Penicyclones A–E, Antibacterial Polyketides from the Deep-Sea

Article pubs.acs.org/jnp

Penicyclones A−E, Antibacterial Polyketides from the Deep-SeaDerived Fungus Penicillium sp. F23‑2 Wenqiang Guo, Zhenzhen Zhang, Tianjiao Zhu, Qianqun Gu, and Dehai Li* Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *

ABSTRACT: Five new ambuic acid analogues, penicyclones A− E (1−5), were isolated from the extract of the deep-sea-derived fungus Penicillium sp. F23-2. The structures including the absolute configurations were established by interpretation of NMR and MS data, as well as the application of ECD, X-ray crystallography, and a chemical conversion, as well as the TDDFT-ECD calculations. Penicyclones A−E (1−5) exhibited antimicrobial activity against the Gram-positive bacterium Staphylococcus aureus with MIC values ranging from 0.3 to 1.0 μg/mL.

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ungi are a promising source of beneficial drug leads due to the unique structures and diverse bioactivities of their secondary metabolites.1 Although thousands of natural products have been discovered from a range of fungal species, most of the genes encoding secondary metabolites are proved to be cryptic under traditional single culture conditions.2 Along with the various efforts to activate the silent genes from genome mining to epigenetic modification, the OSMAC approach (considering the composition of culture medium, aeration, period of cultivation, pH, temperature, etc.) offers an alternative with economic and convenience advantages, based on the knowledge that the types of secondary metabolites are often altered in different cultivation conditions.3 During our efforts to maximize the ability of marine-derived fungi to produce bioactive molecules using the OSMAC approach,4 the deep-sea-derived fungus Penicillium sp. F23-2 was an attractive candidate due to its known production of cytotoxic NRPS alkaloids (meleagrins and roquefortines) and terpenoids in a potato-based medium under static conditions and nitrogen-containing polyketides (sorbicillinoids) in agitated PYG medium culture.5 Inspired by the finding of varied structures, more culture conditions were attempted. During the process, we found the HPLC-UV profiles of the secondary metabolites differed from the previous ones when the fungal strain was cultured on a rice-based solid medium. Further exploration of the components led to the isolation of five new ambuic acid analogues, named penicyclones A−E (1−5), which showed antibacterial activity against Staphylococcus aureus. Herein, we report the details of the isolation, structure elucidation, and biological activities of 1−5.

and HPLC using ODS and chiral-phase columns, yielding compounds 1 (15.0 mg), 2 (9.5 mg), 3 (7.0 mg), 4 (8.0 mg), and 5 (6.5 mg). Penicyclone A (1) was obtained as a white powder. The molecular formula was determined as C12H16O5 on the basis of the protonated molecule peak at m/z 241.1071 by HRESIMS, indicating five degrees of unsaturation. The 1D NMR data (Tables 1 and 2) of 1 suggested the presence of two methyl groups, two methylenes, four methines with two of them oxygenated (δC 69.3 and 70.9) and an sp2 hybrid one (δC 123.4 and δH 5.86), and four nonprotonated carbons including one oxygenated (δC 87.8) and two carbonyl carbons (δC 196.0 and 173.8).

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RESULTS AND DISCUSSION The MeOH extract of the fermentation of Penicillium sp. F23-2 was fractionated by ODS and LH-20 column chromatography © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 26, 2015

A

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Table 1. 1H (500 MHz) NMR Data for Compounds 1−3 in DMSO-d6 no. 1 2 3 4 5

1

2

6 7

5.64, s

5.66, s

4.15, dd (5.7, 4.6)

4.41, dd (7.8, 3.7) 3.76, dd (4.3, 3.7)

4.43, dd (7.8, 3.7)

1.69, m; 1.59, m 1.53, m; 1.39, m 2.23, m 1.89, s 1.03, d (6.9)

1.69, dt (11.8, 2.7); 1.50, dt (11.8, 2.7) 1.60, m; 1.45, m

2.21, dt (14.4, 3.2); 2.00, dt (14.4, 3.2) 1.75, m; 1.24, m

8 9 11 12 13 OH-4 OH-5 OH-6 OH-10

3

5.86, s

3.82, dd (6.3, 4.6)

