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Two new monoterpenoid α-pyrones from a fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa a
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Ting Gong , Xin Zhen , Bing-Juan Li , Jin-Ling Yang & Ping Zhu a
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State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Key Laboratory of Biosynthesis of Natural Products of the National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing100050, China Published online: 22 May 2015.
To cite this article: Ting Gong, Xin Zhen, Bing-Juan Li, Jin-Ling Yang & Ping Zhu (2015): Two new monoterpenoid α-pyrones from a fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1040778 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1040778
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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1040778
Two new monoterpenoid a-pyrones from a fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa Ting Gong, Xin Zhen, Bing-Juan Li, Jin-Ling Yang and Ping Zhu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Key Laboratory of Biosynthesis of Natural Products of the National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
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(Received 4 February 2015; final version received 8 April 2015) Two new monoterpenoid a-pyrones, named nectriapyrones C and D (1 and 2), along with a known a-pyrone (nectriapyrone, 3) were isolated from a marine-derived fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa collected from the South China Sea. Their structures were determined on the basis of 1D NMR, 2D NMR, HR-ESI-MS methods. Keywords: marine fungus; Nectria sp; secondary metabolites; monoterpenoid a-pyrones
1.
Introduction
In the search for novel and bioactive molecules for drug discovery, marine microorganisms are becoming an important and interesting research area, which continue to produce structurally unique secondary metabolites with impressive biological properties [1 – 3]. For these reasons, we investigated the secondary metabolites of the fungal strain Nectria sp. HLS206 (FJ770068.1) isolated from the marine sponge Gelliodes carnosa. This strain belongs to the order of Hypocreales based on phylogenetic analysis of the rDNA ITS sequence [4]. In the earlier report, we described the isolation and elucidation of two mannitol derivatives and three sterols from the EtOAc extract of this strain [5]. During further investigation on active substances, two new monoterpenoid a-pyrones, named as nectriapyrones C and D (1 and 2), together with a known a-pyrone (nectriapyrone, 3) were isolated and their structures were determined by the spectroscopic analysis. We herein report the
details of the isolation and structure elucidation as well as the determination of the anti-bacterial and anti-tumor activities of these compounds.
2. Results and discussion Compound 1 was obtained as a white powder with m.p. 115.2 – 116.38C. The molecular formula was determined to be C11H14O4 by HR-ESI-MS at m/z 211.0967 [M þ H] þ. The UV spectrum of 1 exhibited absorption maxima at 235 and 331 nm, suggesting the presence of a conjugated enone system in its structure. The 1H NMR spectrum of 1 showed two methyl signals at d 1.92 (3H, s) and 1.93 (3H, d, J ¼ 6.0 Hz), a methoxyl signal at d 3.92 (3H, s), an oxygenated methylene signal at d 4.47 (2H, s), two olefinic methane signals at d 6.44 (1H, s), and 6.72 (1H, q, J ¼ 6.0 Hz), which were similar to the 1H NMR data of nectriapyrone (3) [6], except for the absence of a methyl signal at d 1.88 (3H, s) and the presence of an oxygenated methylene signal at d 4.47
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data of 1 and 2 in CDCl3. 1 No. 2 3 4 5 6 7 8
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dH
