 10.8) 4.95 dd (18.0, 1.2) 4.88 dd (10.8, 1.2) 1.00 s 2.08 s 0.98 s 3.73 s

exptl δC

calcd δC

Δδ

45.3

45.20

−0.10

19.3

20.74

1.44

42.1

41.05

−1.05

33.5 44.1

35.14 43.41

1.64 −0.69

17.5

19.00

1.50

27.5

27.39

−0.11

77.5 57.9 38.0 76.6

77.39 57.30 40.31 75.24

−0.11 −0.60 2.31 −1.36

107.4 148.0 80.3 133.4 170.2 8.8 22.4 34.4 16.1 115.5 142.0 144.3 129.5 132.5 27.1 25.3

105.30 147.87 81.25 135.90 168.01 9.62 20.18 31.82 15.56 116.54 144.70 142.48 129.79 132.31 28.38 26.20

−2.10 −0.13 0.95 2.50 −2.19 0.82 −2.22 −2.58 −0.54 1.04 2.70 −1.82 0.29 −0.19 1.28 0.90

36.1 36.8 145.2 33.8

37.13 38.94 144.70 33.32

1.03 2.14 −0.50 −0.48

32.7

28.67

−4.03

36.3 39.5

38.45 39.60

2.15 0.10

150.9

152.03

1.13

109.0

108.76

−0.24

22.7 11.6 21.0

22.05 10.71 19.63

−0.65 −0.89 −1.37

diterpenoid substructures. Based on the 1H−1H COSY spectrum, the following spin systems were established: H-1/ H-2/H-3, H-5/H-6/H-7, and H-9/H-11 for the abietane and H-6′/H-7′/H-8′/H-14′ and H-11′/H-12′ for the 18-nor-rosane (Figure 1). The HMBC spectrum revealed long-range 3219

DOI: 10.1021/acs.jnatprod.7b00595 J. Nat. Prod. 2017, 80, 3218−3223

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Figure 1. Key HMBC (H → C), 1H−1H COSY, and NOESY correlations for 1.

Figure 2. ORTEP drawing of compound 1.

Figure 3. (a) Experimental ECD of compound 2 and calculated ECDs of 2a (5R, 8S, 9R, 10R, 11R, 12R, 14R, 8′S, 9′S, 13′S), 2b (5S, 8R, 9S, 10S, 11S, 12S, 14S, 8′R, 9′R, 13′R); (b) regression analysis of the 13C NMR chemical shift parity plot for 2 (calculated results obtained from gas-phase mPW1PW91/6-311+G(d,p) calculations).

configuration (8β-OH, 11β-OH, 14α-H, 13′α-CH3) of 2 (Figure S1, Supporting Information). The absolute configuration of 2 was determined by calculating the ECD spectrum at the B3LYP/6-311++G(2d,2p)

presence of an additional oxygen linkage between the abietane and nor-rosane subunits. The NOE correlations between H-11 and H3-20; H-14 and H-7α (δH 1.29), H3-17; 8-OH and H-9; and H3-17′ and H-8′ were used to define the relative 3220

DOI: 10.1021/acs.jnatprod.7b00595 J. Nat. Prod. 2017, 80, 3218−3223

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Figure 4. α-Glucosidase inhibition kinetics by compound 1. (a) Dose-dependent inhibition; (b) inhibition kinetics; (c) Lineweaver−Burk plot; (d) determination of the inhibition kinetic parameter (Ki) from the plot of the Lineweaver−Burk slopes vs the concentration of 1. The data points represent the mean values of duplicate experiments.

