Antiviral Merosesquiterpenoids Produced by the Antarctic Fungus

Article pubs.acs.org/jnp

Antiviral Merosesquiterpenoids Produced by the Antarctic Fungus Aspergillus ochraceopetaliformis SCSIO 05702 Junfeng Wang,† Xiaoyi Wei,‡ Xiaochu Qin,§ Xinpeng Tian,† Li Liao,⊥ Kemin Li,§ Xuefeng Zhou,† Xianwen Yang,† Fazuo Wang,† Tianyu Zhang,§ Zhengchao Tu,§ Bo Chen,⊥ and Yonghong Liu*,† †

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica/RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China ‡ Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China § Laboratory of Molecular Engineering and Laboratory of Natural Product Synthesis, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, People’s Republic of China ⊥ SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, People’s Republic of China S Supporting Information *

ABSTRACT: Five new highly oxygenated α-pyrone merosesquiterpenoids, ochraceopones A−E (1−5), together with one new double bond isomer of asteltoxin, isoasteltoxin (6), and two known asteltoxin derivatives, asteltoxin (7) and asteltoxin B (8), were isolated from an Antarctic soil-derived fungus, Aspergillus ochraceopetaliformis SCSIO 05702. Their structures were determined through extensive spectroscopic analysis, CD spectra, quantum mechanical calculations, and X-ray single-crystal diffraction. Ochraceopones A−D (1−4) are the first examples of α-pyrone merosesquiterpenoids possessing a linear tetracyclic carbon skeleton, which has not been previously described. All the isolated compounds were tested for their antiviral, cytotoxic, antibacterial, and antitubercular activities. Among these compounds, ochraceopone A (1), isoasteltoxin (6), and asteltoxin (7) exhibited antiviral activities against the H1N1 and H3N2 influenza viruses with IC50 values of >20.0/12.2 ± 4.10, 0.23 ± 0.05/ 0.66 ± 0.09, and 0.54 ± 0.06/0.84 ± 0.02 μM, respectively. A possible biosynthetic pathway for ochraceopones A−E (1−5) was proposed.

D

broad spectrum of biological activities including inhibition of acetylcholinesterase6,7 and cholesterol acyltransferase,8 as well as antimicrobial,9 phytotoxic,10 and anti-insectan11 activities. The complex polycyclic, highly oxygenated structures and the extraordinary range of biological activities of α-pyrone meroterpenoids have led to many biosynthesis12,13 and total synthesis14−16 programs. As part of our investigations aimed at exploring structurally novel bioactive secondary metabolites from fungal species inhabiting unique environments,17−19 a subculture of an isolate

uring the last century, there were three major influenza pandemics: the 1918 H1N1 Spanish, the 1957 H2N2 Asian, and the 1968 H3N2 Hong Kong outbreaks. In June 2009, the World Health Organization (WHO) identified a new strain of swine origin, H1N1, raising the level of influenza pandemic alert from phase three to phase six.1,2 Natural products have been and continue to be a rich source of antiviral drugs.3 Meroterpenoids are hybrid natural products of both terpenoid and non-terpenoid origin, and more than 330 naturally occurring meroterpenoids have been isolated from various fungal sources.4 Naturally occurring α-pyrone meroterpenoids comprise a diverse group of fungal polyketideterpenoid hybrid metabolites that have attracted a great deal of attention for their unusual structure features4,5 and resulting © 2015 American Chemical Society and American Society of Pharmacognosy

Received: July 24, 2015 Published: December 23, 2015 59

DOI: 10.1021/acs.jnatprod.5b00650 J. Nat. Prod. 2016, 79, 59−65

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Data for 1−4 (500, 125 MHz, CD3OD, TMS, δ ppm) 1 position

δC

1 2 3 4 5 6

167.3, 99.9, 166.4, 109.0, 159.3, 69.2,

C C C C C CH

7 8 9 10 11 12 13 14 15 16

40.4, 83.9, 75.5, 77.0, 76.7, 36.0, 209.9, 56.4, 212.6, 46.9,

CH C CH C C CH C C C CH2

9.7, 17.4, 22.2, 10.4, 23.8, 26.1, 59.4,

CH3 CH3 CH3 CH3 CH3 CH3 CH3

17 18 19 20 21 22 6-OCH3

2 δH (J in Hz)

