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Two new compounds from the plant endophytic fungus Pestalotiopsis versicolor a
a
a
b
Xiao-Long Yang , Le Huang , Hai-Ying Li , Deng-Feng Yang & Zhuang-Zhuang Li
ac
a
College of Pharmaceutical Science, Hebei University, Baoding 071002, China b
National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China c
Department of Pharmacy, Cangzhou Central Hospital, Cangzhou 061001, China Published online: 07 Oct 2014.
To cite this article: Xiao-Long Yang, Le Huang, Hai-Ying Li, Deng-Feng Yang & Zhuang-Zhuang Li (2014): Two new compounds from the plant endophytic fungus Pestalotiopsis versicolor, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.961918 To link to this article: http://dx.doi.org/10.1080/10286020.2014.961918
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Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.961918
Two new compounds from the plant endophytic fungus Pestalotiopsis versicolor Xiao-Long Yanga*, Le Huanga, Hai-Ying Lia, Deng-Feng Yangb and Zhuang-Zhuang Lia,c
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a
College of Pharmaceutical Science, Hebei University, Baoding 071002, China; National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China; cDepartment of Pharmacy, Cangzhou Central Hospital, Cangzhou 061001, China
b
(Received 11 May 2014; final version received 1 September 2014) A new coumarin, 4,6-dihydroxy-7-formyl-3-methylcoumarin (1), and an a-pyrone derivative, 6-[(7S,8R)-8-propyloxiran-1-yl]-4-methoxy-pyran-2-one (2), together with four known a-pyrone derivatives (3 – 6), were isolated from the broth extract of the plant endophytic fungus Pestalotiopsis versicolor. Their structures were elucidated by extensive spectroscopic analysis and by comparison of the chemical shift values with those of related known compounds. Keywords: endophytic fungi; Pestalotiopsis versicolor; coumarin; a-pyrone
1.
Introduction
Fungi that inhabit the normal tissues of host plants without causing apparent pathogenic symptoms are defined as plant endophytic fungi [1], which have been proved to produce a variety of structurally complex secondary metabolites with significant activities [2], as exemplified by antitumor drug taxol produced by Taxomyces andreanae as the first reported endophytic fungus colonizing the inner bark of Pacific yew Taxus brevifolia [3]. Pestalotiopsis genus, as one class of the most widely distributed endophytic fungi, has gained considerable attention in the past several decades, due to the discovery of the anticancer agent taxol from an endophytic fungal strain Pestalotiopsis microspora [4], and the occurrence of numerous compounds with unique structures [5 –7]. Our prior chemical studies of this genus have led to the isolation of a variety of secondary metabolites [8 – 10], suggesting that Pestalotiopsis genus has high biogenetic potential to produce more
structural and biological diverse secondary metabolites. In the course of our search for new bioactive metabolites from this genus, a strain of P. versicolor was subjected to chemical study which led to the isolation of two new compounds (1 and 2) along with four known compounds (3 – 6). Herein, details of the isolation and structural elucidation of two new compounds (1 and 2) are presented. 2.
Compound 1 was assigned the molecular formula of C11H8O5 (eight degrees of unsaturation) on the basis of positive HRTOF-MS analysis at m/z 221.0447 [M þ H] þ. The 1H NMR spectrum (Table 1) showed the presence of two aromatic sharp singlets at dH 6.69 (1H, s) and 7.01 (1H, s), one formyl group at dH 10.33 (1H, s), and one methyl group at dH 2.00 (3H, s). The presence of two sharp singlet aromatic protons was indicative of a 1,2,4,5-tetrasubstituted phenyl ring. While the irradiation of the proton at dH
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Results and discussion
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X.-L. Yang et al.
Table 1. J in Hz).
1
H (500 MHz) and 13C NMR (125 MHz) spectral data for 1 and 2 in DMSO-d6 (d in ppm, 1
No.