level in methanol (Figure 3a). The results showed that the calculated ECD spectrum of the (5R, 8S, 9R, 10R, 11R, 12R, 14R, 8′S, 9′S, 13′S)-isomer resembled the experimental spectrum, whereas the calculated ECD curve of the (5S, 8R, 9S, 10S, 11S, 12S, 14S, 8′R, 9′R, 13′R)-isomer was opposite the experimental spectrum. Therefore, the absolute configuration of 2 was defined as (5R, 8S, 9R, 10R, 11R, 12R, 14R, 8′S, 9′S, 13′S). Additionally, the 13C NMR chemical shifts were also calculated to confirm the structure of 2. The experimental and calculated 13C chemical shifts showed a good linear relationship (R2 = 0.990), as shown by the parity plot in Figure 3b and the results listed in Table 1. Thus, based on the spectroscopic data, the structure of compound 2 was defined as a heterodimeric diterpenoid and was named eupractenoid B. The inhibitory effects of compounds 1 and 2 against αglucosidase were evaluated using p-NPG as the substrate and acarbose as a positive control. Compounds 1 and 2 inhibited αglucosidase activity with IC50 values of 7.94 and 50.36 μM, respectively, compared to an IC50 value of 578.75 μM for acarbose. The inhibition kinetics of compound 1 were studied, as shown in Figure 4. Based on the observed inhibition kinetics (Figure 4b) and the x-intercept of the Lineweaver−Burk plots (Figure 4c), it was concluded that 1 acted via a noncompetitive inhibition mechanism. The inhibition kinetic parameter (Ki) was determined to be 10.8 μM. To investigate the molecular interactions between the potential inhibitor 1 and α-glucosidase, a docking study was performed as shown in Figure 5. When 1 was localized in the αglucosidase hydrophobic pocket, the lactone carbonyl moiety of 1 formed a hydrogen bond with Gln279 (3.3 Å). It was concluded that this interaction was essential for the inhibitory

Figure 5. Docking analysis of compound 1 and α-glucosidase.

effect. Furthermore, hydrophobic interactions were observed between the abietane substructure of 1 and the α-glucosidase Arg315 and Phe303 residues and between the nor-rosane substructure of 1 and the α-glucosidase Tyr158, Phe178, Phe159, Tyr316, and Phe314 residues. 3221