2.38, m 2.01, s 1.11, d (6.9) 5.68, d (5.7) 5.61, d (6.3)

correlation between H-4/H-5 and the HMBC correlations from H-2 to C-1/C-3/C-4, from H-4 to C-5/C-6, from H-5 to C-1/ C-3/C-6, and from H3-11 to C-2/C-3/C-4. The additional COSY correlations H2-7/H2-8/H-9/H3-12 together with the HMBC correlations from H2-7/H2-8 to C-6 and from H2-8/H9/H3-12 to C-10 established the carbon skeleton. The two hydroxy groups were located on C-4 and C-5 based on the COSY correlations from 4-OH/H-4 and 5-OH/H-5. Finally, the gross structure of 1 was established by connecting C-6 (δC 87.8) to C-10 (δC 173.8) to form a δ-valerolactone ring based on the chemical shifts. The relative configuration of 1 was determined by NOESY correlations. The NOEs between H-4 and H-5 suggested they were cis, which agreed with the coupling constant (3JH‑4,H‑5 = 4.6). The NOESY correlations of H-7a (δH 2.21) and H-9 indicated they are on the same face of the lactone ring. The orientation of C-7 was determined to be cofacial on the cyclohexane ring with 5-OH, supported by the correlations between H-7a and OH-5. Finally, the absolute configuration was suggested to be 4R, 5R, 6S, 9R by the X-ray diffraction analysis using Cu Kα radiation [Flack parameter = −0.2(3)] (Figure 3) and further confirmed by good agreement of the ECD spectra between the experimental curve of 1 and the TDDFT-ECD-calculated one for (4R,5R,6S,9R)-1 (Figure 4).

5.08, d (7.8) 4.83, d (4.3) 5.22, s 11.95, brs

3.77, dd (4.3, 3.7)

2.37, 1.90, 1.07, 3.59, 5.08, 4.83, 5.22,

m s d (6.9) s d (7.8) d (4.3) s

Table 2. 13C (125 MHz) NMR Data for Compounds 1−3 in DMSO-d6 no.

1

1 2 3 4 5 6 7 8 9 10 11 12 13

196.0, 123.4, 162.0, 69.3, 70.9, 87.8, 24.6, 24.6, 34.7, 173.8, 21.6, 16.9,

2 C CH C CH CH C CH2 CH2 CH C CH3 CH3

198.3, 123.7, 160.2, 69.0, 74.1, 76.1, 29.3, 26.2, 39.7, 178.1, 20.6, 17.4,

3 C CH C CH CH C CH2 CH2 CH C CH3 CH3

198.2, 123.7, 160.3, 69.0, 74.1, 76.0, 29.1, 26.3, 39.6, 176.8, 20.6, 17.2, 51.7,

C CH C CH CH C CH2 CH2 CH C CH3 CH3 CH3

Figure 2. Key NOESY correlations of compounds 1, 2, 4, and 5.

The planar structure of 1 was deduced from the interpretation of 1D and 2D NMR spectroscopic data (Figure 1). The cyclohexenone moiety was determined by the COSY

Penicyclones B and C (2 and 3) were isolated as colorless oils. The molecular formulas of 2 and 3 were determined as C12H18O6 and C13H20O6 by HRESIMS, respectively. The 1D NMR data of 2 and 3 (Tables 1 and 2) were similar to those of

Figure 1. Key COSY and HMBC correlations of 1−5.

Figure 3. ORTEP drawing of the crystal structure of 1. B

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NMR and DEPT spectroscopic data (Table 3) of 4 revealed the presence of two olefinic methyl singlets, two oxygenated Table 3. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of 4 and 5 in CD3OD 4 no.