2
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dH 2 3 4 5 6 7 8 9 10 11 12
dC 163.6 114.6 167.7 101.4 162.5 114.6 111.0 168.6 125.6 8.2 196.3
6.69 (s) 7.01 (s) 2.00 (s) 10.33 (s)
dH (s) (s) (s) (d) (s) (s) (d) (s) (s) (q) (d)
6.09 (d, J ¼ 2.5 Hz) 3.38 (d, J ¼ 2.3 Hz) 3.18 – 3.20 (m) 1.64 – 1.66 (m) 1.25 – 1.27 (m) 0.84 (t, J ¼ 7.0 Hz) 3.77 (s)
6
O
4
10
11 10
7
OHC
9
8
O
2
O
3
6
9
O
7
1
OH
O
12 4
11
1
8
O 2
1O
3 12
(s) (d) (s) (d) (s) (d) (d) (t) (t) (q) (q)
Pestalotiopsis acaciae except for the presence of formyl group and the absence of hydroxymethyl group in 1 [11], and this suggested that a hydroxymethyl group is replaced by a formyl group supported by the following key HMBC correlations (Figure 2): from H-5 to C-4, C-7 and C9, from H-8 to C-7 and C-12, from H-12 to C-6 and C-8, from H-11 to C-2, C-3 and C4. From the above data, compound 1 was elucidated as 4,6-dihydroxy-7-formyl-3methylcoumarin (Figure 1). Compound 2 was isolated as an optically active white powder with ½a21:8 D 2 98.7 (c 0.6, MeOH). The molecular
OH 5
163.9 88.0 171.5 99.6 167.2 54.7 61.5 33.9 19.2 14.5 56.8
5.49 (d, J ¼ 2.5 Hz)
7.01 (1H, s) displayed a distinct NOE correlation with one formyl proton at d 10.33 (1H, s), which further confirmed the presence of a 1,2,4,5-tetrasubstituted phenyl ring. The interpretation of its 13C NMR and HSQC spectroscopic data displayed 11 carbon signals accounting for 1 ester carbonyl carbon, 1 formyl group, 1 1,2,4,5-tetrasubstituted phenyl ring, 1 tetrasubstituted double bond, and 1 methyl group (Table 1). Detailed analysis of its NMR data exhibited similarities to the known compound 4,6-dihydroxy-7-hydroxymethyl-3-methylcoumarin previously isolated from another endophytic fungi
HO
dC
5
OCH3
OCH3 OH
2
3
O O
O
O O O
OCH3
O
O
O
O OCH3
OH
OCH3
OH
HO 4
Figure 1. The structures of compounds 1 – 6.
5
6
Journal of Asian Natural Products Research
3
O
OH HO
O
OHC
O
OCH3
O O
1
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Figure 2.
1
2
H, 1H COSY and selected key HMBC correlations of compounds 1 and 2.
formula of 2 was established as C11H14O4 on the basis of positive HR-TOF-MS analysis at m/z 211.0973 [M þ H]þ. In 1H NMR spectrum (Table 1), the signals at dH 5.49 (1H, d, J ¼ 2.5 Hz) and 6.09 (1H, d, J ¼ 2.5 Hz) due to olefinic protons, and at dH 3.77 (3H, s) due to ZOCH3, along with IR absorption bands at 1738, 1654, 1576, 1235 cm21 and a UV maximum at 282 nm, revealed the presence of a 4-methoxy-6substituted a-pyrone moiety in 2 [12]. The cross-peaks between H-7 and H-8/H-9/H10/H-11 were observed in the 1H, 1H COSY spectrum (Figure 2), which allowed us to establish one spin system from C-7 to C-11. Careful analysis of its NMR data revealed features very closely resembled those of a coexisting known compound 3 except for 18 mass less than 3 [13], indicating that compound 3 lost water molecule to form epoxide 2. Finally, the planar structure of 2 was constructed as shown in Figure 1. Comparing the structure of 2 with that of one known compound, 6-[20 -propyloxiran-1-yl]-2Hpyran-2-one isolated from Trichoderma viride UCA 06 [14], we found that both compounds have the same 2-propyloxiran1-yl moiety attached to C-6 position. Significantly, the absolute configuration of 2-propyloxiran-1-yl moiety in 6-[20 propyloxiran-1-yl]-2H-pyran-2-one has been determined to be 1S, 2R through chemical synthesis of its stereoisomers. Thus, the absolute configuration of 2 was finally determined to be 7S and 8R by comparing its optical rotation (½a21:8 D 2 98.78 (c 0.6, MeOH)) and NMR data with those of 6-[(10 S, 20 R)-20 -propyloxiran-
1-yl]-2H-pyran-2-one (½a25 D 2 908 (c 0.45, CHCI3)), which is identical with that of coisolated known compound 3 in which its absolute configuration was also determined to be 7S and 8R by chemical synthesis [13]. Compounds 3 –6 were identified as LL-P880g (3) [13], scirpyrone A (4) [15], scirpyrone B (5) [15], and 6-pentyl-4methoxy-pyran-2-one (6) [16] by comparison of their NMR and MS data with those reported. Compounds 1 – 6 were tested for antifungal activities against three fungal strains including Fusarium solani, Ustilago maydis, and Candida albicans. However, all compounds were proved to be devoid of significant antifungal activity in the bioassays used.
