Note pubs.acs.org/jnp
Sorbicatechols A and B, Antiviral Sorbicillinoids from the MarineDerived Fungus Penicillium chrysogenum PJX-17 Jixing Peng, Xiaomin Zhang, Lin Du, Wei Wang, 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: Two novel sorbicillinoids combining a bicyclo[2.2.2]octane with a 2-methoxyphenol moiety, named sorbicatechols A (1) and B (2), were isolated from the culture of the marine sediment-derived fungus Penicillium chrysogenum PJX-17, together with the known protocatechuic acid methyl ester and caffeic acid methyl ester (3). Their structures, including absolute configurations, were assigned by analysis of NMR, MS data, and TDDFT ECD calculations. Compounds 1 and 2 exhibited activities against influenza virus A (H1N1), with IC50 values of 85 and 113 μ M, respectively.
S
ester). Structurally, compounds 1 and 2 are formally derived from a sorbicillin unit and a styrene moiety by endo- and exoDiels−Alder cycloadditions, respectively. In this paper, we describe the isolation, structure elucidation, and anti-influenza A (H1N1) activities for compounds 1 and 2. Compound 1 was obtained as a yellow oil. The molecular formula was established as C23H26O6 on the basis of the HRESIMS ion at m/z 399.1801 [M + H]+, indicating 11 degrees of unsaturation. The major UV absorption at 361 nm suggested the presence of a polyene conjugated carbonyl chromophore. The IR absorptions at 3408, 1720, 1629, and 1519 cm−l indicated the presence of hydroxy and enolized βdiketone groups. Analysis of the 1D NMR data (Table 1) in CDCl3 suggested the presence of 23 carbon resonances including four methyls (one oxygenated), one methylene, nine methines (seven olefinic), nine quaternary carbons (two carbonyls), and one enol, suggesting 23 nonexchangeable protons, and one exchangeable proton in the molecule. Accordingly, two additional hydroxy groups in 1 are needed to satisfy the molecular formula. Two additional exchangeable protons (δH 6.04, 5-OH; 8.91, 18-OH) were observed in the 1H NMR spectrum in DMSO-d6 (Table 1). The characteristic proton signals at δH 7.38, 6.33, 6.29, 6.23, and 1.91 suggested the existence of a sorbyl group,3,4 which was further confirmed by the COSY correlation from H-10 to H-14
orbicillinoids are members of a large family of polyketides with highly diverse carbon skeletons and bioactivities that have been isolated from both marine and terrestrial fungi.1 Since 1948, more than 50 members of the family have been found. These members can be further divided into four subfamilies corresponding to common structural features: monomeric sorbicillinol derivatives, bisorbicillinoids, trisorbicillinoids, and hybrids of different biosynthetic precursors.1 Hybrid sorbicillinoids are a new subfamily and are proposed to be derived from either a Diels−Alder or a Michael reaction of a monomeric sorbicillinoid diene and a second non-sorbicillinoid dienophile. The rezishanones, isolated from Penicillium notatum in 2005, were the first members of this new group,2 with 27 members in the group to date.1,3,4 Many hybrid sorbicillinoids possess attractive structural features and interesting biological properties.1,3,4 Exploring the biosynthesis of hybrid precursors has inspired new avenues of research for adding chemical diversity to natural product libraries. In previous studies, our research group has reported 20 new sorbicillinoids from marine-derived fungi, including a monomeric sorbicillinoid, seven bisorbicillinoids, four trisorbicillinoids, and eight hybrid polycyclic sorbicillinoids.3,4 In the course of our ongoing search for sorbicillin derivatives using the characteristic UV absorption at ∼350 nm, we targeted a marinederived fungus with anti-influenza activity, identified as a Penicillium chrysogenum strain. Further chemical analysis of the EtOAc extracts of the broth led to the isolation of two new sorbicillin derivatives (1 and 2), together with two known phenolic acid methyl esters (3 and protocatechuic acid methyl © 2014 American Chemical Society and American Society of Pharmacognosy
Received: November 24, 2013 Published: February 4, 2014 424
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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for 1 and 2 in CDCl3 1 position
δC, type
1 2 3 4
65.1, 197.9, 112.0, 40.5,
5 6 7
74.6, C 211.9, C 47.8, CH
8
2 δH (J in Hz)
C C C CH
3.29, dd (2.8, 2.8)
3.01, m
31.4, CH2
1.84, m
δC, type 65.0, 199.7, 110.5, 40.6,
C C C CH
75.9, C 212.1, C 49.9, CH 30.2, CH2
3.05, m