4.43, d (3.4) 2.13, dd (11.9, 3.4) 4.33, s

2.20, m

2.83, d (17.2); 2.72, d (17.2) 1.92, s 2.24, s 1.52, s 1.09, d (6.5) 1.30, s 1.35, s 3.51, s

δC 165.9, 103.3, 165.1, 109.1, 159.2, 64.6,

C C C C C CH

41.3, 85.6, 74.7, 77.0, 76.8, 40.6, 209.7, 56.3, 212.6, 46.6,


9.5, 17.3, 18.3, 11.4, 24.0, 25.9,

CH3 CH3 CH3 CH3 CH3 CH3

3 δH (J in Hz)

4.57, d (9.4) 2.33, dd (11.7, 9.4) 4.33, s

2.03, m

2.79, d (17.4); 2.71, d (17.4) 1.93, s 2.25, s 1.39, s 1.24, d (6.9) 1.33, s 1.36, s

of the fungal strain Aspergillus ochraceopetaliformis SCSIO 05702, obtained from a soil sample that was collected near the Great Wall station (Chinese Antarctic station), was grown in a nutrient-deprived culture medium. Its ethyl acetate extract displayed significant in vitro antiviral activity against H1N1 influenza virus and contained a variety of secondary metabolites with similar UV absorptions at 207, 290, and 340 nm, as shown by HPLC analysis with a photodiode array. Further chemical investigations of the culture extract afforded five new α-pyrone merosesquiterpenoids, which we have named ochraceopones A−E (1−5), together with one new double-bond isomer of asteltoxin, isoasteltoxin (6), and two known asteltoxin derivatives, asteltoxin (7) 20 and asteltoxin B (8).21,22 Ochraceopones A−D (1−4) are the first examples of α-pyrone merosesquiterpenoids possessing a linear tetracyclic carbon skeleton, which has not been previously described. Compounds 1−8 were tested for their antiviral, cytotoxic, antibacterial, and antitubercular activities. Details of the isolation, structure elucidation, and bioactivities of these compounds are reported herein.

δC 166.9, 98.9, 165.4, 109.2, 157.4, 21.4,

C C C C C CH2

34.7, 83.4, 74.3, 77.0, 76.3, 39.7, 209.8, 56.5, 212.4, 46.6,


9.6, 17.2, 18.6, 10.3, 23.8, 26.1,


4 δH (J in Hz)

2.60, dd (16.2, 4.8); 2.01, d (16.2) 2.11, m 4.42, s

1.82, m

2.82, d (17.2); 2.69, d (17.2) 1.93, s 2.23, s 1.33, s 1.04, d (6.8) 1.31, s 1.36, s

δC 164.7, 97.7, 164.7, 108.4, 159.7, 114.3,

C C C C C CH

134.2, 87.7, 76.5, 76.9, 77.4, 39.7, 209.8, 56.8, 212.0, 46.4,

C C CH C C CH C C C CH2

9.2, 17.5, 26.1, 9.3, 23.5, 26.1,


δH (J in Hz)

6.23, s

4.28, s

2.88, m

2.93, d (17.0); 2.62, d (17.0) 1.91, s 2.22, s 1.73, s 1.15, d (6.7) 1.28, s 1.73, s