Figure 4. B3LYP/6-31+G(d)-calculated ECD spectrum of (4R,5R,6S,9R)-1 (red) and the experimental spectrum of 1 (black) (σ = 0.28 eV).

the coisolated 1, indicating that they shared the same carbon structure. The major differences are the appearance of two additional exchangeable protons (δH 5.22 and 11.95) in 2 and of one exchangeable proton (δH 5.22) and a methoxy group (δC 51.7, δH 3.59) in 3. The planar structures were further established by COSY and HMBC correlations (Figure 1). The coupling constants between H-4 and H-5 (3.7 Hz) of 2 and 3 indicated that they are cis, which agreed with the NOESY correlations of H-4/H-5 (Figure 2). The similar ECD spectra of 1−3 (Figure 5) indicated that they share the same 4R, 5R, 6S

δC, type

1 2 3 4 5 6 7

194.2, 123.4, 156.2, 60.5, 67.4, 61.4, 26.4,

C CH C CH CH C CH2

8 9 10

34.3, CH2 138.7, C 116.1, CH

11 12 13 14

33.3, 177.3, 14.9, 19.0,

CH2 C CH3 CH3

5 δH (J in Hz)

5.70, brs 3.66, d (3.0) 4.52, d (3.0) 2.28, ddd (5.7, 5.7, 1.6); 1.58, ddd (5.7, 5.7, 1.6) 2.16, 2.14, m 5.28, t (6.6) 3.00, m 1.65, s 1.97, s

δC, type 194.5, 122.2, 158.2, 59.2, 66.8, 60.3, 25.8,

C CH C CH CH C CH2

117.1, CH 137.7, C 34.7, CH2 29.4, 178.3, 14.9, 19.0,

CH2 C CH3 CH3

δH (J in Hz) 5.71, s 3.64, d (3.0) 4.51, d (3.0) 2.73, dd (15.4, 8.2); 2.41, dd (15.4, 8.2) 5.11, t (7.1) 2.29, dd (13.6, 7.1) 2.37, brs 1.67, s 1.98, s

methines, one oxygenated nonprotonated carbon, two olefinic methines, two olefinic quaternary carbons, and two carbonyl carbons. The planar structure of 4 was deduced from interpretation of the COSY and HMBC spectroscopic data. The cyclohexenone moiety was determined by comparison of the NMR data with those of the reported deacetoxyyanuthone A6 and confirmed by the COSY and HMBC correlations (Figure 1). The additional COSY correlations between H2-7/ H2-8 and H-10/H2-11 together with the HMBC corrections from H-7 to C-1 and C-6, from H-14 to C-9, C-10, and C-11, and from H-11 to C-12 indicated a branched-chain carboxylic acid, which was located on the cyclohexenone at C-6. Finally, the planar structure of 4 was deduced by attaching two methyl groups to C-3/C-9 based on the HMBC correlations from H313 to C-2/C-3/C-4 and from H3-14 to C-8/C-9/C-10. Penicyclone E (5) was obtained as a colorless oil. The 1D NMR data of 5 (Table 3) were similar to those of 4, except the main differences of H-8/10 (4: δH 2.16, 5.28; 5: δH 5.11, 2.29) and C-8/10 (4: δC 34.3, 116.1; 5: δC 117.1, 34.7) indicated that the positions of the double bonds in the side chains were exchanged (Δ9 in 4 vs Δ8 in 5). This conclusion was further supported by the COSY (H-7/H-8 and H-10/H-11) and HMBC correlations (Figure 1). The relative configurations of 4 and 5 were determined by analysis of the coupling constants and NOESY correlations. The side chain double bonds in 4 and 5 were both assigned the E-geometry on the basis of the NOESY correlations of H-8a (δH 2.16) with H-10 (δH 5.28) in 4 and H-8 (δH 5.11) with H10a (δH 2.29) in 5, respectively (Figure 2). The small vicinal coupling constant (3JH‑4, H‑5 = 3.0 Hz) in compounds 4 and 5 suggested the cis relationship of H-4 and H-5, which agreed with their NOESY correlations (Figure 2). The NOESY correlation of H-7 (δH 2.28 in 4, δH 2.73 in 5) with H-5 (δH 4.52 in 4, δH 4.51 in 5) indicated that they are cofacial relative to the epoxide ring and the cyclohexenone ring. The absolute configuration of 4 was deduced as 2R, 3R, 4R by comparison of the calculated ECD spectrum of (2R,3R,4R)4 and the experimental one (Figure 6). Compounds 4 and 5

Figure 5. Experimental ECD curves of 1−3 in CH3OH.