DOI: 10.1021/acs.jnatprod.7b00595 J. Nat. Prod. 2017, 80, 3218−3223

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Eupractenoid B (2): white, amorphous solid; [α]25 D −11.4 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 201.8 (4.43), 220.5 (2.24), 242.0 (2.00), 275.1 (1.55) nm; ECD (CH3OH) Δε203.0 +11.44, Δε222.0 −12.05, Δε233.0 +1.69, Δε259.5 +2.92; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 1; HRESIMS m/z 651.3461 (calcd for C39H52O6Cl 651.3452). ECD Calculations. A conformational search was performed by Monte Carlo using the MMFF94 molecular mechanics force field in the Spartan 10 software. The conformers were reoptimized in methanol at the rcam-B3LYP/6-311++G(2d,2p) level using the Gaussian 09 program. The B3LYP/6-311++G(2d,2p) harmonic vibrational frequencies were determined to verify the stability of the optimized conformers. The solvent energies, oscillator strengths, and rotational strengths of the first 20 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311+ +G(2d,2p) level. The ECD spectra were simulated using the GaussSum 2.25 program (σ = 0.25 eV). All quantum computations were performed using the Gaussian 09 program package on an IBM cluster machine at the High Performance Computing Center of Peking Union Medical College.16,17 Calculated 13C NMR Data. Conformational analyses were performed by random searching with an energy cutoff of 2.5 kcal/ mol using the SYBYL-X 2.0 software. The MMFF94S force field was employed. Owing to the rigid skeleton and the limitations of the NOESY correlations, two lowest energy conformers were found for 2. The conformers were reoptimized in the gas phase at the DFT/ B3LYP/6-31G* level using the Gaussian 09 program. The 13C NMR shielding constants of compound 2 were calculated by the GIAO method at the MPW1PW91/6-31G(d,p) level of theory in the gas phase. The computational 13C NMR data were obtained by linear regression.18 X-ray Crystallographic Analysis of Compound 1. The crystal structure and absolute configuration of compound 1 were determined by single-crystal X-ray diffraction. A suitable crystal obtained from CH2Cl2 was selected and examined on an Xcalibur Onyx Nova diffractometer at 100.00(10) K. Crystal data for compound 1: C39H54O7 (M = 634.82), monoclinic, space group I2, a = 18.06063(19) Å, b = 9.22305(12) Å, c = 20.6152(2) Å, α = 90°, β = 91.6998(9)°, γ = 90°, volume = 3432.44(7) Å3, Z = 4, T = 100.00(10) K, μ(Cu Kα) = 0.661 mm−1, Dcalc = 1.228 g/cm3, 17 055 reflections collected, 6184 unique reflections [Rint = 0.0235, Rsigma = 0.0236] used in all the calculations, R1(final) = 0.0288, wR2 = 0.0735 (all data), Flack parameter = −0.00(5)/0.03(5). The crystallographic data for 1 were deposited at the Cambridge Crystallographic Data Centre under the reference number CCDC 1554072. Molecular Docking Simulations. Molecular docking simulations of compound 1 in the crystallographic asymmetric unit were performed to determine the conformation of compound 1 in the binding pocket. For the structure of α-glucosidase, the molecular docking analysis was carried out by using a high sequence homology (PDB ID: 3A4A; gi number: 411229) of α-glucosidase through searching the Protein Data Bank (PDB). The initial structure was constructed based on comparison of the geometrical parameters of the calculated model and crystal structure. The pdbqt files were prepared by a standard procedure using the AutoDock Tools 1.5.6 software. The docking procedures were performed using the AutoDock Vina program with the default scoring function. The exhaustiveness was set to 100, and the number of output conformations was set to 20. The searching seed was random. The calculated geometries were ranked by the free energy of binding, and the best poses were selected for further analysis.19,20 α-Glucosidase Inhibitory Activity. The abilities of compounds 1 and 2 to inhibit α-glucosidase were tested according to a previously described assay.21 Inhibitory Effects on Mycobacterium tuberculosis GlmU. A colorimetric acetyltransferase assay was performed using 5,5′-dithiobis(2-nitrobenzoic acid) and the purified M.tb. GlmU protein.22