moiety (A ring). Furthermore, the HMBC spectrum showed the following correlations: H3-19 with C-7, C-8, and C-9, H-9 with C-7, C-11, and C-13, H-12 with C-8, C-10, and C-16, H320 with C-7, C-11, and C-12, H3-21/H3-22 with C-13, C-14, and C-15, and H2-16 with C-10, C-11, C-12, and C-15. This evidence, as well as one proton spin system deduced from 1 H−1H COSY correlations, H-7/H-12/H3-20, led to the establishment of a highly oxygenated decalin system (C and D rings). The α-pyrone moiety (A ring) and decalin system (C and D rings) were found to be directly connected via one oxygenated methine (C-6), which was further supported by COSY correlations of H-6/H-7/H-12/H3-20 and by the key HMBC correlation from H-6 to C-1 and C-3 (Figure 1). However, the 2D NMR spectra did not provide sufficient information to elucidate the unambiguous connecting pattern between C-3 and C-8. If the carbons C-3 and C-8 were connected via one oxygen bridge, this kind of connecting pattern was consistent with the degrees of unsaturation and molecular formula. The NOESY correlations of H-7/H3-20 and H-12/H3-19 indicated a trans-diaxial-like relationship of H-7/ H3-19. Additional NOESY correlations of H-6/H-7, H-6/H320, H-7/H3-20, and H-9/H3-19 located H-6, H-7, and H3-20 on the same face, which positioned 6-OCH3, H-9, H-12, and H3-19 on the opposite face (Figure 1). In order to verify the proposed structure, compound 1 was subjected to single-crystal X-ray diffraction analysis (Figure 2). With the molecule bearing nine oxygen atoms, the final refinement on the Cu Kα data resulted in a Flack parameter23−25 of −0.2(4), which not only indicated the connection of C-3 to C-8 through an ether linkage but also determined the absolute stereochemistry of 1 to be 6R, 7S, 8S, 9R, 10R, 11R, and 12R. Compound 2 was obtained as a yellow, amorphous powder. Its molecular formula was determined to be C22H28O9 on the

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RESULTS AND DISCUSSION Compound 1 was obtained as an amorphous powder and crystallized from methanol to give yellow crystals. The molecular formula of 1 was determined to be C23H30O9 from the HRESIMS peak at m/z 473.1791 [M + Na]+. Its IR spectrum exhibited absorptions at 3368 cm−1 (hydroxy) and 1684 and 1647 cm−1 (carbonyl). The 13C NMR (Table 1), DEPT, and HMQC spectra revealed the presence of 23 carbons, namely, seven methyls, one methylene, four methines, and 11 nonprotonated carbons. The HMBC correlations from H-6 to C-1, C-2, and C-3, H3-17 to C-3, C-4, and C-5, and H318 to C-4 and C-5 suggested the presence of an α-pyrone 60



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Compound 3 was isolated as a yellow, amorphous solid, and its molecular formula was established as C22H28O8 by an HRESIMS peak at m/z 421.1849 [M + H]+ (calcd, 421.1862). The 1H and 13C NMR data of 3 were almost the same as those of compounds 1 and 2. Comparison of their NMR data revealed the difference occurred at the B ring. One methylene signal at δC 21.4 for C-6 was observed in 3, instead of the oxygenated methine between C-2 and C-7 in 1 and 2. This deduction was further supported by the COSY correlations of H2-6/H-7/H-12/H3-20 and by the HMBC correlation of C-6 with C-1, C-3, C-8, and C-12. The NOESY correlations of H7/H3-20 and H-12/H3-19 also indicated the trans-diaxial-like relationship of H-7/H3-19. Additional NOESY correlations of H-9/H3-19 and H3-19/H-12 located H-9, H-12, and H3-19 on the same face, consistent with the NOESY correlations of the C ring in 1 and 2 (Figure 3). This assignment was further confirmed by ECD calculations, and the absolute configurations of 3 were determined as 7R, 8S, 9R, 10R, 11R, 12R, respectively (Figure 4). The molecular formula of compound 4 was determined to be C22H26O8 by HRESIMS, indicating 18 amu less than 2 and 10 degrees of unsaturation. Further comparison of the NMR data of 4 with those of 2 revealed a Δ6 double bond for 4 as a replacement for the oxygenated methine (C-6) and methine (C-7) in 2, which was supported by HMBC correlations from olefinic methine H-6 (δH 6.23, 1H, s) to C-1, C-3, C-8, and C12. The NOESY correlations of H-9/H3-19 and H-12/H3-19 indicated H-9, H-12, and H3-19 were on the same face (Figure 3). The absolute configurations of 4 were further determined as 8S, 9R, 10R, 11R, 12R, respectively, by comparing the calculated ECD curve with its experimental values (Figure 4), consistent with the corresponding absolute configurations of the C ring in compounds 1−3. Compound 5 was isolated as a yellow crystal. The molecular formula was established to be C22H30O6 (eight degrees of unsaturation) by HRESIMS with analysis of the 1H and 13C NMR spectroscopic data. Comparison of UV−vis and 1H and 13 C NMR data with those of compound 3 (Tables 1 and S1) revealed a high degree of similarity, indicating the same αpyrone moiety (A ring). The main differences between 5 and 3 were the connecting pattern of the decalin system (C and D rings). Detailed analysis of the 1D- and 2D-NMR spectral data revealed that the decalin ring of 5 is quite similar to those of arisugacin C,7 which was further supported by the 1H−1H COSY, HMBC, and NOESY experiments. This deduction was unambiguously confirmed by the X-ray crystallographic diffraction analysis (Figure 2), which determined the absolute

