Three new 2-pyranone derivatives, namely Norcardiatones A (1), B (2) and C (3), were isolated from the agar cultures of the strain Nocardiopsis sp. A00203, a mangrove endophytic actinomycete. Their structures were elucidated by spectroscopic and mass-spectrometric analyses, including 1D-, 2D-NMR and HR Q-TOF-MS. Compound 1 showed week cytotoxicity against HeLa cells in MTT assay.National Natural Science Fund for Distinguished Young Scholars [30325044]; Chinese Ministry of Education [306010]; China Ocean Resource RD Association [DYXM-115-02-2-13]; 973 Program [2010CB833802

Chunhua Lu

Lin, Cheng

Yuemao Shen

沈月毛

鲁春华

Records of Natural Products

National Natural Science Fund for Distinguished Young Scholars [30325044]; Chinese Ministry of Education [306010]; China Ocean Resource RD Association [DYXM-115-02-2-13]; 973 Program [2010CB833802]Three new 2-pyranone derivatives, namely Norcardiatones A (1), B (2) and C (3), were isolated from the agar cultures of the strain Nocardiopsis sp. A00203, a mangrove endophytic actinomycete. Their structures were elucidated by spectroscopic and mass-spectrometric analyses, including 1D-, 2D-NMR and HR Q-TOF-MS. Compound 1 showed week cytotoxicity against HeLa cells in MTT assay

Lin, C.

Lu, C. H.

Shen, Y. M.

Xiamen University Institutional Repository

Three New 2-pyranone Derivatives from Mangrove Endophytic Actinomycete Strain Nocardiopsis sp A00203