The inhibitory effects of compounds 1 and 2 against on M.tb. GlmU, a tuberculosis treatment target, were also investigated. Compound 2 moderately inhibited the acetyl transfer activity of M.tb. GlmU (IC50 = 41.85 μM); however, compound 1 did not inhibit M.tb. GlmU (IC50 > 100 μM). Neither 1 nor 2 exhibited cytotoxic activity against five human cancer cell lines (IC50 values >100 μM), that is, the A549 (human lung carcinoma), Bel-7402 (human liver carcinoma), BGC-823 (human stomach carcinoma), HCT-8 (human colon carcinoma), and A2780 (human ovarian carcinoma) cell lines, as measured by the MTT method. In conclusion, two heterodimeric diterpenoids (1 and 2) were isolated from E. ebracteolata roots, and their structures were determined by various spectroscopic techniques, including UV, HRESIMS, 1D NMR, 2D NMR, X-ray diffraction, and ECD and by calculation of the ECD and 13C NMR spectra of 2. Compound 1 exhibited a moderate inhibitory effect on αglucosidase. Additionally, compound 2 inhibited the acetyl transfer activity of M.tb. GlmU, a novel tuberculosis treatment target.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. UV spectra were collected on a JASCO V-650 spectrophotometer. NMR spectra were obtained with a Bruker 600 NMR spectrometer using various solvents with tetramethylsilane as an internal standard. ESIMS data were recorded on an API 3200 mass spectrometer (AB Sciex, Framingham, MA, USA). HRESIMS were collected on an Agilent 1290 Infinity system connected to a 6540 UHD Accurate-Mass QTOF system. Analytical HPLC experiments were conducted on a Dionex UltiMate 3000 instrument (Thermo Scientific) equipped with a diode array detector and a Waters XBridge RP C18 column (250 × 4.6 mm, 5 μm). Preparative HPLC was performed on an Agel instrument equipped with a UV detector and YMC C18 column (250 × 10 mm, 5 μm). Column chromatography was performed using silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China). TLC was performed using precoated silica gel GF254 plates (5 × 10 cm, 2.5 × 7.5 cm, Qingdao Marine Chemical Inc.). The spots were visualized under UV light or by spraying the plates with 10% sulfuric acid in EtOH, followed by heating at 105 °C. Plant Material. E. ebracteolata roots were collected from Bozhou in Anhui Province, People’s Republic of China, and identified by Prof. Qing-Shan Yang of Anhui University of Chinese Medicine. A voucher specimen (No. P-231) was deposited in the College of Pharmacy, Dalian Medical University. Extraction and Isolation. The powdered roots of E. ebracteolata (15 kg) were extracted with 80% EtOH (3 × 50 L, each 1.5 h). After EtOH evaporation in vacuo, the aqueous residue was diluted with H2O and sequentially partitioned with petroleum ether, EtOAc, and nBuOH. The EtOAc extract (504 g) was separated into 55 fractions by silica gel column chromatography using petroleum ether/acetone (50:1−2:1) mixtures as the eluent. Subfraction 10 (6 g) was further separated into 23 fractions (A1−A23) by MPLC (ODS) using MeOH/ H2O (40:60−95:5) mixtures as the eluent. Subfraction A17 was purified by preparative HPLC (RP C18 column, 8 mL/min, detected at 210 and 250 nm) with a 75:25 MeOH/H2O mixture as the eluent to yield compounds 1 (tR = 67.5 min, 18 mg) and 2 (tR = 84.5 min, 3 mg). Eupractenoid A (1): colorless crystals (CH2Cl2); [α]25 D −8 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 201.4 (4.40), 221.3 (2.37), 243.5 (2.05), 276.0 (1.87) nm; ECD (CH3OH) Δε207.0 +16.05, Δε220.5 −2.26, Δε232.5 +15.86, Δε255.0 +24.47, Δε283 −3.79, Δε314.0 −2.64; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 1; HRESIMS m/z 615.3694 (calcd for C39H51O6 615.3686). 3222

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(16) Che, C. T.; Zhou, T. X.; Ma, Q. G.; Qin, G. W.; Williams, I. D.; Wu, H. M.; Shi, Z. S. Phytochemistry 1999, 52, 117−121. (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T., Jr.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Rev. C 01; Gaussian, Inc.: Wallingford, CT, 2009. (18) Stephens, P. J.; Harada, N. Chirality 2010, 22, 229−233. (19) Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. J. Chem. Rev. 2012, 112, 1839−1862. (20) Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.; Harrell, P. M.; Trinh, Y. T.; Zhang, Q. H.; Urbatsch, I. L.; Chang, G. Science 2009, 323, 1718−1722. (21) Hu, X. J.; Wang, X. B.; Kong, L. Y. J. Agric. Food Chem. 2013, 61, 1501−1508. (22) Zhang, W. L.; Jones, V. C.; Scherman, M. S.; Mahapatra, S.; Crick, D.; Bhamidi, S.; Xin, Y.; McNeil, M. R.; Ma, Y. F. Int. J. Biochem. Cell Biol. 2008, 40, 2560−2571.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00595. Spectra of the new compounds (PDF) X-ray data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-411-86110419. Fax: +86-411-86110408. E-mail: [email protected] (L. Feng). *E-mail: [email protected] (X.-C. Ma). ORCID

Xiaochi Ma: 0000-0003-4397-537X Author Contributions #

Y. Wei, C. Wang, and Z. Cheng contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Nos. 81503201 and 81622047), Dalian Outstanding Youth Science and Technology Talent awards (2014J11JH132 and 2015J12JH201), and Program for Distinguished Professor of Liaoning Province. The calculations were performed by Minghua Chen (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China). The authors thank Prof. Q.-S. Yang of Anhui University of Chinese Medicine for the assistance.

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DOI: 10.1021/acs.jnatprod.7b00595 J. Nat. Prod. 2017, 80, 3218−3223