Figure 1. Key 1H−1H COSY (bold), HMBC (arrows), and NOESY (dashed arrows) correlations of 1.

Figure 2. ORTEP drawing of compounds 1 and 5 (Cu Kα).

basis of HRESIMS at m/z 459.1624 [M + Na]+ (calcd, 459.1631), with 9 degrees of unsaturation. The similar UV and 1D NMR spectra to those of 1 suggested that they shared the same skeleton. However, the methoxyl group (6-OCH3) in the 1 H NMR spectrum of 1 was absent in that of 2. In the 13C NMR spectrum, one oxygenated methine at δC 64.6 for C-6 was still observed in 2, supporting the substitution of a methoxyl group (6-OCH3) in the B ring in 1 by a hydroxy group in 2, which was consistent with the molecular formula. The key COSY, HMBC, and NOESY correlations (Figure 3) for the highly oxygenated decalin ring and α-pyrone moiety were in good agreement with the data for 1. Additionally, the absolute configurations of 2 were determined as 6R, 7S, 8S, 9R, 10R, 11R, 12R, respectively, by comparing the calculated ECD spectrum with its experimental values (Figure 4).

Figure 3. Key NOESY correlations of compounds 2−6. 61



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Figure 4. Comparison between calculated (CAM-B3LYP/TZVP) and experimental ECD spectra of 2−4 in MeOH.

Scheme 1. Postulated Biogenetic Pathway for Ochraceopones A−E (1−5)

stereochemistry of 5 to be 7R, 8S, 9R, 11S, and 12R. α-Pyrone merosesquiterpenoids possessing an angular tetracyclic carbon skeleton are frequently isolated from the genera Penicillium and Aspergillus as anti-cholinesterase active constituents.4,5 Compound 6 was obtained as a yellow oil with the molecular formula C23H30O7 as determined by HRESIMS, indicating 9 degrees of unsaturation. Its 1H NMR and 13C NMR (DEPT) spectra include signals for six nonprotonated carbons, seven unsaturated methine carbons, four oxygenated methine carbons, one methylene carbon, and five methyl carbons, including one methoxyl group. The connectivity of the protons and C atoms was established by the 1H, 13C, and HMQC spectra (Table S2). The general features of its NMR spectroscopic data (Table S2) closely resembled those of asteltoxin (7).20 Detailed comparison of NMR data of these two compounds suggested that compound 6 had the same 2,8dioxabicyclo[3.3.0]octane and α-pyrone units. Significant differences in NMR spectra were observed in the signals for the polyene chain. These differences suggested that compound 6 and asteltoxin (7) differed only in the geometry of the double bond in the polyene chain. The double bond between C-11 and