consistent with a molecular formula of C11H14O4. The 1H and 13C NMR (Table 1) spectral data suggested that 2 also had the a-pyrone skeleton as 1 with differences at 2-butylene side chain. The quartet signal at d 6:72 (H-8) and the singlet at d 4.47(2H, H-12) of 1 were replaced by the triplet signal at d 6.69 (H-8) and the doublets at d 4.40 (2H, H-9) of 2, indicated that the hydroxy group substitued at C-9. The 13C NMR, HMBC, and NOE data (Table 1 and Figure 1) also confirmed this structure of 2, and it was assigned as 9-hydroxy nectriapyrone and named nectriapyrone D. The known compound nectriapyrone (3) was identified by comparison of its spectral data (1H and 13C NMR) with those reported in the literature [6]. It was well known that the a-pyrones were biosynthesized by polyketide synthetase (PKS) pathway primed with acetyl units which can either be derived from malonyl-CoA or methyl malonyl-CoA. The polyketide chain was assembled, modified and released as the a-pyrone spontaneously [7]. We proposed two new a-pyrones (1 and 2) were produced from nectriapyrone (3) by the hydroxylation of the allylic postion in the side chain (Figure 2). Nectriapyrone has already been isolated as an antibiotic against Staphylococcus aureus (MIC 30 mg/ml) [8]. In addition, nectriapyrone presented weak cytotoxic activities toward human T leukemia and melanoma tumor cells (IC 50s were 2.27 £ 103 and 6.97 £ 103 mM, respectively) [9]. Hence, antibacterial and anti-tumor activities of our isolated compounds (1 –3) in vitro were
2
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165.4 102.4 166.4 6.44 (1H, s) 92.9 6.20 (1H, s) 158.7 131.5 6.72 (1H, q, 133.1 6.69 (1H, t, 6.0) 6.5) 1.93 (3H, d, 14.0 4.40 (2H, d, 6.0) 6.5) 1.92 (3H, s) 8.6 1.94 (3H, s) 3.92 (3H, s) 56.3 3.90 (3H, s) 4.47 (2H, s) 56.4 1.92 (3H, s)
164.8 102.8 165.6 92.6 159.0 127.6 132.6 59.6 8.6 56.1 12.7
(2H, s). Thus, one hydroxylated methyl could be suggested, which was also confirmed by the 13C NMR spectrum (Table 1) and the formula of 1. The hydroxyl located at C-12 could be ascertained on the basis of HMBC data (H-12 at d 4.47 to C-6 at d 158.7 and C-8 at d 133.1; H-8 at d 6.72 to C-12 at d 56.4) and also by comparison with the data of 3. The C-7/C-8 double bond was assigned the E configuration on the basis of the NOE experiments. Irradiation of the H-12 at d 4.47 produced an enhancement of 9-CH3 at d 1.93 but no enhancement of H-8 at d 6.72. Therefore, the structure of 1 was determined to be 12-hydroxy nectriapyrone and named nectriapyrone C. Compound 2 was isolated as a yellow powder with m.p. 120.5– 122.08C. The HR-ESI-MS of 2 exhibited a pseudomolecular ion at m/z 211.0970 [M þ H]þ, 11
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Figure 1. Structures of compounds 1 –3 and key HMBC correlations of 1 and 2.
Journal of Asian Natural Products Research O
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Figure 2. Proposed biosynthetic pathway of compounds 1 – 3.
examined. Unfortunately, none of them exhibited anti-bacterial activity at the dose of 500 mg/disc against S. aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa using the paper disc diffusion method. Furthermore, they were all inactive against six human tumor cell lines (HCT116, HepG2, BGC823, NCIH1650, A2780, and A375) in vitro by MTT method (IC50 . 10 mM). Further pharmacological studies of isolated compounds are currently underway.
3.
Experimental
3.1. General experimental procedures Melting points were determined on a XT Digital Melting-Point Apparatus with Microscope and uncorrected (Tsigtao Unicom, Linyi, China). UV spectra were obtained on a JASCO U-650 spectrophotometer (Jasco, Tokyo, Japan). 1H NMR (500 MHz), 13C NMR (125 MHz), NOE difference, DEPT, HMBC spectra were run on Mercury-500 (Varian, Palo Alto, CA, USA) with TMS (tetramethylsilane) as internal standard. HR-ESI-MS and ESI-
MS were performed on an Agilent 1100 LC/MSD Trap-SL mass spectrometer (Agilent, Santa Clara, CA, USA). Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and RP-18 (Merck, 40 –60 mm; Darmstadt, Germany) were used for column chromatography (CC) and silica gel GF-254 (Qingdao Marine Chemical Factory, Qingdao, China) was used for thin-layer chromatography. Medium pressure liquid chromatography (MPLC) was employed on LC2000 MPLC equipment (Separation technology, Beijing, China). Semi-preparative high performance liquid chromatography (HPLC) was carried out on a preparation Agilent ODS Eclipse XDB C-18 column (10 mm, 250 mm £ 20 mm, Agilent, USA) with Shimadzu pump LC-6AD and UV detector SPD-20A (set at 230 nm, Shimadzu, Japan) at a flow rate of 4.0 ml/min. 3.2.