absolute configurations. The configurations of C-9 of 2 and 3 were indicated as R by formation of 3 via the alcoholysis of 1 (Scheme 1), together with biogenetic considerations based on their coisolation. In addition, it is possible that similar conversions among compounds 1−3 may occur during isolation or fermentation. Penicyclone D (4) was isolated as a colorless oil. The molecular formula of C14H18O5, which gave six degrees of unsaturation, was established by HRESIMS. The 1H and 13C Scheme 1. Alcoholysis of 1

C

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min. After cooling to room temperature, each flask was inoculated with spores inoculum and incubated at 15 °C for 60 days. The fermentation broth was extracted with MeOH and then was concentrated under reduced pressure to give an extract (15.0 g). Extraction and Purification. The extract was separated by ODS column chromatography with a gradient elution step of MeOH/H2O (10% to 100%) to give six fractions (fraction 1 to fraction 6). Fraction 2 (30% MeOH/H2O) was further separated on a Sephadex LH-20 column with MeOH to provide six subfractions (fraction 2-1 to fraction 2-6). Fraction 2-3 was separated by MPLC and then semipreparative HPLC eluting with MeOH/H2O (25:75 + 0.2% TFA) to obtain a mixture of 1/2/3 (45.5 mg, tR = 17.5 min) together with a mixture of 4/5 (18.0 mg, tR = 19.0 min). Employing a chiralphase column (DAICEL IA), the mixtures were further fractionated with 2-propanol/hexane (15:85) to yield compounds 1 (15.0 mg, tR = 12.0 min), 2 (9.5 mg, tR = 12.5 min), and 3 (7.0 mg, tR = 13.0 min) and with 2-propanol/hexane (10:90) to yield compounds 4 (8.0 mg, tR = 11.6 min) and 5 (6.5 mg, tR = 13.6 min). Penicyclone A (1): white powder; [α]20D +72.8 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 220 (3.12), 230 (3.05) nm; ECD (c 4.17 × 10−3 M, MeOH) λmax (Δε) 218 (+1.74), 242 (−3.78) nm; IR (KBr) νmax 3419, 2931, 1712, 1683, 1222 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 241.1071 [M + H]+ (calcd for C12H17O5, 241.1071). Penicyclone B (2): colorless oil; [α]20D +83.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 221 (3.23), 231 (3.12) nm; ECD (c 3.88 × 10−3 M, MeOH) λmax (Δε) 216 (+1.39), 245 (−2.56) nm; IR (KBr) νmax 3429, 2935, 1720, 1670, 1200 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 259.1178 [M + H]+ (calcd for C12H19O6, 259.1176). Penicyclone C (3): colorless oil; [α]20D +89.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 220 (3.10), 231 (3.05) nm; ECD (c 3.68 × 10−3 M, MeOH) λmax (Δε) 218 (+1.53), 248 (−2.11) nm; IR (KBr) νmax 3429, 2945, 1723, 1675, 1208 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 295.1153 [M + Na]+ (calcd for C13H20O6Na, 295.1152). Penicyclone D (4): colorless oil; [α]20D +84.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 225 (2.93), 230 (3.23) nm; ECD (c 3.76 × 10−3 M, MeOH) λmax (Δε) 322 (+0.64), 240 (−0.90) nm; IR (KBr) νmax 3425, 2933, 1704, 1680, 1325, 1232 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 289.1049 [M + Na]+ (calcd for C14H18O5Na, 289.1046). Penicyclone E (5): colorless oil; [α]20D +83.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 225 (2.93), 230 (3.22) nm; ECD (c 3.76 × 10−3 M, MeOH) λmax (Δε) 322 (+0.21), 242 (−0.41) nm; IR (KBr) νmax 3420, 2934, 1705, 1681, 1325, 1232 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 289.1049 [M + Na]+ (calcd for C14H18O5Na, 289.1046). Bioactivity Assays. Antimicrobial activity was measured by the broth microdilution method against Staphylococcus aureus,9 with penicillin G as the positive control. Cytotoxic activities were evaluated on the HeLa (human cervical cancer) and BEL-7402 (human hepatocellular carcinoma) cell lines by the MTT method10 and the HEK-293 (human embryonic kidney), HCT-116 (human colon cancer), and A549 (lung cancer) cell lines by using the SRB method,11 with doxorubicin as positive control. X-ray Crystallographic Analysis of Compound 1. An optically active white crystal of 1 was obtained in MeOH/H2O. The crystal data were recorded with an Xcalibur Eos Gemini single-crystal diffractometer with Cu Kα radiation (λ = 1.5418 Å). The structures were solved by audit creation method (SHELXL-97) and refined using SHELXL-97 (Sheldrick, 1997). Crystallographic data have been deposited in the Cambridge Crystallographic Data Center with the deposition number CCDC 1061526. A copy of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, U.K. (fax +44(0)-1233-336033; email [email protected]). Crystal data of 1: clear, light, colorless crystal, monoclinic, space group P21, a = 9.2138(9) Å, b = 7.3606(6) Å, c = 16.9811(12) Å, β = 102.272(8)°, V = 1125.34(16) Å3, Z = 2, μ(Cu Kα) = 3.8975, T =