C-12 possessed the Z-geometry, which was further deduced by the key ROESY correlation between H-10 (δH 7.04) and H-13 (δH 7.66) (Figure 3). The ROESY correlations of H-6/H3-21 indicated a cis-diaxial-like relationship of the 2,8dioxabicyclo[3.3.0]octane ring in 6. Additional NOESY correlations of H2-2/H-6, H-6/H-8, H-6/H3-21, and H-7/H321 located H2-2, H-6, H-7, and H3-21 on the same face, which positioned H-3 and H3-20 on the opposite face (Figure 3). The key NOESY correlations for the junction of the 2,8dioxabicyclo[3.3.0]octane ring in 6 were in good agreement with the data for asteltoxin (7). Furthermore, the absolute configurations of asteltoxin (7) were revealed as 3R, 4R, 5R, 6S, 7R, 8R, 9E, 11E, 13E by total synthesis in 2003.20 Thus, the structure of 6 was suggested as the Δ11 double-bond isomerism of asteltoxin (7) and named isoasteltoxin. The five α-pyrone merosesquiterpenoids 1−5 are probably biosynthesized via polyketide and mevalonate hybrid biogenetic pathways12,13 (Scheme 1). In the proposed biogenesis of 1−5, an all-trans farnesyl pyrophosphate is produced via the mevalonate pathway, which condenses with acetyl-CoA and malonyl-CoA to form an intermediate a, which further 62



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Table 2. Antiviral Activity of 1, 6, and 7 against H1N1 and H3N2 Virusesa H1N1

H3N2

compound

CC50 (μM)

IC50 (μM)

SI

CC50 (μM)

IC50 (μM)

SI

1 6 7

0.54 ± 0.14 0.24 ± 0.01

>20.0 0.23 ± 0.05 0.54 ± 0.06

2.35 0.44

20.9 ± 2.77 0.54 ± 0.14 0.24 ± 0.01

12.2 ± 4.10 0.66 ± 0.09 0.84 ± 0.02

1.71 0.81 0.29

a

Cytotoxicity (CC50) and antiviral activity (IC50) were determined by the CPE inhibition assay on MDCK cells. Selectivity index (SI) is the ratio of CC50 to IC50. Data are expressed as means ± SD of three independent experiments. Tamiflu was used as the positive control, with IC50 values of 16.9 and 18.5 nM, respectively. Microbiological Culture Collection Center with a deposition number of CGMCC 10279. A reference culture is deposited in our laboratory at −80 °C. The producing strain was prepared on potato dextrose agar slants at 3.3% salt concentration and stored at 4 °C. Fermentation and Extraction. A. ochraceopetaliformis SCSIO 05702 was grown under static conditions at 24 °C for 70 days in one hundred 1000 mL conical flasks containing liquid medium (300 mL/ flask) composed of soluble starch (10 g/L), polypeptone (1 g/L), and tap water after adjusting its pH to 7.5. The fermented whole broth (30 L) was filtered through cheesecloth to separate it into filtrate and mycelia. The filtrate was concentrated under vacuum to about a quarter of the original volume and then extracted three times with EtOAc to give an EtOAc solution, while the mycelia were extracted three times with acetone. The acetone solution was evaporated under reduced pressure to afford an aqueous solution. The aqueous solution was extracted three times with EtOAc to give another EtOAc solution. Both EtOAc solutions were combined and concentrated under reduced pressure to give a dark brown gum (37.2 g). Purification. The EtOAc extract (37.2 g) was subjected to vacuum liquid chromatography (VLC) on a silica gel column using step gradient elution with MeOH−CH2Cl2 (0−100%) to separate it into four fractions based on TLC properties. Fraction 1 (3.7 g) was separated into four subfractions (Frs. 1-1−1-4) by Sephadex LH-20 eluting with MeOH−CH2Cl2 (1:1). Fr. 1-1 (73 mg) was directly separated by HPLC (53% MeOH−H2O) to yield 4 (1.4 mg, tR 7.9 min), 5 (16.8 mg, tR 15.1 min), and subfraction 1-1-4. Subfraction 1-14 was further purified by HPLC (35% acetonitrile−H2O) to yield 8 (5.7 mg, tR 13.6 min), 7 (36.7 mg, tR 21.3 min), and 6 (1.7 mg, tR 23.9 min), respectively. Fraction 2 was divided into five parts (Frs. 2-1−25) by Sephadex LH-20 (MeOH). Fr. 2-2 was further purified by HPLC (33% acetonitrile−H2O) to yield 2 (18.5 mg, tR 8.3 min), 3 (21.0 mg, tR 11.7 min), and 1 (15.8 mg, tR 19.4 min), respectively. X-ray Crystallographic Data of 1. Ochraceopone A (1) was crystallized from methanol to give faintly yellow crystals. Crystal data: Monoclinic, space group P2(1) with a = 15.1132(12) Å, b = 7.0509(5) Å, c = 22.3790(2) Å, V = 2325.2(3) Å3, Z = 2, Dcalc = 1.358 g/cm3, R = 0.0614, wR2 = 0.1535. The absolute configuration was determined on the basis of a Flack parameter of −0.2(4), refined using 5213 Friedel pairs. Crystallographic data (excluding structure factors) for structure 1 in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1062873. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44 (0)-1223-336033 or e-mail: [email protected]]. X-ray Crystallographic Data of 5. Ochraceopone E (5) was crystallized from methanol to give faintly yellow platelets. Crystal data: Triclinic, space group P1 with a = 7.4876(11) Å, b = 7.5455(12) Å, c = 10.2187(16) Å, V = 504.00(12) Å3, Z = 1, Dcalc = 1.286 g/cm3, R = 0.0470, wR2 = 0.1210. The absolute configuration was determined on the basis of a Flack parameter of 0.0(5), refined using 2008 Friedel pairs. Crystallographic data (excluding structure factors) for structure 5 in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1062874. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44 (0)-1223-336033 or e-mail: [email protected]]. Ochraceopone A (1): yellow crystal; [α]25D +20.4 (c 1.6, MeOH); UV (MeOH) λmax (log ε) 207 (4.14), 289 (3.58), 356 (3.11) nm; IR