The fungal strain
The fungal strain HLS206 was isolated from the marine sponge Gelliodas carnosa, which was collected from the South China Sea and identified by Professor
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Jin-He Li at Institute of Oceanology, Chinese Academy of Science, Qingdao. This strain belongs to the order of Hypocreales, with 88% sequence identity to Nectria coryli (HM534895.1) based on phylogenetic analysis of the rDNA ITS sequence [5]. This fungus has been deposited at our laboratory in the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. 3.3. Fermentation, extraction, and isolation The fungal strain was first cultivated on the YPD agar plates (yeast extract peptone dextrose medium, 1% yeast extract, 2% peptone, 2% glucose, 2% agar) at 258C for 3 d, then mycelium was inoculated into the seed medium (0.4% glucose, 1.0% malt extract, 0.4% yeast extract; 50 ml of the medium in 250 ml Erlenmeyer flask) and cultivated on a rotary shaker at 170 r/min, 258C for 3 d. The seed culture (10 ml) was transferred into the solid rice medium (1 g rice/ml distilled H2O, 100 g rice in 500 ml flask; the rice was soaked in the water overnight before autoclaving at 1218C for 30 min) and statically cultivated at 258C for 45 d. The solid cultures (5 kg) were extracted repeatedly with EtOAc and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (28.2 g). The EtOAc extract was subjected to ODS-C18 CC (30 mm £ 350 mm) eluted with MeOH – H2O (20:80 –30:70 – 40:60 – 50:50 – 60:40 –70:30 – 80:20 – 100:0, v/v). The eluted fractions were combined to eight fractions (Fr.1 –Fr.8) on basis of HPLC behavior. Fr.1 (2.5 g) was separated by Sephadex LH-20 CC using MeOH as eluent to give 15 fractions (Fr.1.1 – Fr.1.15), Fr.1.6 (1.0 g) purified first by preparative HPLC (MeOH:H2O ¼ 38:62) and again by preparative HPLC (CH3CN: H2O ¼ 18:72) to afford 1 (20.5 mg, flow rate 4.0 ml/min, tR ¼ 23.2 min) and 2
(25.5 mg, flow rate 4.0 ml/min, tR ¼ 25.2 min). Fr.3 (1.5 g) was separated by MPLC (MeOH:H2O ¼ 65:35) to yield four fractions (Fr.3.1 – Fr.3.4). Fr.3.4 (860 mg) was purified by Sephadex LH20 CC to yield 3 (85.0 mg).
3.3.1.
Nectriapyrone A (1)
White powder; mp 115.2– 116.38C. UV (MeOH) lmax (nm): 235, 331; IR (KBr) n max: 3200, 2960, 1703, 1619, 1455, 1356, 800 and 703 cm21; 1H and 13C NMR spectral data see Table 1; HR-ESI-MS m/z: 211.0967 [M þ H]þ(calcd for C11H15O4, 211.0965).
3.3.2. Nectriapyrone B (2) Yellow powder; mp 120.5 –122.08C. UV (MeOH) lmax (nm): 235, 330; IR (KBr) nmax: 3230, 2952, 1718, 1658, 1462, 1387, 822 and 756 cm21; 1H and 13C NMR spectral data see Table 1; HR-ESI-MS m/z: 211.0970 [M þ H]þ(calcd for C11H15O4, 211.0965).
3.4. Biological assays Anti-bacterial and anti-tumor bioassays were undertaken with compounds of purity . 90% by HPLC.
3.4.1.
Anti-bacterial bioassays
The anti-bacterial assays were performed as previously described [4]. The tested compounds dissolved in methanol at a concentration of 5 mg/ml were screened for anti-bacterial activities against S. aureus 29213, B. subtilis 63501, E. coli 25922, and P. aeruginosa 27853. The paper disc (6 mm diam.) was dropped the solution of tested compound and then dried in the air (100 ml). Ampicillin (Sigma, SantaClara; CA; USA; 98% pure) was included as a positive control.