were proved to share the same 2R, 3R, 4R absolute configurations, evidenced by the similar Cotton effects around 240 (negative) and 320 (positive) nm.

Figure 6. B3LYP/6-31+G(d)-calculated ECD spectrum of (4R,5R,6R)-4 (red) and the experimental spectrum of 4 (black) (σ = 0.25 eV).

Compounds 1−5 were not cytotoxic against the HeLa, BEL7402, HEK-293, HCT-116, and A549 cell lines (IC50 > 50 μM), but they showed antimicrobial activity against the bacterium Staphylococcus aureus, with MIC values of 0.3, 1.0, 0.9, 0.8, and 0.5 μg/mL, respectively. Ambuic acids are highly functionalized cyclohexenones that have been isolated from some tropical plant endophytic fungi such as Pestalotiopsis sp. and Monochactia sp.7 All the reported ambuic acid analogues have similar carboxylic acid side chains and differ in the hexasubstituted cyclohexenone. As a series of widely distributed compounds, they were discovered to have cytotoxic8 and antibacterial activities.7,9 Penicyclones A−E (1− 5) have new carboxylic acid side chains, and 1 possesses a new spiro-δ-valerolactone ring system.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Waters 2487, while ECD spectra were measured on a JASCO J-715 spectra polarimeter. IR spectra were recorded on a Nicolet NEXUS 470 spectrophotometer on KBr discs. 1H NMR, 13C NMR, DEPT, and 2D NMR spectra were recorded on an Agilent 500 MHz DD2 spectrometer using TMS as an internal standard. HRESIMS and ESIMS data were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. X-ray crystal data were measured on an Xcalibur Eos Gemini single-crystal diffractometer (Cu Kα radiation). Column chromatography (CC) was performed on silica gel (100−400 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (Amersham Biosciences), and ODS resin (50 mm, Merck). Analytical HPLC used a C18 column (YMC-Pack ODS-A, 4.6 × 250 mm, S-5 μm, 1 mL/min). Preparative HPLC separation used a C18 column (YMC-Pack ODS-A, 10 × 250 mm, S-5 μm, 3 mL/min); DAICEL IA (4.6 × 250 mm, 5 μm, 1 mL/min). Fungal Material. The working stocks were preserved on potato dextrose agar slants stored at 4 °C. The isolation and identification of Penicillium sp. F23-2 had been previously described.5 Fermentation. Penicillium sp. F23-2 was cultured on slants with PDA at 28 °C for 5 days. Fermentation was carried out in Erlenmeyer flasks (1000 mL) containing 80 g of rice and naturally collected seawater (120 mL per flask) from Jiaozhou Bay, Qingdao, China. The contents were soaked overnight before autoclaving at 121 °C for 20 D