undergoes epoxidation, polyene cyclization, and retro-aldol/ aldol rearrangement26,27 to form the core skeleton, featuring a tetracyclic structure core. This pathway involves oxidation, epoxidation, cyclization, retro-aldol/aldol rearrangement, methylation, and dehydration to form ochraceopones A−E (1−5). Previously, α-pyrone merosesquiterpenoids were reported to be composed of a highly oxygenated trans-decalin system and an α-pyrone moiety, featuring an angular tetracycle structure.1,2 Compounds 1−4 are the new skeletal class of the α-pyrone merosesquiterpenoids, and the linear tetracyclic system found in ochraceopones A−D (1−4) has not been previously described. All the isolated compounds 1−8 were tested for their antiviral (H1N1 and H3N2), cytotoxic (the K562, MCF7, A549, U937, HeLa, DU145, HL60, and HT29 cell lines), antibacterial (Escherichia coli and Staphylococcus aureus), and antituberculosis activities. Among these compounds, ochraceopone A (1), isoasteltoxin (6), and asteltoxin (7) exhibited antiviral activities against the H1N1 and H3N2 influenza viruses with IC50 values of >20.0/12.2 ± 4.10, 0.23 ± 0.05/0.66 ± 0.09, and 0.54 ± 0.06/0.84 ± 0.02 μM, respectively (Table 2). It was noteworthy that the selectivity indexes (SI) of antiH1N1 activity of 6 and 7 were 2.35 and 0.44, respectively. These results indicated that the geometry of the Δ11 double bond in the polyene chain might contribute to the anti-H1N1 activity and selectivity index. However, none of compounds displayed cytotoxic, antibacterial, or antitubercular activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a PerkinElmer 341 polarimeter. UV spectra were recorded on a Shimadzu UV-2401PC spectrometer. IR spectra were measured on a JASCO FT/IR-480 Plus spectrometer with KBr pellets. Circular dichroism spectra were recorded with a Chirascan circular dichroism spectrometer (Applied Photophysics, Ltd.). 1H and 13C NMR, DEPT, and 2D-NMR spectra were recorded on a Bruker DRX500 spectrometer using TMS as internal standard, and chemical shifts were recorded as δ-values. HRESIMS (including ESIMS) spectra were recorded on an Applied Biosystems Mariner 5140 spectrometer. X-ray diffraction intensity data were collected on a CrysAlis PRO CCD area detector diffractometer with graphite-monochromated Cu Kα radiation (λ = 1.541 78 Å). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm) and over silica gel (200−300 mesh) (Qingdao Marine Chemical Factory, China) and Sephadex LH-20 (Amersham Biosciences, Sweden), respectively. All solvents used were of analytical grade (Tianjin Fuyu Chemical and Industry Factory). Semipreparative HPLC was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min). Fungal Material. The fungal strain A. ochraceopetaliformis SCSIO 05702 was isolated from a soil sample that was collected near the Great Wall station (Chinese Antarctic station) and identified by one of our authors (X.T.). It was identified according to its morphological characteristics and ITS gene sequences (GenBank Accession No. KR653311). This strain has been deposited in China General 63