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3.4.2. Anti-tumor bioassays The MTT assay was used for the antitumor bioassay in vitro in human cancer cell lines HCT116 (colon cancer), HepG2 (liver cancer), BGC823 (liver cancer), NCI-H1650 (nonsmall-cell lung cancer), A2780 (ovarian cancer), and A375 (lung cancer) as reported [10], and the doseresponse curves were fitted with Sigma plot. IC50 s were determined. Taxol (Sigma; 98% pure) was included as a positive control. Acknowledgments We acknowledge the Research Team of Professor Xiao-guang Chen, Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College for cytotoxicity bioassays. We are grateful to the Department of Instrumental Analysis of our Institute for measurement of the IR, NMR, and MS data.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was financially supported by grant from the Mega-project for Inovative Drugs [grant number 2012ZX09301002-001-005] and National Natural Science Foundation of China [grant number 81402846].
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References [1] G. Chen, H.F. Wang, and Y.H. Pei, J. Asian Nat. Prod. Res. 16, 105 (2014). doi:10.1080/10286020.2013.855202. [2] A. Evidente, A. Kornienko, A. Cimmino, A. Andolfi, F. Lefranc, V. Mathieu, and R. Kiss, Nat. Prod. Rep. 31, 617 (2014). doi:10.1039/c3np70078j. [3] D. Skropeta and L.Q. Wei, Nat. Prod. Rep. 31, 999 (2014). doi:10.1039/ C3NP70118B. [4] W.C. Liu, C.Q. Li, P. Zhu, J.L. Yang, and K.D. Cheng, Fungal Divers. 42, 1 (2010). doi:10.1007/s13225-010-0022-8. [5] R.S. Wang, T. Gong, P. Zhu, and K.D. Cheng, J. Chin. Pharm. Sci. 21, 183 (2012). dio:10.5246/jcps.2012.02. 024. [6] A.G. Avent, J.R. Hanson, and A. Truneh, Phytochemistry 31, 1065 (1992). doi:10. 1016/0031-9422(92)80079-T. [7] Y. Zou, W. Xu, Y.T. Tsunematsu, M.C. Tang, K.J. Watanabe, and Y. Tang, Org. Lett. 16, 6390 (2014). doi:10.1021/ ol503179v. [8] M.S.R. Nair and S.T. Carey, Tetrahedron Lett. 16, 1655 (1975). [9] D.O. Guimara˜es, W.S. Borges, C.Y. Kawano, P.H. Ribeiro, G.H. Goldman, A. Nomizo, O.H. Thiemann, G. Oliva, N.P. Lopes, and M.T. Pupo, FEMS Immunol. Med. Microbiol. 52, 134 (2008). doi:10.1111/j.1574-695X.2007.00354.x. [10] G. Ni, Q.J. Zhang, Z.F. Zheng, R.Y. Chen, and D.Q. Yu, J. Nat. Prod. 72, 966 (2009). doi:10.1021/np800789y.
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Two new monoterpenoid α-pyrones from a fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa a
a
a
a
a
Ting Gong , Xin Zhen , Bing-Juan Li , Jin-Ling Yang & Ping Zhu a
Click for updates
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Key Laboratory of Biosynthesis of Natural Products of the National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing100050, China Published online: 22 May 2015.
To cite this article: Ting Gong, Xin Zhen, Bing-Juan Li, Jin-Ling Yang & Ping Zhu (2015): Two new monoterpenoid α-pyrones from a fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1040778 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1040778
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1040778
Two new monoterpenoid a-pyrones from a fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa Ting Gong, Xin Zhen, Bing-Juan Li, Jin-Ling Yang and Ping Zhu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Key Laboratory of Biosynthesis of Natural Products of the National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Downloaded by [New York University] at 01:00 11 June 2015
(Received 4 February 2015; final version received 8 April 2015) Two new monoterpenoid a-pyrones, named nectriapyrones C and D (1 and 2), along with a known a-pyrone (nectriapyrone, 3) were isolated from a marine-derived fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa collected from the South China Sea. Their structures were determined on the basis of 1D NMR, 2D NMR, HR-ESI-MS methods. Keywords: marine fungus; Nectria sp; secondary metabolites; monoterpenoid a-pyrones
1.