and the HMBC correlation between H-10 and C-9 (Figure 1). The E configurations of these double bonds in the sorbyl moiety were determined from the large coupling constants (3JH‑10,H‑11 = 14.8 Hz, 3JH‑12,H‑13 = 12.9 Hz) and further confirmed by the observed correlation between H-11 and H-13 in the NOESY spectrum (Figure 2). The Z configuration for the C-3/C-9 double bond was supported by the downfield chemical shift (δH 14.32) of the intramolecularly hydrogen bonded 9-OH. The structure of the bicyclo[2.2.2]octane moiety was confirmed by the HMBC correlations from H-4 to C-2, C-3, C-5, C-6, and C-7, from 1-CH3 to C-1, C-2, C-6, and C-7, and from 5-CH3 to C-4, C-5, and C-6, as well as COSY correlations (H-7/H2-8/H-4), which agreed with the reported data.3,4 A hydroxy group was assigned at C-5 based upon its chemical shift (δC 74.6), as well as the comparison with those of reported sorbicillin derivatives.3,4 The connection of C-3 and C-10 via C-9 was deduced from HMBC correlations from H-10 to C-9, H-11 to C-9, and H-4 to C-9. Aside from the above structure units, a subunit consisting of seven carbons remained. COSY correlations (H-19/H-20) and HMBC correlations from H-16 to C-18 and C-20, H-19 to C-15 and C-17, H-20 to C-16 and C-18, and 17-OCH3 to C-17 established a methoxy-substituted phenolic moiety. The position of the methoxy in 1 was determined by a NOESY correlation between 17-OCH3 and H-16. In the phenolic moiety, HMBC correlations from both H-16 and H-20 to C-7 revealed the connection to the bicyclo[2.2.2]octane with C-15 linking to C-7, establishing the full carbon skeleton. Furthermore, hydroxy groups were assigned to C-9 and C-18, respectively, according to the molecular formula and chemical shifts of C-9 (δC 167.1) and C-18 (δC 144.9), completing the planar structure of 1. The assignment of the stereoconfiguration of 1 is discussed below. Compound 2 had the same molecular formula, C23H26O6, as that of 1, established on the basis of the HRESIMS ion detected at m/z 399.1803 [M + H]+. Its UV and IR spectra were almost identical to those of 1. Analysis of its NMR data (Table 1), in
9 10 11
167.1, C 118.0, CH 142.5, CH
12
130.9, CH
13 14 15 16 17 18 19 20
139.9, 18.9, 133.2, 110.2, 146.5, 144.9, 114.3, 121.5,
1-CH3 5-CH3 17-OCH3 5-OH 9-OH 18-OH a
CH CH3 C CH C C CH CH
10.5, CH3 24.3, CH3 55.7, CH3
6.29, d (14.8) 7.38, dd (14.8, 12.9) 6.33, dd (12.9, 12.9) 6.23, m 1.91, d (6.6) 6.45, d (1.7)
6.79, d (8.2) 6.49, dd (8.2, 1.7) 0.93, s 1.26, s 3.79, s 6.04,a s 14.32, s 8.91,a s
167.3, C 118.1, CH 142.2, CH 131.0, CH 139.7, CH 19.0, CH3 132.0, C 111.2, CH 146.4, C 144.9, C 114.1,CH 122.4, CH 10.3, CH3 25.2, CH3 55.9, CH3
δH (J in Hz)
3.23, brs
3.03, dd (10.9, 7.7) 2.25, td (10.9, 3.3) 2.57, dd (12.7, 7.7) 6.25, d (14.8) 7.33, dd (14.8,11.0) 6.30, dd (11.0, 11.0) 6.21, m 1.91, d (6.6) 6.71, brs
6.80, d (8.2) 6.60, brd (8.2) 0.84, s 1.28, s 3.82, s 5.30, s 13.93, s
Recorded in DMSO-d6.
particular the COSY and HMBC correlations, revealed that compounds 2 and 1 shared the same planar structure. The 1H and 13C NMR spectra of 1 and 2 are very similar (Table 1). The 1H NMR spectrum of 1 has resonances of H2-8 at δH 1.84 and 3.05 and H-16 at δH 6.45, while the corresponding signals of H2-8 in 2 are at δH 2.25 and 2.57 and H-16 in 2 at δH 6.71. The largest carbon shift differences center around C-7 (Table 1). These differences implied that compound 1 was an epimer of 2 at C-7. The absolute configurations of 1 and 2 were determined by the analysis of their CD spectra, which agreed with the biosynthetic pathway of this compound class. All of the known hybrid sorbicillinoids, such as chloctanspirones A and B4 from Penicillium terrestre, spirosorbicillinols A−C5 from Trichoderma sp., and sorbicillactones A and B6 and sorbifuranones A−C7 from P. chrysogenum, were derived from sorbicillinol with another non-sorbicillinoid precursor by either a Diels−Alder reaction or a Michael addition. In each case, the S configuration at C-5 of sorbicillinol was not modified during the biosynthesis. It is likely that compounds 1 and 2 are also generated by a Diels−Alder reaction between sorbicillinol and a styrene dienophile (Scheme 1). In the proposed biosynthetic pathway, three asymmetric centers of 1 and 2 are created during the [4+2] cycloaddition reaction, with an endo-transition giving two 425
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bicyclo[2.2.2]octane ring. On the basis of the DFT-optimized structures for low-energy conformers for both endo- and exoproducts, the distances between 17-OCH3 and 1-CH3 are less than 5 Å (4.114−4.858 Å) in all the exo-orientations for the aromatic ring to the C-2/C-3 bond, while two conformers are over 5 Å (5.093 and 6.360 Å) and two are near 5 Å (4.696 and 4.789 Å) in the endo-orientation (Figures E1−E8, Supporting Information). According to the NOESY correlations between 17-OCH3 and 1-CH3, compound 2 was tentatively assigned as the exo-product (Figure 2). Consequently, compound 1, which showed no NOESY correlations between 17-OCH3 and 1-CH3, was assigned an endo-orientation. In order to confirm the assignment and their respective absolute configurations, we employed TDDFT CD calculations to compare theoretical with experimentally derived data for all the possible isomers. Density functional theory (DFT) calculations performed at the B3LYP/ 6-311+G** level were used to generate ECD spectra for a set of the lowest-energy conformers of each isomer. The resulting ECD spectra were combined by Boltzmann weighting to give a composite spectrum for each diastereomer. The calculated ECD curves (Figures 3 and 4) of (1R,4S,5S,7R)-1 and
Figure 1. Key COSY and HMBC correlations of 1 and 2.