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(KBr) νmax 3368, 1684, 1647, 1558, 1541, 1456, 1418, 1250, 1105, 1067, 1022, 870 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 473.1791 [M + Na]+ (calcd for C23H30NaO9, 473.1782). Ochraceopone B (2): yellow, amorphous powder; [α]25D +17.7 (c 1.9, MeOH); UV (MeOH) λmax (log ε) 207 (4.05), 289 (3.47), 340 (3.17) nm; IR (KBr) νmax 3370, 1674, 1643, 1572, 1408, 1385, 1280, 1250, 1173, 1126, 1065, 1020 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 459.1624 [M + Na]+ (calcd for C22H28NaO9, 459.1631). Ochraceopone C (3): yellow, amorphous solid; [α]25D +23.4 (c 2.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.04), 289 (3.46), 340 (3.15) nm; IR (KBr) νmax 3134, 1682, 1636, 1568, 1456, 1410, 1250, 1119, 1067, 1022 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 421.1849 [M + H]+ (calcd for C22H29O8, 421.1862). Ochraceopone D (4): yellow, amorphous solid; [α]25D −82.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.16), 239 (3.97), 340 (3.66) nm; IR (KBr) νmax 3393, 1695, 1678, 1636, 1558, 1541, 1456, 1418, 1375, 1254, 1117, 1042 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 441.1525 [M + Na]+ (calcd for C22H26NaO8, 441.1525). Ochraceopone E (5): yellow crystal; [α]25D −57.9 (c 1.7, MeOH); UV (MeOH) λmax (log ε) 207 (4.08), 289 (3.53), 354 (2.87) nm; IR (KBr) νmax 3393, 1663, 1636, 1570, 1418, 1240, 1144, 1105, 1096, 1074 cm−1; 1H NMR and 13C NMR data, see Table S1; HRESIMS m/ z 389.1971 [M − H]− (calcd for C22H29O6, 389.1970). Isoasteltoxin (6): yellow oil; [α]25D +31.4 (c 0.2, CH3OH); UV (MeOH) λmax (log ε) 221 (3.70), 268 (4.13), 273 (4.13), 365 (4.08) nm; 1H NMR and 13C NMR data, see Table S2; HRESIMS m/z 441.1887 [M + Na]+ (calcd for C23H30NaO7, 441.1884). Computational Details. Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with the Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian09 program package,28 respectively, using default grids and convergence criteria. An MMFF conformational search generated lowenergy conformers within a 10 kcal/mol energy window, which were subjected to geometry optimization using the DFT method at the B3LYP/6-31G(d) level. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (ΔG) at 298.15 K. Energies of the low-energy conformers in MeOH were recalculated at the B3LYP/def2-TZVP level. Solvent effects were taken into account by using the polarizable continuum model (PCM). The TDDFT calculations were performed using the hybrid B3LYP and CAMB3LYP functionals and the Ahlrichs’ basis set TZVP (triple-ζ valence plus polarization).29 The number of excited states was 40 for all compounds. The CD spectra were generated by the program SpecDis30 using a Gaussian band shape with 0.28 or 0.30 eV exponential half-width from dipole-length dipolar and rotational strengths. The equilibrium population of each conformer at 298.15 K was calculated from its relative free energies using Boltzmann statistics. The calculated spectra were generated from the low-energy conformers according to the Boltzmann weighting of each conformer in MeOH solution. Biological Assays. The antiviral activities against H1N1 and H3N2 were evaluated by the CPE inhibition assay.31 Confluent MDCK cell monolayers were incubated with influenza virus at 37 °C for 1 h. After removing the virus, cells were maintained in infecting media (RPMI 1640, 4 μg/mL of trypsin) containing different concentrations of test compounds. After 48 h incubation at 37 °C, the cells were fixed with 100 μL of 4% formaldehyde for 20 min at room temperature. After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for 30 min. The plates were washed and dried, and the intensity of crystal violet staining for each well was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. The IC50 was calculated as the compound concentration required to inhibit influenza virus yield at 48 h postinfection by 50%. Tamiflu was used as the positive control, with IC50 values of 16.9 and 18.5 nM, respectively.