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100.00(10) K, F(000) = 512, crystal size 0.46 × 0.04 × 0.03 mm3; Rint (R factor for symmetry-equivalent intensities) = 0.0439, R factors (0.0471) and goodness-of-fit (1.046), Flack parameter = −0.2(3). Computation Section. Conformational searches were run employing the “systematic” procedure implemented in Spartan’14,12 using MMFF (Merck molecular force field). All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian09 program.13 The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT (TDDFT) calculations were performed on the three lowestenergy conformations for 1 and two lowest-energy conformations for 4 (>5% population) for each configuration using 20 excited states and using a polarizable continuum model (PCM) for MeOH. ECD spectra were generated using the program SpecDis14 by applying a Gaussian band shape with 0.28 and 0.25 eV width for 1 and 4, from dipolelength rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for about 99% of the weights. The calculated spectrum was blue-shifted by 8 nm for 4 to facilitate comparison to the experimental data. Alcoholysis Reaction of 1. To a solution of 1 (0.5 mg) in MeOH (0.5 mL) was added 0.5 mg of 4-dimethylaminopyridine. After stirring at 60 °C for 10 h, solvent was removed via a stream of N2. The reaction product was analyzed by reversed-phase HPLC using a gradient solvent system of MeCN/H2O (5:95 → 50:50, 0.2% TFA). The HPLC analysis of the alcoholysis product of 1 displayed a peak at 14.9 min, and retention times for 1 and 3 were 15.7 and 14.9 min, respectively.

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(3) (a) Yin, W.; Keller, N. P. J. Microbiol. 2011, 49, 329−339. (b) Saleem, T. S. M.; Ravi, V.; Gauthaman, K.; Saisivam, S. J. Pharm. Sci. 2009, 1, 57−60. (c) Bugni, T. S.; Ireland, C. M. Nat. Prod. Rep. 2004, 2, 143−163. (d) Bode, H. B.; Bethe, B.; Hofs, R.; Zeeck, A. ChemBioChem 2002, 3, 619−627. (4) (a) Cai, S.; Sun, S.; Peng, J.; Kong, X.; Zhou, H.; Zhu, T.; Gu, Q.; Li, D. Tetrahedron 2015, 72, 3715−3719. (b) Luan, Y.; Wei, H.; Zhang, Z.; Che, Q.; Zhu, T.; Gu, Q.; Li, D. J. Nat. Prod. 2014, 77, 1718−1723. (c) Zhang, G.; Wu, G.; Zhu, T.; Kurtán, T.; Li, J.; Qi, X.; Gu, Q.; Li, D. J. Nat. Prod. 2013, 76, 1946−1957. (5) (a) Guo, W.; Gu, Q.; Li, D. J. Nat. Prod. 2013, 76, 2106−2112. (b) Du, L.; Feng, T.; Zhao, B.; Li, D.; Cai, S.; Zhu, T.; Wang, F.; Xiao, X.; Gu, Q. J. Antibiot. 2010, 63, 165−170. (c) Du, L.; Li, D.; Zhu, T.; Cai, S.; Wang, F.; Xiao, X.; Gu, Q. Tetrahedron 2009, 65, 1033−1039. (6) Li, X. F.; Choi, H. D.; Kang, J. S.; Lee, C.; Son, B. W. J. Nat. Prod. 2003, 66, 1499−1500. (7) Li, J. Y.; Harper, J. K.; Grant, D. M.; Strobel, G. A. Phytochemistry 2001, 56, 463−468. (8) Xie, J.; Li, J.; Yang, Y.; Chen, Y.; Zhao, P. Phytochem. Lett. 2014, 10, 291−294. (9) (a) Ding, G.; Li, Y.; Fu, S.; Liu, S.; Wei, J.; Che, Y. J. Nat. Prod. 2009, 72, 182−186. (b) Li, X.; Choi, H.; Kang, J.; Lee, C.; Son, B. J. Nat. Prod. 2003, 66, 1499−1500. (10) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (11) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (12) Spartan’14; Wavefunction Inc.: Irvine, CA, 2013. (13) 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.; Montgomery, J. A., Jr.; 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, Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009. (14) Bruhn, T.; Hemberger, Y.; Schaumlöffel, A.; Bringmann, G. SpecDis, Version 1.53; University of Wuerzburg, Germany, 2011.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00655. MS and NMR spectra for compounds 1−5, HPLC analysis of the alcoholysis reaction of 1, and ECD calculations of compounds 1 and 4 (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 41176120, 21542001, and 21372208), the Shandong Provincial Natural Science Fund for Distinguished Young Scholars (JQ201422), the NSFCShandong Joint Fund (No. U1406402), the National High Technology Research and Development Program of China (No. 2013AA092901), and the Program for New Century Excellent Talents in University (No. NCET-12-0499).

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