Figure 3. B3LYP/6-311+G(d,p)-calculated ECD spectra of (1R,4S,5S,7R)-1 (red) and (1S,4R,5S,7S)-iso-1 (blue) and the experimental ECD spectrum of 1 (black) (σ = 0.28 eV).
Figure 2. Key NOESY correlations of 1 and 2.
possible endo-products (the aromatic ring is endo to the C-2/C3 bridge) with the absolute configurations (1R,4S,5S,7R) and (1S,4R,5S,7S) or an exo-transition state giving (1R,4S,5S,7S) and (1S,4R,5S,7R), with the consideration of the rigidity of the
(1R,4S,5S,7S)-2 gave the best agreement with the experimental data of compounds 1 and 2, respectively. Thus the absolute
Scheme 1. Possible Biosynthetic Route to Sorbicatechols A (1) and B (2)
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mannitol (20.0 g/L), MgSO4·7H2O (0.1 g/L), KH2PO4 (0.5 g/L), CaCl2 (0.1 g/L), FeSO4 (0.002 g/L), MnCl2 (0.002 g/L), ZnSO4 (0.002 g/L), and seawater (Huiquan Bay, Yellow Sea). After 30 days of cultivation, 20 L of whole broth was filtered through cheesecloth to separate it into supernatant and mycelia. The former was extracted three times with EtOAc, while the latter was extracted three times with acetone and concentrated under reduced pressure to afford an aqueous solution, which was extracted three times with EtOAc. Both EtOAc solutions were combined and concentrated under reduced pressure to give an organic extract (6.0 g). Purification. The extract was subjected to vacuum liquid chromatography over a silica gel column using a gradient elution with petroleum ether−CHCl3−MeOH to give three fractions. Fraction 2 was rechromatographed on a silica gel column with petroleum ether−acetone to provide two subfractions (fractions 2.1 and 2.2). Fraction 2.1 was subjected to Sephadex LH-20 column chromatography eluting with MeOH and then purified by semipreparative HPLC (70:30 MeOH−H2O with 0.2% TFA, 4 mL/min) to give compounds 1 (12 mg, tR 8 min) and 2 (5.0 mg, tR 16 min). Fraction 2.2 was separated by Sephadex LH-20 eluting with CHCl3−MeOH (1:1) and then by semipreparative HPLC (45:55 MeOH−H2O with 0.2% TFA, 4 mL/min) to afford protocatechuic acid methyl ester (3 mg, tR 7 min) and compound 3 (6 mg, tR 10 min). Sorbicatechol A (1): yellow oil; [α]20D +19 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 361 (4.42) nm; CD (0.88 × 10−3 M, MeOH) λmax (Δε) 354.5 (+3.58), 311 (−10.34), 279sh (−3.27), 245 (+4.05), 220 (+0.56), 204.5 (+2.81) nm; IR (KBr) νmax 3408, 2925, 1720, 1629, 1519, 1247, 998 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 399.1801 [M + H]+ (calcd for C23H26O6, 399.1808). Sorbicatechol B (2): yellow oil; [α]20D +120 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 356 (4.44) nm; CD (0.88 × 10−3 M, MeOH) λmax (Δε) 355 (+15.41), 313.5 (−17.38), 282sh (−4.15), 244.5 (+4.34), 207 (−3.87) nm; IR (KBr) νmax 3400, 2925, 1720, 1629, 1519, 1247, 998 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 399.1803 [M + H]+ (calcd for C23H26O6, 399.1808). Anti-influenza A (H1N1) Assay. The antiviral activity against H1N1 was evaluated by the CPE inhibition assay. Confluent MDCK cell monolayers were first incubated with influenza virus (A/Puerto Rico/8/34 (H1N1), PR/8) at 37 °C for 1 h. After removing the virus dilution, cells were maintained in infecting media (RPMI 1640, 4 μg/ mL of trypsin) containing different test compounds at 37 °C. After a 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 (BioRad) at 570 nm. The IC50 was calculated as the compound concentration required to inhibit influenza virus yield at 48 h postinfection by 50%. Ribavirin was used as the positive control in the CPE inhibition assay. 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/6-311+G(d,p) 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 calculations were performed on the lowest-energy conformations (>5% population) for each configuration using 30 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 0.28−0.36 eV width, from dipole-length 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 weight. The calculated spectrum was blue-shifted by 15−25 nm to facilitate comparison to the experimental data.
Figure 4. B3LYP/6-311+G(d,p)-calculated ECD spectra of (1R,4S,5S,7S)-2 (red) and (1S,4R,5S,7R)-iso-2 (blue) and the experimental ECD spectrum of 2 (black) (σ = 0.36 eV).
configurations of 1 and 2 could be determined as (1R,4S,5S,7R) and (1R,4S,5S,7S), respectively. Protocatechuic acid methyl ester8 and caffeic acid methyl ester (3)9 were identified by comparison of their spectroscopic data (NMR and MS) with those in the literature. The antiviral activities of compounds 1 and 2 against influenza A virus (H1N1) were evaluated by the cytopathic effect (CPE) inhibition assay.10 They showed inhibitory effects with IC50 values of 85 and 113 μM, respectively (ribavirin as a positive control, IC50 84 μM). Sorbicatechols A (1) and B (2) are presumably formed by endo and exo intermolecular Diels−Alder reactions between sorbicillinol (common precursor) as the diene and 4-hydroxy-3methoxystyrene11 (proposed hybrid precursor) as the dienophile. Unfortunately, the two key precursors have not been isolated. However, two related analogues, protocatechuic acid methyl ester and caffeic acid methyl ester (3), have been isolated from this strain. Sorbicatechol B (2), a natural exoaddition product of sorbicillinol with a dienophile not related to the sorbicillinoids, is quite rare in nature, and only a few cases have been reported.3f,5 Compounds 1 and 2 represent the first sorbicillin derivatives with anti-H1N1 activity.