3. Experimental 3.1 General experimental procedures Optical rotation spectra were recorded on JASCO P-1020 spectropolarimeter (Shimadzu, Tokyo, Japan). IR spectra were measured on a Perkin-Elmer 577 spectrometer (Perkin-Elmer, Waltham, MA, USA). UV spectra were measured on Shimadzu UV-2401PC spectrometer (Shimadzu, Kyoto, Japan), lmax (log 1) in nm. 1D and 2D NMR spectra were recorded on a Bruker AM-600 spectrometer (Bruker, Faellanden, Switzerland) with TMS as an internal standard, d in ppm, J in Hz. The HR-TOF-MS data were measured on a Bruker apex-ultra 7.0 T spectrometer (Bruker).
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3.2 Fungal material and cultivation conditions Pestalotiopsis versicolor NBRC 6319 was obtained from NITE Biological Resource Center. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 288C for 7 days and then inoculated into 200 ml of potato dextrose broth (PDB) in 500 ml Erlenmeyer flasks under shaking conditions at 308C for 14 days. 3.3
Extraction and isolation
The culture broths (4.0 l) were extracted three times with ethyl acetate. After evaporation in vacuo, the crude extract (1.3 g) was subjected to ODS column chromatography (Cosmosil 75C18-PREP, Nacalai Tesque, Kyoto, Japan) eluted with a stepwise gradient system from water to methanol (0%, 25%, 50%, 75%, and 100% methanol (v/v)) to afford five fractions 1 – 5. The major peaks of fractions 1 and 2 are similar, and thus we combined them and further separated by preparative HPLC (Shimadzu, LC-20AD and SPD-20A Prominence Diode Array Detector) using Cosmosil 5C18-PAQ Waters (10 mm £ 250 mm, Nacalai Tesque) at a flow rate of 2.5 ml/min with a mixture of H2O and CH3CN, both containing 0.05% trifluoroacetic acid (TFA). Separation was performed with a linear gradient from solvent B/solvent A of 10:90 to 100:0 for 30 min, 100:0 for an additional 20 min, 100:0 to 10:90 for 5 min, 10:90 for an additional 5 min, which resulted in the isolation of 1 (1.5 mg, tR 22.3 min), 2 (2.0 mg, tR 16.1 min), 3 (6.8 mg, tR 15.5 min), 4 (37.0 mg, tR 24.2 min), 5 (7.0 mg, tR 21.3 min), and 6 (2.0 mg, tR 23.1 min). 3.3.1 4,6-Dihydroxy-7-formyl-3methylcoumarin (1) White powder; UV (MeOH) lmax (log 1): 354 (3.78), 281 (4.18), 254 (3.17) nm. IR (KBr) vmax: 3389, 1743, 1726, 1651, 1546,
1215 cm21. 1H and 13C NMR spectral data, see Table 1. HR-TOF-MS (positive): m/z 221.0447 [M þ H] þ (calcd for C11H9O5, 221.0444).