Cytotoxicity was assayed with the CCK-8 (Dojindo, Japan) method.17 Cell lines K562, MCF-7, A549, U937, HeLa, DU145, HL60, and HT29 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells were routinely grown and maintained in RPMI or DMEM media with 10% fetal bovine serum and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/ Forma Scientific CO2 water-jacketed incubator with 5% CO2 in air at 37 °C. A cell viability assay was determined with the CCK-8 (Dojindo, Japan) assay. Cells were seeded at a density of 400−800 cells/well in 384-well plates and treated with various concentrations of compounds or solvent control. After 72 h incubation, CCK-8 reagent was added, and absorbance was measured at 450 nm using an Envision 2104 multilabel reader (PerkinElmer, USA). Dose−response curves were plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA). Taxol was used as the positive control, with IC50 values of 7.17, 7.32, 2.12, 1.66, 3.01, 2.30, 1.99, and 3.04 nM, respectively. The antimicrobial activities against Staphyloccocus aureus and Escherichia coli were evaluated by an agar dilution method.32 The tested strains were cultivated in LB agar plates for bacteria at 37 °C. Compounds and positive control were dissolved in DMSO at different concentrations from 100 to 0.01 μg/mL by the continuous 2-fold dilution method. A 5 μL quantity of test solution was absorbed by a paper disk (5 mm diameter) and placed on the assay plates. After 24 h incubation, zones of inhibition (mm in diameter) were recorded. The minimum inhibitory concentrations (MICs) were defined as the lowest concentration at which no microbial growth could be observed. Ciprofloxacin lactate was used as a positive control for Staph. aureus and E. coli, with MIC values of 1.25 and 0.02 μg/mL, respectively. The H37Ra strain of M. tuberculosis was used for the anti-TB bioassay.33,34 The test materials were dissolved in DMSO at 10 mg/ mL and tested in a series of 2-fold dilutions with the highest concentration of 100 μg/mL (and 1% v/v DMSO). Samples were incubated for 7 days with M. tuberculosis in a 96-well plate, and then cell growth was determined using the Alamar Blue dye with fluorometric detection. The MIC was defined as the lowest concentration resulting in 90% or greater inhibition of fluorescence compared to bacteria-only controls. INH (isoniazid) was used as the positive control, with an MIC value of 2.04 μM.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00650. ITS gene sequence data of SCSIO 05702, ECD computational details of compounds 2−4, and NMR and HRESIMS spectra of 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail (Y. Liu): [email protected]. Tel/Fax: +86-0208902-3244. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Key Basic Research Program of China (973)’s Project (2011CB915503), the Open Foundation of the SOA Key Laboratory for Polar Science, Polar Research Institute of China (KP201305), the National High Technology Research and Development Program (863 Program, 2012AA092104), the National Natural Science Foundation of China (Nos. 21502204, 31270402, 21172230, 41476135, and 41476136), the Strategic Priority Research Program of the Chinese Academy of Sciences 64



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(XDA11030403), and Guangdong Marine Economic Development and Innovation of Regional Demonstration Project (GD2012-D01-001).

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