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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 Beckman DU 640 spectrophotometer. CD spectra were measured on a JASCO J-715 (JASCO) spectropolarimeter. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer in KBr discs. NMR spectra were recorded on a JEOL JNMECP 600 spectrometer using TMS as an internal standard. HRESIMS spectra were measured on a Micromass EI-4000 (Autospec-Ultima-TOF). Semipreparative HPLC was performed using an ODS column (YMCPack ODS-A, 5 μm, 10 × 250 mm, 4 mL/min). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm) or over silica gel (200−300 mesh, Qingdao Marine Chemical Factory). Size exclusion chromatography was performed using Sephadex LH-20 (GE Healthcare). Fungal Material. The fungal strain Penicillium chrysogenum PJX-17 was isolated from sediments collected in the South China Sea and was identified by its ITS sequence. The ITS1-5.8S-ITS2 rDNA sequence of the fungus PJX-17 has been submitted to GenBank with the accession number KF318778. A voucher specimen is deposited in our laboratory at −20 °C. The working strain was prepared on potato dextrose agar slants and stored at 4 °C. Fermentation and Extraction. The fungus PJX-17 was cultured under static conditions at 28 °C in 1 L Erlenmeyer flasks containing 300 mL of liquid culture medium, composed of bran (10.0 g/L), 427
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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 *
Computational data, HPLC analysis of the EtOAc extract of P. chrysogenum PJX-17, as well as NMR spectra for compounds 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.
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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 and 21372208), the National High Technology Research and Development Program of China (No. 2013AA092901), the Program for New Century Excellent Talents in University (No. NCET-12-0499), the Public Projects of State Oceanic Administration (No. 2010418022-3), and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0944).
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REFERENCES
(1) Harned, A. M.; Volp, K. A. Nat. Prod. Rep. 2011, 28, 1790−1810. (2) Maskey, R. P.; Grun-Wollny, I.; Laatsch, H. J. Nat. Prod. 2005, 68, 865−870. (3) (a) Liu, W. Z.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T. J. Antibiot. 2005, 58, 441−446. (b) Liu, W. Z.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T. J. Antibiot. 2005, 58, 621−624. (c) Li, D. H.; Wang, F. Z.; Cai, S. X.; Zeng, X.; Xiao, X.; Gu, Q. Q.; Zhu, W. M. J. Antibiot. 2007, 60, 317−320. (d) Li, D. H.; Wang, F. Z.; Xiao, X.; Fang, Y. C.; Zhu, T. J.; Gu, Q. Q.; Zhu, W. M. Tetrahedron Lett. 2007, 48, 5235−5238. (e) Du, L.; Zhu, T. J.; Li, L. Y.; Cai, S. X.; Zhao, B. Y.; Gu, Q. Q. Chem. Pharm. Bull. 2009, 57, 220−223. (f) Li, D. H.; Cai, S. X.; Zhu, T. J.; Wang, F. Z.; Xiao, X.; Gu, Q. Q. Tetrahedron 2010, 66, 5101−5106. (g) Chen, L.; Zhu, T. J.; Ding, Y. Q.; Khan, I. A.; Gu, Q. Q.; Li, D. H. Tetrahedron Lett. 2012, 53, 325−328. (h) Guo, W. Q.; Peng, J. X.; Zhu, T. J.; Gu, Q. Q.; Keyzers, R. A.; Li, D. H. J. Nat. Prod. 2013, 76, 2016−2112. (4) Li, D. H.; Chen, L.; Zhu, T. J.; Kurtán, T.; Mándi, A.; Zhao, Z. M.; Li, J.; Gu, Q. Q. Tetrahedron 2011, 67, 7913−7918. (5) Washida, K.; Abe, N.; Sugiyama, Y.; Hirota, A. Biosci. Biotechnol. Biochem. 2009, 73, 1355−1361. (6) Bringmann, G.; Lang, G.; Gulder, T. A. M.; Tsuruta, H.; Muhlbacher, J.; Maksimenka, K.; Steffens, S.; Schaumann, K.; Stohr, R.; Wiese, J.; Imhoff, J. F.; Perovic-Ottstadt, S.; Boreiko, O.; Muller, W. E. G. Tetrahedron 2005, 61, 7252−7265. (7) Bringmann, G.; Lang, G.; Bruhn, T.; Schaffler, K.; Steffens, S.; Schmaljohann, R.; Wiese, J.; Imhoff, J. F. Tetrahedron 2010, 66, 9894− 9901. (8) Miyazawa, M.; Oshima, T.; Koshio, K.; Itsuzaki, Y.; Anzai, J. J. Agric. Food Chem. 2003, 51, 6953−6956. (9) Zhu, Y.; Zhang, L. X.; Zhao, Y.; Huang, G. D. Food Chem. 2010, 118, 228−238. (10) (a) Peng, J. X.; Jiao, J. J; Li, J.; Wang, W.; Gu, Q. Q.; Zhu, T. J.; Li, D. H. Bioorg. Med. Chem. Lett. 2012, 22, 3188−3190. (b) Hung, H. C.; Tseng, C. P.; Yang, J. M.; Ju, Y. W.; Tseng, S. N.; Chen, Y. F.; Chao, Y. S.; Hsieh, H. P.; Shih, S. R.; Hsu, J. T. A. Antiviral Res. 2009, 81, 123−131. (11) Takemoto, M.; Achiwa, K. Tetrahedron Lett. 1999, 40, 6595− 6598. (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, 428
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Sorbicatechols A and B, Antiviral Sorbicillinoids from the MarineDerived Fungus Penicillium chrysogenum PJX-17 Jixing Peng, Xiaomin Zhang, Lin Du, Wei Wang, 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: Two novel sorbicillinoids combining a bicyclo[2.2.2]octane with a 2-methoxyphenol moiety, named sorbicatechols A (1) and B (2), were isolated from the culture of the marine sediment-derived fungus Penicillium chrysogenum PJX-17, together with the known protocatechuic acid methyl ester and caffeic acid methyl ester (3). Their structures, including absolute configurations, were assigned by analysis of NMR, MS data, and TDDFT ECD calculations. Compounds 1 and 2 exhibited activities against influenza virus A (H1N1), with IC50 values of 85 and 113 μ M, respectively.