3.3.2 6-[(7S,8R)-8-propyloxiran-1-yl]4-methoxy-pyran-2-one (2) White powder, ½a21:8 2 98.7 (c 0.6, D MeOH). UV (MeOH) lmax (log 1): 226 (3.58), 282 (3.97) nm. IR (KBr) vmax: 3084, 2973, 1738, 1654, 1576, 1235 cm21. 1 H and 13C NMR spectral data, see Table 1. HR-TOF-MS (positive): m/z 211.0973 [M þ H] þ (calcd for C11 H15O 4, 211.0965).
3.4
Antifungal assays
Antifungal bioassays were conducted as described previously in the literature [17]. The fungal strains, F. solani, U. maydis, and C. albicans, were obtained from NITE Biological Resource Center. The strains were grown on PDA. Targeted microbes (3 – 4 colonies) were prepared from broth culture (288C for 72 h), and the final spore suspensions of fungal (in PDB) were 104 mycelial fragments/ml. Test samples (1 mg/ml as stock solution in DMSO and serial dilutions) were transferred to 96well clear plate in triplicate, and the suspension of the test organisms was added to each well, achieving a final volume of 120 ml (ketoconazole was used as the positive control). After incubation, the minimum inhibitory concentration was defined as the lowest test concentration that completely inhibited the growth of the test organisms.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21202033), the Natural Science Foundation of Hebei Province (C2012201047) and Foundation of Hebei University (179).
Journal of Asian Natural Products Research
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References [1] W.S. Borges, K.B. Borges, P.S. Bonato, S. Said, and M.T. Pupo, Curr. Org. Chem. 13, 1137 (2009). [2] H.W. Zhang, Y.C. Song, and R.X. Tan, Nat. Prod. Rep. 23, 753 (2003). [3] A. Stierle, G. Strobel, and D. Stierle, Science 260, 214 (1993). [4] G. Strobel, X. Yang, J. Sears, R. Kramer, R.S. Sidhu, and W.M. Hess, Microbiology 142, 435 (1996). [5] X.L. Yang, J.Z. Zhang, and D.Q. Luo, Nat. Prod. Rep. 29, 622 (2012). [6] L. Liu, T. Bruhn, L.D. Guo, D.C.G. R. Gotz, A. Brun, Y. Stich, S. Che, and G. Bringmann, Chem. Eur. J. 17, 2604 (2011). [7] L. Liu, S.B. Niu, X.H. Lu, X.L. Chen, H. Zhang, L.D. Guo, and Y.S. Che, Chem. Commun. 46, 460 (2010). [8] X.L. Yang, S. Zhang, H.J. Zhu, and D. Q. Luo, Molecules 16, 1910 (2011). [9] X.L. Yang, S. Zhang, Q.B. Hu, D.Q. Luo, and Y. Zhang, J. Antibiot. 64, 723 (2011).
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[10] X.L. Yang, S. Zhang, H.J. Zhu, D.Q. Luo, and Y. Gao, Helv. Chim. Acta 94, 1463 (2011). [11] X.L. Yang, T. Awakawa, T. Wakimoto, and I. Abe, Tetrahedron Lett. 54, 5814 (2013). [12] W.J. McGahren, G.A. Ellestad, G.O. M, P. Kunstmann, and P. Mullen, J. Org. Chem. 38, 3542 (1973). [13] Y. Kimura, T. Hamasaki, and H. Nakajima, Agric. Biol. Chem. 50, 1649 (1986). [14] M. Daoubi, C. Pinedo-Rivilla, M. B. Rubio, R. Hermosa, E. Monte, J. Aleu, and I.G. Collado, Tetrahedron 65, 4834 (2009). [15] J. Li, X.F. Wu, G. Ding, Y. Feng, X. J. Jiang, L.D. Guo, and Y.S. Che, Eur. J. Org. Chem. 12, 2445 (2012). [16] A. Evidente, M.C. Zonno, A. Andolfi, C. Troise, A. Cimmino, and M. Vurro, J. Antibiot. 65, 203 (2012). [17] D.T. Wicklow, B.K. Joshi, W.R. Gamble, and J.B. Gloer, Appl. Environ. Microbiol. 64, 4482 (1998).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Two new compounds from the plant endophytic fungus Pestalotiopsis versicolor a
a
a
b
Xiao-Long Yang , Le Huang , Hai-Ying Li , Deng-Feng Yang & Zhuang-Zhuang Li
ac
a
College of Pharmaceutical Science, Hebei University, Baoding 071002, China b
National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China c
Department of Pharmacy, Cangzhou Central Hospital, Cangzhou 061001, China Published online: 07 Oct 2014.