ORIGINAL ARTICLE     The article was published by Academy of Chemistry of Globe Publications www.acgpubs.org/RNP © Published 10/05/2010 EISSN:1307-6167     Rec. Nat. Prod. 4:4 (2010) 176-179   Three New 2-pyranone Derivatives from Mangrove Endophytic Actinomycete Strain Nocardiopsis sp. A00203  Cheng Lin, Chunhua Lu * and Yuemao Shen   Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering; Xiamen Engineering Research Center of Marine Microbial Drug Discovery; Fujian Engineering Laboratory of Pharmaceuticals; School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, P. R. China   (Received May 21, 2010; Revised August 25, 2010; Accepted August 31, 2010)  Abstract:  Three new 2-pyranone derivatives, namely Norcardiatones A (1), B (2) and C (3), were isolated from the agar cultures of the strain Nocardiopsis sp. A00203, a mangrove endophytic actinomycete. Their structures were elucidated by spectroscopic and mass-spectrometric analyses, including 1D-, 2D-NMR and HR Q-TOF-MS. Compound 1 showed week cytotoxicity against HeLa cells in MTT assay.  Keywords: 2-pyranone derivatives; Nocardiopsis sp. A00203; spectroscopic analyses.   1. Introduction Mangrove and semi-mangrove plants are mostly distributed in the tropical and subtropical coastal regions of the world. Because of their special ecosystems that straddle the land and the sea, mangrove plants are found to be a rich source of endophytic fungal species [1–2]. More than 200 species of endophytic fungi have been isolated and identified from mangrove plants, being the second largest community of marine fungi. Studies revealed that the endophytic fungi of mangrove can produce many kinds of metabolites with great potential for anti-microbial and anti-tumor medicinal use [3]. Several actinomycetes isolated from root soil of mangrove have been studied, and several antimicrobial and cytotoxic compounds were obtained [4,5]. However, the mangrove ecosystem is a largely unexplored source for actinomycetes with the potential to produce biologically active secondary metabolites. In this paper, three new 2-pyranone derivatives (1-3) were isolated and identified from the mangrove actinomycetes Nocardiopsis sp. A00203                                                   * Corresponding author: E-Mail: ahua0966@xmu.edu.cn; Phone:086-592-2184180 Fax:086-592-2181722  Lin et.al., Rec. Nat. Prod. (2010) 4:4 176-179   177 2. Materials and Methods  2.1. Microorganism Material   The strain A00203 was isolated from the leaves of Aegiceras corniculatum collected from Jimei, Fujian Province, China. Both a traditional morphological assessment and 16S rDNA sequencing (GenBank accession number: AJ539401.1) were performed to characterize it as Nocardiopsis sp., and named as A00203. A nucleotide-to-nucleotide BLAST query of the NCBI database yielded Nocardiopsis aegyptica strain DSM44442 as the closest match to the 16S rDNA of A00203 (98%).   2.2 Fermentation and Isolation   The fermentation of Nocardiopsis sp. A00203 was performed on YMG (5 L) and YEME (10 L) agar plates for 7 d at 28°C. The cultures of two different media were diced and extracted with EtOAc/MeOH/AcOH (80 : 15 : 5, v/v/v), respectively. The two crude extracts were partitioned between EtOAc and H2O (1 : 1) until the EtOAc layer was colorless. The combined organic layers were dried over sodium sulfate (anhydrous) and concentrated in vacuo to afford EtOAc extracts 1.38 g (YMG medium) and 2.10 g (YEME medium), respectively. The EtOAc extract (1.38 g) was subjected to MPLC (RP-18 (145 g), MeOH/H2O (1 : 1)) to afford Fr.1. Fr.1 (97 mg) was further subjected to Sephadex LH-20 column (in MeOH) to afford two fractions Fr.1.1 – Fr.1.2. Fr.1.2 (27 mg) was separated to Fr.1.2.1 and Fr.1.2.2 by MPLC (RP-18 (30 g), MeOH/H2O (9 : 11) ). Fr.1.2.2 was subjected to silica gel chromatography (petroleum ether/acetone 100:1 and 50:1) to yield 1 (2.0 mg). The EtOAc extract (2.10 g) was subjected to MPLC (RP-18 (130 g), MeOH/H2O (1 : 1)) to afford two fractions Fr.1 - Fr.2. Fr.2 (80 mg) was subjected to Sephadex LH-20 column (in MeOH) to afford Fr.2.1 (18 mg), which was further separated to Fr.2.1.1 and Fr.2.1.2 by MPLC (RP-18 (30 g), MeOH/H2O (1 : 1)). Fr.2.1.2 was subjected to silica gel chromatography (petroleum ether/acetone 100:1 and 80:1) to yield 2/3 (2.0 mg).     Figure 1. 1H-1H COSY correlations and the selected HMBC correlations of compound 1 and the structures of compounds 1-3.  3.  Results and Discussion   3.1. Structure elucidation  Compound 1 was isolated as yellow oil. The molecular formula was determined to be C12H16O3 with five degrees of unsaturation in the molecule by HR Q-TOF-MS (m/z 231.1001 [M + Na]+, calcd. 