S
ester). Structurally, compounds 1 and 2 are formally derived from a sorbicillin unit and a styrene moiety by endo- and exoDiels−Alder cycloadditions, respectively. In this paper, we describe the isolation, structure elucidation, and anti-influenza A (H1N1) activities for compounds 1 and 2. Compound 1 was obtained as a yellow oil. The molecular formula was established as C23H26O6 on the basis of the HRESIMS ion at m/z 399.1801 [M + H]+, indicating 11 degrees of unsaturation. The major UV absorption at 361 nm suggested the presence of a polyene conjugated carbonyl chromophore. The IR absorptions at 3408, 1720, 1629, and 1519 cm−l indicated the presence of hydroxy and enolized βdiketone groups. Analysis of the 1D NMR data (Table 1) in CDCl3 suggested the presence of 23 carbon resonances including four methyls (one oxygenated), one methylene, nine methines (seven olefinic), nine quaternary carbons (two carbonyls), and one enol, suggesting 23 nonexchangeable protons, and one exchangeable proton in the molecule. Accordingly, two additional hydroxy groups in 1 are needed to satisfy the molecular formula. Two additional exchangeable protons (δH 6.04, 5-OH; 8.91, 18-OH) were observed in the 1H NMR spectrum in DMSO-d6 (Table 1). The characteristic proton signals at δH 7.38, 6.33, 6.29, 6.23, and 1.91 suggested the existence of a sorbyl group,3,4 which was further confirmed by the COSY correlation from H-10 to H-14
orbicillinoids are members of a large family of polyketides with highly diverse carbon skeletons and bioactivities that have been isolated from both marine and terrestrial fungi.1 Since 1948, more than 50 members of the family have been found. These members can be further divided into four subfamilies corresponding to common structural features: monomeric sorbicillinol derivatives, bisorbicillinoids, trisorbicillinoids, and hybrids of different biosynthetic precursors.1 Hybrid sorbicillinoids are a new subfamily and are proposed to be derived from either a Diels−Alder or a Michael reaction of a monomeric sorbicillinoid diene and a second non-sorbicillinoid dienophile. The rezishanones, isolated from Penicillium notatum in 2005, were the first members of this new group,2 with 27 members in the group to date.1,3,4 Many hybrid sorbicillinoids possess attractive structural features and interesting biological properties.1,3,4 Exploring the biosynthesis of hybrid precursors has inspired new avenues of research for adding chemical diversity to natural product libraries. In previous studies, our research group has reported 20 new sorbicillinoids from marine-derived fungi, including a monomeric sorbicillinoid, seven bisorbicillinoids, four trisorbicillinoids, and eight hybrid polycyclic sorbicillinoids.3,4 In the course of our ongoing search for sorbicillin derivatives using the characteristic UV absorption at ∼350 nm, we targeted a marinederived fungus with anti-influenza activity, identified as a Penicillium chrysogenum strain. Further chemical analysis of the EtOAc extracts of the broth led to the isolation of two new sorbicillin derivatives (1 and 2), together with two known phenolic acid methyl esters (3 and protocatechuic acid methyl © 2014 American Chemical Society and American Society of Pharmacognosy
Received: November 24, 2013 Published: February 4, 2014 424
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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for 1 and 2 in CDCl3 1 position
δC, type
1 2 3 4
65.1, 197.9, 112.0, 40.5,
5 6 7
74.6, C 211.9, C 47.8, CH
8
2 δH (J in Hz)
C C C CH
3.29, dd (2.8, 2.8)
3.01, m
31.4, CH2
1.84, m
δC, type 65.0, 199.7, 110.5, 40.6,
C C C CH
75.9, C 212.1, C 49.9, CH 30.2, CH2
3.05, m
and the HMBC correlation between H-10 and C-9 (Figure 1). The E configurations of these double bonds in the sorbyl moiety were determined from the large coupling constants (3JH‑10,H‑11 = 14.8 Hz, 3JH‑12,H‑13 = 12.9 Hz) and further confirmed by the observed correlation between H-11 and H-13 in the NOESY spectrum (Figure 2). The Z configuration for the C-3/C-9 double bond was supported by the downfield chemical shift (δH 14.32) of the intramolecularly hydrogen bonded 9-OH. The structure of the bicyclo[2.2.2]octane moiety was confirmed by the HMBC correlations from H-4 to C-2, C-3, C-5, C-6, and C-7, from 1-CH3 to C-1, C-2, C-6, and C-7, and from 5-CH3 to C-4, C-5, and C-6, as well as COSY correlations (H-7/H2-8/H-4), which agreed with the reported data.3,4 A hydroxy group was assigned at C-5 based upon its chemical shift (δC 74.6), as well as the comparison with those of reported sorbicillin derivatives.3,4 The connection of C-3 and C-10 via C-9 was deduced from HMBC correlations from H-10 to