Introduction
In the search for novel and bioactive molecules for drug discovery, marine microorganisms are becoming an important and interesting research area, which continue to produce structurally unique secondary metabolites with impressive biological properties [1 – 3]. For these reasons, we investigated the secondary metabolites of the fungal strain Nectria sp. HLS206 (FJ770068.1) isolated from the marine sponge Gelliodes carnosa. This strain belongs to the order of Hypocreales based on phylogenetic analysis of the rDNA ITS sequence [4]. In the earlier report, we described the isolation and elucidation of two mannitol derivatives and three sterols from the EtOAc extract of this strain [5]. During further investigation on active substances, two new monoterpenoid a-pyrones, named as nectriapyrones C and D (1 and 2), together with a known a-pyrone (nectriapyrone, 3) were isolated and their structures were determined by the spectroscopic analysis. We herein report the
details of the isolation and structure elucidation as well as the determination of the anti-bacterial and anti-tumor activities of these compounds.
2. Results and discussion Compound 1 was obtained as a white powder with m.p. 115.2 – 116.38C. The molecular formula was determined to be C11H14O4 by HR-ESI-MS at m/z 211.0967 [M þ H] þ. The UV spectrum of 1 exhibited absorption maxima at 235 and 331 nm, suggesting the presence of a conjugated enone system in its structure. The 1H NMR spectrum of 1 showed two methyl signals at d 1.92 (3H, s) and 1.93 (3H, d, J ¼ 6.0 Hz), a methoxyl signal at d 3.92 (3H, s), an oxygenated methylene signal at d 4.47 (2H, s), two olefinic methane signals at d 6.44 (1H, s), and 6.72 (1H, q, J ¼ 6.0 Hz), which were similar to the 1H NMR data of nectriapyrone (3) [6], except for the absence of a methyl signal at d 1.88 (3H, s) and the presence of an oxygenated methylene signal at d 4.47
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data of 1 and 2 in CDCl3. 1 No. 2 3 4 5 6 7 8
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9 10 11 12
dH
consistent with a molecular formula of C11H14O4. The 1H and 13C NMR (Table 1) spectral data suggested that 2 also had the a-pyrone skeleton as 1 with differences at 2-butylene side chain. The quartet signal at d 6:72 (H-8) and the singlet at d 4.47(2H, H-12) of 1 were replaced by the triplet signal at d 6.69 (H-8) and the doublets at d 4.40 (2H, H-9) of 2, indicated that the hydroxy group substitued at C-9. The 13C NMR, HMBC, and NOE data (Table 1 and Figure 1) also confirmed this structure of 2, and it was assigned as 9-hydroxy nectriapyrone and named nectriapyrone D. The known compound nectriapyrone (3) was identified by comparison of its spectral data (1H and 13C NMR) with those reported in the literature [6]. It was well known that the a-pyrones were biosynthesized by polyketide synthetase (PKS) pathway primed with acetyl units which can either be derived from malonyl-CoA or methyl malonyl-CoA. The polyketide chain was assembled, modified and released as the a-pyrone spontaneously [7]. We proposed two new a-pyrones (1 and 2) were produced from nectriapyrone (3) by the hydroxylation of the allylic postion in the side chain (Figure 2). Nectriapyrone has already been isolated as an antibiotic against Staphylococcus aureus (MIC 30 mg/ml) [8]. In addition, nectriapyrone presented weak cytotoxic activities toward human T leukemia and melanoma tumor cells (IC 50s were 2.27 £ 103 and 6.97 £ 103 mM, respectively) [9]. Hence, antibacterial and anti-tumor activities of our isolated compounds (1 –3) in vitro were