231.0997) and NMR data (Tables 1 and 2). The IR spectra showed the absorptions for OH groups (3285 cm-1), and conjugated carbonyl (1726 cm-1 and 1691 cm-1) groups. The 13C NMR spectrum of 1 displayed twelve signals corresponding to four methyls, three methylenes (one being oxygenated), and New 2-pyranone derivatives  178 five quaternary carbon atoms, including a carbonyl carbon at δ 163.3 (C(2)). The HMBC correlations from H-C(3a) to C(2), C(3) and C(4), from H-C(5a) to C(4), C(5) and C(6), from H-C(1’a) to C(1’), C(2’) and C(6), along with 1H-1H COSY correlations H-C(2’)↔H-C(3’)↔H-C(4’) established the crude structure of  1 (Figure 1). One carbonyl and three double-bond groups can explain four degrees of unsaturation, and the remaining one  indicated the linkage of C(2) and C(6) via oxygen due to the chemical shift of C(6) (δ(C) 157.3), which was relatively upfield shifted compared to regular COOH groups. It was consistent with the skeleton of pyranone [6, 7]. The configuration of C=C between C(1’) and C(2’) was determined to be (E) by the NOE correlations H-C(3’)↔H-C(1’a)↔H-C(2’). Therefore, compound 1 was determined to be 6-(4-hydroxypent-2E-en-2-yl)-3, 5-dimethyl-2H-pyran-2-one, namely norcardiatone A (Figure 1). Compounds 2/3 were isolated as yellow oil. The IR absorptions indicated the presence of OH groups (3353 cm-1), carbonyl (1667 cm-1) and unsaturated carbon/carbon double bonds (1633 and 1596 cm-1). Analysis of the 13C NMR spectrum of 2, twenty four carbon signals for six methyls (at δ  18.9, 16.5, 16.4, 14.5, 13.9, 13.5), five methylenes (at δ 60.3, 59.7, 36.7, 21.7, 20.8), one methane (at δ 34.4), four carbonyl or oxygenated double bond carbons (at δ 164.3, 163.8, 163.2, 161.1) and eight unsaturated carbons (at δ 141.9, 141.8, 138.7, 126.7, 122.8, 123.5, 114.0, 113.9) were observed (Tables 1 and 2). Careful inspection of the HMBC correlations from six methyls to corresponding carbons and 1H-1H COSY correlations, two independent structures were deduced to be 2 and 3 as shown (Figure 1). The HR Q-TOF-MS gave quasi molecular ion peaks at m/z 209.1180 [M + H]+ (calcd. 209.1178), 231.0994 [M + Na]+ (calcd. 231.0997)), 211.1330 [M + H]+ (calcd. 211.1334), and 233.1150 [M + Na]+ (calcd. 233.1154), which also indicated a mixture 2/3. The ratio of 2/3 was about 1 : 1 based on the integral of protons in 1H NMR. Compared NMR and MS data with those of 1, compounds 2 and 3 were determined to be 5-(hydroxymethyl)-3-methyl-6-(pent-2E-en-2-yl)-2H-pyran-2-one and 5-(hydroxymethyl)-3-methyl-6-(pentan-2-yl)-2H-pyran-2-one, namely norcardiatones B (2) and C (3).        Table 1. 1H NMR data for compounds 1-3 (at 600 MHz in CDCl3, δ in ppm, J in Hz).  Position (H) 1 2 3 3a 2.10 (3H, s) 2.12 (3H, s) 2.09 (3H, s) 4 7.04 (1H, s) 7.33 (1H, s) 7.20 (1H, s) 5a 2.06 (3H, s) 4.46 (2H, s) 4.42 (2H, s) 1’   2.89(1H, m) 1’a 1.97 (3H, s) 1.93 (3H, s) 1.24 (3H, d, J = 6.8) 2’ 5.69 (1H, d, J = 6.4) 5.71 (1H ,t , J = 7.3) 1.75 (1H, m)  1.53 (1H, m) 3’ 4.74 (1H, quint., J = 6.4) 2.22 (2H, m) 1.29 (1H, m) 1.22 (1H, m) 4’ 1.34 ( 3H, d, J = 6.4) 1.06 (3H, t, J = 7.5) 0.89(3H, t, J = 7.3)  3.2 Cytotoxicity and Antimicrobial activity  HeLa cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum and 2 mM L-glutamine. The cells were maintained at 37oC in a humidified atmosphere at 95% air and 5% CO2. Cell viability was measured by MTT assay[8]. Compound 1 showed no effect on the growth of Escherichia coli, Bacillus subtilis, Staphylococcus aureus and yeasts (Candida albicaus and Aspergillus niger) at the concentration of 50 µg/disc. Compound 1 exhibited week cytotoxicity against HeLa cells (10 µg/mL, 2.73% and 20 µg/mL, 7.39%) in MTT assay.     Lin et.al., Rec. Nat. Prod. (2010) 4:4 176-179   179                           Table 2. 13C NMR data for compounds 1-3 (at 150 MHz in CDCl3  δ in ppm). Position (C) 1 2 3 2 163.3 (C) 163.2 (C) 163.8 (C) 3 123.6 (C) 123.5 (C) 122.8 (C) 3a 16.3 (CH3) 16.5a (CH3) 16.4a (CH3) 4 144.5 (CH) 144.9b (CH) 144.8b (CH) 5 110.4 (C) 114.0c (C) 113.9c (C) 5a 16.6 (CH3) 60.3 (CH2) 59.7 (CH2) 6 157.3 (C) 161.1 (C) 164.3 (C) 1’ 128.4 (C) 126.7 (C) 34.4 (CH) 1’a 14.9 (CH3) 14.5 (CH3) 18.9 (CH3) 2’ 138.8 (CH) 138.7 (CH) 36.7 (CH2) 3’ 64.7 (CH) 21.7(CH2) 20.8(CH2) 4’ 23.1 (CH3) 13.5d (CH3) 13.9d (CH3) a, b, c, and d Interchangeable.  Acknowledgments This work was partially supported by the National Natural Science Fund for Distinguished Young Scholars to Y.S. (30325044), the Key Grant of Chinese Ministry of Education (306010), China Ocean Resource R&D Association (Grant DYXM-115-02-2-13) and 973 Program (2010CB833802).  Supporting Information  Supporting Information accompanies this paper on http://www.acgpubs.org/RNP   References [1] P. Lin (2001). A review on mangrove research in China. J. Xiamen Univer. (Nat. Sci) 40, 592–603. [2] J. Wu, Q. Xiao, J. Xu, M.-Y. Li, J.-Y. Pan and M.-H. Yang (2008). Natural products from true mangrove flora; source, chemistry and bioactivities, Nat Prod Rep. 25,955-981. [3] A.-R. Liu, X.-P. Wu and T. Xu (2007). Research advances in endophytic fungi of mangrove, Chin. J. Appl. Ecol.  18, 912–918. [4] K. Hong, A.-H. Gao, H. Gao, L. Zhang, H.-P. Lin, J. Li, X.-S. Yao, M. Goodfellow and J.-S. Ruan (2009). Actinomycetes for marine drug discovery isolated from mangrove soils and plants in China, Mar Drugs 7, 24–44. [5] L. Han, X.-S. Huang, I. Sattler, H.-Z. Fu, S. Grabley and W.-H. Lin (2007). Two new constituents from mangrove Bruguiera gymnorrhiza, J. Asian Nat. Prod. Res. 9, 327–331. [6] A. J. Cavelheiro and M. Yoshida (2000). 6-[ω-[arylaakenyl]-5,6-dihydro-α-pyrones from Cryptocarya moschata (Lauraceae), Phytochemistry 53, 811–819. [7] X. Yong, J.-F. Zhao, X.-D. Yang, R. Huang and L. Li (2005). Chemical Constituents of Litsea euosma, Acta Bot. Yunn. 27, 695-697. [8] T. Mosmann (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, Immunol. Methods 65, 55-63.  © 2010 Reproduction is free for scientific studies  

Three New 2-pyranone Derivatives from Mangrove Endophytic Actinomycete Strain Nocardiopsis sp. A00203

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Three New 2-pyranone Derivatives from Mangrove Endophytic Actinomycete Strain Nocardiopsis sp. A00203

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