C-9, H-11 to C-9, and H-4 to C-9. Aside from the above structure units, a subunit consisting of seven carbons remained. COSY correlations (H-19/H-20) and HMBC correlations from H-16 to C-18 and C-20, H-19 to C-15 and C-17, H-20 to C-16 and C-18, and 17-OCH3 to C-17 established a methoxy-substituted phenolic moiety. The position of the methoxy in 1 was determined by a NOESY correlation between 17-OCH3 and H-16. In the phenolic moiety, HMBC correlations from both H-16 and H-20 to C-7 revealed the connection to the bicyclo[2.2.2]octane with C-15 linking to C-7, establishing the full carbon skeleton. Furthermore, hydroxy groups were assigned to C-9 and C-18, respectively, according to the molecular formula and chemical shifts of C-9 (δC 167.1) and C-18 (δC 144.9), completing the planar structure of 1. The assignment of the stereoconfiguration of 1 is discussed below. Compound 2 had the same molecular formula, C23H26O6, as that of 1, established on the basis of the HRESIMS ion detected at m/z 399.1803 [M + H]+. Its UV and IR spectra were almost identical to those of 1. Analysis of its NMR data (Table 1), in
9 10 11
167.1, C 118.0, CH 142.5, CH
12
130.9, CH
13 14 15 16 17 18 19 20
139.9, 18.9, 133.2, 110.2, 146.5, 144.9, 114.3, 121.5,
1-CH3 5-CH3 17-OCH3 5-OH 9-OH 18-OH a
CH CH3 C CH C C CH CH
10.5, CH3 24.3, CH3 55.7, CH3
6.29, d (14.8) 7.38, dd (14.8, 12.9) 6.33, dd (12.9, 12.9) 6.23, m 1.91, d (6.6) 6.45, d (1.7)
6.79, d (8.2) 6.49, dd (8.2, 1.7) 0.93, s 1.26, s 3.79, s 6.04,a s 14.32, s 8.91,a s
167.3, C 118.1, CH 142.2, CH 131.0, CH 139.7, CH 19.0, CH3 132.0, C 111.2, CH 146.4, C 144.9, C 114.1,CH 122.4, CH 10.3, CH3 25.2, CH3 55.9, CH3
δH (J in Hz)
3.23, brs
3.03, dd (10.9, 7.7) 2.25, td (10.9, 3.3) 2.57, dd (12.7, 7.7) 6.25, d (14.8) 7.33, dd (14.8,11.0) 6.30, dd (11.0, 11.0) 6.21, m 1.91, d (6.6) 6.71, brs
6.80, d (8.2) 6.60, brd (8.2) 0.84, s 1.28, s 3.82, s 5.30, s 13.93, s
Recorded in DMSO-d6.
particular the COSY and HMBC correlations, revealed that compounds 2 and 1 shared the same planar structure. The 1H and 13C NMR spectra of 1 and 2 are very similar (Table 1). The 1H NMR spectrum of 1 has resonances of H2-8 at δH 1.84 and 3.05 and H-16 at δH 6.45, while the corresponding signals of H2-8 in 2 are at δH 2.25 and 2.57 and H-16 in 2 at δH 6.71. The largest carbon shift differences center around C-7 (Table 1). These differences implied that compound 1 was an epimer of 2 at C-7. The absolute configurations of 1 and 2 were determined by the analysis of their CD spectra, which agreed with the biosynthetic pathway of this compound class. All of the known hybrid sorbicillinoids, such as chloctanspirones A and B4 from Penicillium terrestre, spirosorbicillinols A−C5 from Trichoderma sp., and sorbicillactones A and B6 and sorbifuranones A−C7 from P. chrysogenum, were derived from sorbicillinol with another non-sorbicillinoid precursor by either a Diels−Alder reaction or a Michael addition. In each case, the S configuration at C-5 of sorbicillinol was not modified during the biosynthesis. It is likely that compounds 1 and 2 are also generated by a Diels−Alder reaction between sorbicillinol and a styrene dienophile (Scheme 1). In the proposed biosynthetic pathway, three asymmetric centers of 1 and 2 are created during the [4+2] cycloaddition reaction, with an endo-transition giving two 425
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bicyclo[2.2.2]octane ring. On the basis of the DFT-optimized structures for low-energy conformers for both endo- and exoproducts, the distances between 17-OCH3 and 1-CH3 are less than 5 Å (4.114−4.858 Å) in all the exo-orientations for the aromatic ring to the C-2/C-3 bond, while two conformers are over 5 Å (5.093 and 6.360 Å) and two are near 5 Å (4.696 and 4.789 Å) in the endo-orientation (Figures E1−E8, Supporting Information). According to the NOESY correlations between 17-OCH3 and 1-CH3, compound 2 was tentatively assigned as the exo-product (Figure 2). Consequently, compound 1, which showed no NOESY correlations between 17-OCH3 and 1-CH3, was assigned an endo-orientation. In order to confirm the assignment and their respective absolute configurations, we employed TDDFT CD calculations to compare theoretical with experimentally derived data for all the possible isomers. Density functional theory (DFT) calculations performed at the B3LYP/ 6-311+G** level were used to generate ECD spectra for a set of the lowest-energy conformers of each isomer. The resulting ECD spectra were combined by Boltzmann weighting to give a composite spectrum for each diastereomer. The calculated ECD curves (Figures 3 and 4) of (1R,4S,5S,7R)-1 and
Figure 1. Key COSY and HMBC correlations of 1 and 2.