2
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165.4 102.4 166.4 6.44 (1H, s) 92.9 6.20 (1H, s) 158.7 131.5 6.72 (1H, q, 133.1 6.69 (1H, t, 6.0) 6.5) 1.93 (3H, d, 14.0 4.40 (2H, d, 6.0) 6.5) 1.92 (3H, s) 8.6 1.94 (3H, s) 3.92 (3H, s) 56.3 3.90 (3H, s) 4.47 (2H, s) 56.4 1.92 (3H, s)
164.8 102.8 165.6 92.6 159.0 127.6 132.6 59.6 8.6 56.1 12.7
(2H, s). Thus, one hydroxylated methyl could be suggested, which was also confirmed by the 13C NMR spectrum (Table 1) and the formula of 1. The hydroxyl located at C-12 could be ascertained on the basis of HMBC data (H-12 at d 4.47 to C-6 at d 158.7 and C-8 at d 133.1; H-8 at d 6.72 to C-12 at d 56.4) and also by comparison with the data of 3. The C-7/C-8 double bond was assigned the E configuration on the basis of the NOE experiments. Irradiation of the H-12 at d 4.47 produced an enhancement of 9-CH3 at d 1.93 but no enhancement of H-8 at d 6.72. Therefore, the structure of 1 was determined to be 12-hydroxy nectriapyrone and named nectriapyrone C. Compound 2 was isolated as a yellow powder with m.p. 120.5– 122.08C. The HR-ESI-MS of 2 exhibited a pseudomolecular ion at m/z 211.0970 [M þ H]þ, 11
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Figure 1. Structures of compounds 1 –3 and key HMBC correlations of 1 and 2.
Journal of Asian Natural Products Research O
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Figure 2. Proposed biosynthetic pathway of compounds 1 – 3.
examined. Unfortunately, none of them exhibited anti-bacterial activity at the dose of 500 mg/disc against S. aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa using the paper disc diffusion method. Furthermore, they were all inactive against six human tumor cell lines (HCT116, HepG2, BGC823, NCIH1650, A2780, and A375) in vitro by MTT method (IC50 . 10 mM). Further pharmacological studies of isolated compounds are currently underway.
3.
Experimental
3.1. General experimental procedures Melting points were determined on a XT Digital Melting-Point Apparatus with Microscope and uncorrected (Tsigtao Unicom, Linyi, China). UV spectra were obtained on a JASCO U-650 spectrophotometer (Jasco, Tokyo, Japan). 1H NMR (500 MHz), 13C NMR (125 MHz), NOE difference, DEPT, HMBC spectra were run on Mercury-500 (Varian, Palo Alto, CA, USA) with TMS (tetramethylsilane) as internal standard. HR-ESI-MS and ESI-
MS were performed on an Agilent 1100 LC/MSD Trap-SL mass spectrometer (Agilent, Santa Clara, CA, USA). Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and RP-18 (Merck, 40 –60 mm; Darmstadt, Germany) were used for column chromatography (CC) and silica gel GF-254 (Qingdao Marine Chemical Factory, Qingdao, China) was used for thin-layer chromatography. Medium pressure liquid chromatography (MPLC) was employed on LC2000 MPLC equipment (Separation technology, Beijing, China). Semi-preparative high performance liquid chromatography (HPLC) was carried out on a preparation Agilent ODS Eclipse XDB C-18 column (10 mm, 250 mm £ 20 mm, Agilent, USA) with Shimadzu pump LC-6AD and UV detector SPD-20A (set at 230 nm, Shimadzu, Japan) at a flow rate of 4.0 ml/min. 3.2.