Figure 3. B3LYP/6-311+G(d,p)-calculated ECD spectra of (1R,4S,5S,7R)-1 (red) and (1S,4R,5S,7S)-iso-1 (blue) and the experimental ECD spectrum of 1 (black) (σ = 0.28 eV).
Figure 2. Key NOESY correlations of 1 and 2.
possible endo-products (the aromatic ring is endo to the C-2/C3 bridge) with the absolute configurations (1R,4S,5S,7R) and (1S,4R,5S,7S) or an exo-transition state giving (1R,4S,5S,7S) and (1S,4R,5S,7R), with the consideration of the rigidity of the
(1R,4S,5S,7S)-2 gave the best agreement with the experimental data of compounds 1 and 2, respectively. Thus the absolute
Scheme 1. Possible Biosynthetic Route to Sorbicatechols A (1) and B (2)
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mannitol (20.0 g/L), MgSO4·7H2O (0.1 g/L), KH2PO4 (0.5 g/L), CaCl2 (0.1 g/L), FeSO4 (0.002 g/L), MnCl2 (0.002 g/L), ZnSO4 (0.002 g/L), and seawater (Huiquan Bay, Yellow Sea). After 30 days of cultivation, 20 L of whole broth was filtered through cheesecloth to separate it into supernatant and mycelia. The former was extracted three times with EtOAc, while the latter was extracted three times with acetone and concentrated under reduced pressure to afford an aqueous solution, which was extracted three times with EtOAc. Both EtOAc solutions were combined and concentrated under reduced pressure to give an organic extract (6.0 g). Purification. The extract was subjected to vacuum liquid chromatography over a silica gel column using a gradient elution with petroleum ether−CHCl3−MeOH to give three fractions. Fraction 2 was rechromatographed on a silica gel column with petroleum ether−acetone to provide two subfractions (fractions 2.1 and 2.2). Fraction 2.1 was subjected to Sephadex LH-20 column chromatography eluting with MeOH and then purified by semipreparative HPLC (70:30 MeOH−H2O with 0.2% TFA, 4 mL/min) to give compounds 1 (12 mg, tR 8 min) and 2 (5.0 mg, tR 16 min). Fraction 2.2 was separated by Sephadex LH-20 eluting with CHCl3−MeOH (1:1) and then by semipreparative HPLC (45:55 MeOH−H2O with 0.2% TFA, 4 mL/min) to afford protocatechuic acid methyl ester (3 mg, tR 7 min) and compound 3 (6 mg, tR 10 min). Sorbicatechol A (1): yellow oil; [α]20D +19 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 361 (4.42) nm; CD (0.88 × 10−3 M, MeOH) λmax (Δε) 354.5 (+3.58), 311 (−10.34), 279sh (−3.27), 245 (+4.05), 220 (+0.56), 204.5 (+2.81) nm; IR (KBr) νmax 3408, 2925, 1720, 1629, 1519, 1247, 998 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 399.1801 [M + H]+ (calcd for C23H26O6, 399.1808). Sorbicatechol B (2): yellow oil; [α]20D +120 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 356 (4.44) nm; CD (0.88 × 10−3 M, MeOH) λmax (Δε) 355 (+15.41), 313.5 (−17.38), 282sh (−4.15), 244.5 (+4.34), 207 (−3.87) nm; IR (KBr) νmax 3400, 2925, 1720, 1629, 1519, 1247, 998 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 399.1803 [M + H]+ (calcd for C23H26O6, 399.1808). Anti-influenza A (H1N1) Assay. The antiviral activity against H1N1 was evaluated by the CPE inhibition assay. Confluent MDCK cell monolayers were first incubated with influenza virus (A/Puerto Rico/8/34 (H1N1), PR/8) at 37 °C for 1 h. After removing the virus dilution, cells were maintained in infecting media (RPMI 1640, 4 μg/ mL of trypsin) containing different test compounds at 37 °C. After a 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 (BioRad) at 570 nm. The IC50 was calculated as the compound concentration required to inhibit influenza virus yield at 48 h postinfection by 50%. Ribavirin was used as the positive control in the CPE inhibition assay. 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/6-311+G(d,p) 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 calculations were performed on the lowest-energy conformations (>5% population) for each configuration using 30 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 0.28−0.36 eV width, from dipole-length 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 weight. The calculated spectrum was blue-shifted by 15−25 nm to facilitate comparison to the experimental data.
Figure 4. B3LYP/6-311+G(d,p)-calculated ECD spectra of (1R,4S,5S,7S)-2 (red) and (1S,4R,5S,7R)-iso-2 (blue) and the experimental ECD spectrum of 2 (black) (σ = 0.36 eV).
configurations of 1 and 2 could be determined as (1R,4S,5S,7R) and (1R,4S,5S,7S), respectively. Protocatechuic acid methyl ester8 and caffeic acid methyl ester (3)9 were identified by comparison of their spectroscopic data (NMR and MS) with those in the literature. The antiviral activities of compounds 1 and 2 against influenza A virus (H1N1) were evaluated by the cytopathic effect (CPE) inhibition assay.10 They showed inhibitory effects with IC50 values of 85 and 113 μM, respectively (ribavirin as a positive control, IC50 84 μM). Sorbicatechols A (1) and B (2) are presumably formed by endo and exo intermolecular Diels−Alder reactions between sorbicillinol (common precursor) as the diene and 4-hydroxy-3methoxystyrene11 (proposed hybrid precursor) as the dienophile. Unfortunately, the two key precursors have not been isolated. However, two related analogues, protocatechuic acid methyl ester and caffeic acid methyl ester (3), have been isolated from this strain. Sorbicatechol B (2), a natural exoaddition product of sorbicillinol with a dienophile not related to the sorbicillinoids, is quite rare in nature, and only a few cases have been reported.3f,5 Compounds 1 and 2 represent the first sorbicillin derivatives with anti-H1N1 activity.
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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 Beckman DU 640 spectrophotometer. CD spectra were measured on a JASCO J-715 (JASCO) spectropolarimeter. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer in KBr discs. NMR spectra were recorded on a JEOL JNMECP 600 spectrometer using TMS as an internal standard. HRESIMS spectra were measured on a Micromass EI-4000 (Autospec-Ultima-TOF). Semipreparative HPLC was performed using an ODS column (YMCPack ODS-A, 5 μm, 10 × 250 mm, 4 mL/min). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm) or over silica gel (200−300 mesh, Qingdao Marine Chemical Factory). Size exclusion chromatography was performed using Sephadex LH-20 (GE Healthcare). Fungal Material. The fungal strain Penicillium chrysogenum PJX-17 was isolated from sediments collected in the South China Sea and was identified by its ITS sequence. The ITS1-5.8S-ITS2 rDNA sequence of the fungus PJX-17 has been submitted to GenBank with the accession number KF318778. A voucher specimen is deposited in our laboratory at −20 °C. The working strain was prepared on potato dextrose agar slants and stored at 4 °C. Fermentation and Extraction. The fungus PJX-17 was cultured under static conditions at 28 °C in 1 L Erlenmeyer flasks containing 300 mL of liquid culture medium, composed of bran (10.0 g/L), 427
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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 *
Computational data, HPLC analysis of the EtOAc extract of P. chrysogenum PJX-17, as well as NMR spectra for compounds 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.
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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 and 21372208), the National High Technology Research and Development Program of China (No. 2013AA092901), the Program for New Century Excellent Talents in University (No. NCET-12-0499), the Public Projects of State Oceanic Administration (No. 2010418022-3), and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0944).
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REFERENCES
(1) Harned, A. M.; Volp, K. A. Nat. Prod. Rep. 2011, 28, 1790−1810. (2) Maskey, R. P.; Grun-Wollny, I.; Laatsch, H. J. Nat. Prod. 2005, 68, 865−870. (3) (a) Liu, W. Z.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T. J. Antibiot. 2005, 58, 441−446. (b) Liu, W. Z.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T. J. Antibiot. 2005, 58, 621−624. (c) Li, D. H.; Wang, F. Z.; Cai, S. X.; Zeng, X.; Xiao, X.; Gu, Q. Q.; Zhu, W. M. J. Antibiot. 2007, 60, 317−320. (d) Li, D. H.; Wang, F. Z.; Xiao, X.; Fang, Y. C.; Zhu, T. J.; Gu, Q. Q.; Zhu, W. M. Tetrahedron Lett. 2007, 48, 5235−5238. (e) Du, L.; Zhu, T. J.; Li, L. Y.; Cai, S. X.; Zhao, B. Y.; Gu, Q. Q. Chem. Pharm. Bull. 2009, 57, 220−223. (f) Li, D. H.; Cai, S. X.; Zhu, T. J.; Wang, F. Z.; Xiao, X.; Gu, Q. Q. Tetrahedron 2010, 66, 5101−5106. (g) Chen, L.; Zhu, T. J.; Ding, Y. Q.; Khan, I. A.; Gu, Q. Q.; Li, D. H. Tetrahedron Lett. 2012, 53, 325−328. (h) Guo, W. Q.; Peng, J. X.; Zhu, T. J.; Gu, Q. Q.; Keyzers, R. A.; Li, D. H. J. Nat. Prod. 2013, 76, 2016−2112. (4) Li, D. H.; Chen, L.; Zhu, T. J.; Kurtán, T.; Mándi, A.; Zhao, Z. M.; Li, J.; Gu, Q. Q. Tetrahedron 2011, 67, 7913−7918. (5) Washida, K.; Abe, N.; Sugiyama, Y.; Hirota, A. Biosci. Biotechnol. Biochem. 2009, 73, 1355−1361. (6) Bringmann, G.; Lang, G.; Gulder, T. A. M.; Tsuruta, H.; Muhlbacher, J.; Maksimenka, K.; Steffens, S.; Schaumann, K.; Stohr, R.; Wiese, J.; Imhoff, J. F.; Perovic-Ottstadt, S.; Boreiko, O.; Muller, W. E. G. Tetrahedron 2005, 61, 7252−7265. (7) Bringmann, G.; Lang, G.; Bruhn, T.; Schaffler, K.; Steffens, S.; Schmaljohann, R.; Wiese, J.; Imhoff, J. F. Tetrahedron 2010, 66, 9894− 9901. (8) Miyazawa, M.; Oshima, T.; Koshio, K.; Itsuzaki, Y.; Anzai, J. J. Agric. Food Chem. 2003, 51, 6953−6956. (9) Zhu, Y.; Zhang, L. X.; Zhao, Y.; Huang, G. D. Food Chem. 2010, 118, 228−238. (10) (a) Peng, J. X.; Jiao, J. J; Li, J.; Wang, W.; Gu, Q. Q.; Zhu, T. J.; Li, D. H. Bioorg. Med. Chem. Lett. 2012, 22, 3188−3190. (b) Hung, H. C.; Tseng, C. P.; Yang, J. M.; Ju, Y. W.; Tseng, S. N.; Chen, Y. F.; Chao, Y. S.; Hsieh, H. P.; Shih, S. R.; Hsu, J. T. A. Antiviral Res. 2009, 81, 123−131. (11) Takemoto, M.; Achiwa, K. Tetrahedron Lett. 1999, 40, 6595− 6598. (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, 428
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