The fungal strain
The fungal strain HLS206 was isolated from the marine sponge Gelliodas carnosa, which was collected from the South China Sea and identified by Professor
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Jin-He Li at Institute of Oceanology, Chinese Academy of Science, Qingdao. This strain belongs to the order of Hypocreales, with 88% sequence identity to Nectria coryli (HM534895.1) based on phylogenetic analysis of the rDNA ITS sequence [5]. This fungus has been deposited at our laboratory in the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. 3.3. Fermentation, extraction, and isolation The fungal strain was first cultivated on the YPD agar plates (yeast extract peptone dextrose medium, 1% yeast extract, 2% peptone, 2% glucose, 2% agar) at 258C for 3 d, then mycelium was inoculated into the seed medium (0.4% glucose, 1.0% malt extract, 0.4% yeast extract; 50 ml of the medium in 250 ml Erlenmeyer flask) and cultivated on a rotary shaker at 170 r/min, 258C for 3 d. The seed culture (10 ml) was transferred into the solid rice medium (1 g rice/ml distilled H2O, 100 g rice in 500 ml flask; the rice was soaked in the water overnight before autoclaving at 1218C for 30 min) and statically cultivated at 258C for 45 d. The solid cultures (5 kg) were extracted repeatedly with EtOAc and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (28.2 g). The EtOAc extract was subjected to ODS-C18 CC (30 mm £ 350 mm) eluted with MeOH – H2O (20:80 –30:70 – 40:60 – 50:50 – 60:40 –70:30 – 80:20 – 100:0, v/v). The eluted fractions were combined to eight fractions (Fr.1 –Fr.8) on basis of HPLC behavior. Fr.1 (2.5 g) was separated by Sephadex LH-20 CC using MeOH as eluent to give 15 fractions (Fr.1.1 – Fr.1.15), Fr.1.6 (1.0 g) purified first by preparative HPLC (MeOH:H2O ¼ 38:62) and again by preparative HPLC (CH3CN: H2O ¼ 18:72) to afford 1 (20.5 mg, flow rate 4.0 ml/min, tR ¼ 23.2 min) and 2
(25.5 mg, flow rate 4.0 ml/min, tR ¼ 25.2 min). Fr.3 (1.5 g) was separated by MPLC (MeOH:H2O ¼ 65:35) to yield four fractions (Fr.3.1 – Fr.3.4). Fr.3.4 (860 mg) was purified by Sephadex LH20 CC to yield 3 (85.0 mg).
3.3.1.
Nectriapyrone A (1)
White powder; mp 115.2– 116.38C. UV (MeOH) lmax (nm): 235, 331; IR (KBr) n max: 3200, 2960, 1703, 1619, 1455, 1356, 800 and 703 cm21; 1H and 13C NMR spectral data see Table 1; HR-ESI-MS m/z: 211.0967 [M þ H]þ(calcd for C11H15O4, 211.0965).
3.3.2. Nectriapyrone B (2) Yellow powder; mp 120.5 –122.08C. UV (MeOH) lmax (nm): 235, 330; IR (KBr) nmax: 3230, 2952, 1718, 1658, 1462, 1387, 822 and 756 cm21; 1H and 13C NMR spectral data see Table 1; HR-ESI-MS m/z: 211.0970 [M þ H]þ(calcd for C11H15O4, 211.0965).
3.4. Biological assays Anti-bacterial and anti-tumor bioassays were undertaken with compounds of purity . 90% by HPLC.
3.4.1.
Anti-bacterial bioassays
The anti-bacterial assays were performed as previously described [4]. The tested compounds dissolved in methanol at a concentration of 5 mg/ml were screened for anti-bacterial activities against S. aureus 29213, B. subtilis 63501, E. coli 25922, and P. aeruginosa 27853. The paper disc (6 mm diam.) was dropped the solution of tested compound and then dried in the air (100 ml). Ampicillin (Sigma, SantaClara; CA; USA; 98% pure) was included as a positive control.
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3.4.2. Anti-tumor bioassays The MTT assay was used for the antitumor bioassay in vitro in human cancer cell lines HCT116 (colon cancer), HepG2 (liver cancer), BGC823 (liver cancer), NCI-H1650 (nonsmall-cell lung cancer), A2780 (ovarian cancer), and A375 (lung cancer) as reported [10], and the doseresponse curves were fitted with Sigma plot. IC50 s were determined. Taxol (Sigma; 98% pure) was included as a positive control. Acknowledgments We acknowledge the Research Team of Professor Xiao-guang Chen, Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College for cytotoxicity bioassays. We are grateful to the Department of Instrumental Analysis of our Institute for measurement of the IR, NMR, and MS data.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was financially supported by grant from the Mega-project for Inovative Drugs [grant number 2012ZX09301002-001-005] and National Natural Science Foundation of China [grant number 81402846].
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