To cite this article: Xiao-Long Yang, Le Huang, Hai-Ying Li, Deng-Feng Yang & Zhuang-Zhuang Li (2014): Two new compounds from the plant endophytic fungus Pestalotiopsis versicolor, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.961918 To link to this article: http://dx.doi.org/10.1080/10286020.2014.961918
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, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.961918
Two new compounds from the plant endophytic fungus Pestalotiopsis versicolor Xiao-Long Yanga*, Le Huanga, Hai-Ying Lia, Deng-Feng Yangb and Zhuang-Zhuang Lia,c
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a
College of Pharmaceutical Science, Hebei University, Baoding 071002, China; National Engineering Research Center for Non-Food Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China; cDepartment of Pharmacy, Cangzhou Central Hospital, Cangzhou 061001, China
b
(Received 11 May 2014; final version received 1 September 2014) A new coumarin, 4,6-dihydroxy-7-formyl-3-methylcoumarin (1), and an a-pyrone derivative, 6-[(7S,8R)-8-propyloxiran-1-yl]-4-methoxy-pyran-2-one (2), together with four known a-pyrone derivatives (3 – 6), were isolated from the broth extract of the plant endophytic fungus Pestalotiopsis versicolor. Their structures were elucidated by extensive spectroscopic analysis and by comparison of the chemical shift values with those of related known compounds. Keywords: endophytic fungi; Pestalotiopsis versicolor; coumarin; a-pyrone
1.
Introduction
Fungi that inhabit the normal tissues of host plants without causing apparent pathogenic symptoms are defined as plant endophytic fungi [1], which have been proved to produce a variety of structurally complex secondary metabolites with significant activities [2], as exemplified by antitumor drug taxol produced by Taxomyces andreanae as the first reported endophytic fungus colonizing the inner bark of Pacific yew Taxus brevifolia [3]. Pestalotiopsis genus, as one class of the most widely distributed endophytic fungi, has gained considerable attention in the past several decades, due to the discovery of the anticancer agent taxol from an endophytic fungal strain Pestalotiopsis microspora [4], and the occurrence of numerous compounds with unique structures [5 –7]. Our prior chemical studies of this genus have led to the isolation of a variety of secondary metabolites [8 – 10], suggesting that Pestalotiopsis genus has high biogenetic potential to produce more
structural and biological diverse secondary metabolites. In the course of our search for new bioactive metabolites from this genus, a strain of P. versicolor was subjected to chemical study which led to the isolation of two new compounds (1 and 2) along with four known compounds (3 – 6). Herein, details of the isolation and structural elucidation of two new compounds (1 and 2) are presented. 2.
Compound 1 was assigned the molecular formula of C11H8O5 (eight degrees of unsaturation) on the basis of positive HRTOF-MS analysis at m/z 221.0447 [M þ H] þ. The 1H NMR spectrum (Table 1) showed the presence of two aromatic sharp singlets at dH 6.69 (1H, s) and 7.01 (1H, s), one formyl group at dH 10.33 (1H, s), and one methyl group at dH 2.00 (3H, s). The presence of two sharp singlet aromatic protons was indicative of a 1,2,4,5-tetrasubstituted phenyl ring. While the irradiation of the proton at dH
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Results and discussion
2
X.-L. Yang et al.
Table 1. J in Hz).
1
H (500 MHz) and 13C NMR (125 MHz) spectral data for 1 and 2 in DMSO-d6 (d in ppm, 1
No.
2
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dH 2 3 4 5 6 7 8 9 10 11 12
dC 163.6 114.6 167.7 101.4 162.5 114.6 111.0 168.6 125.6 8.2 196.3
6.69 (s) 7.01 (s) 2.00 (s) 10.33 (s)
dH (s) (s) (s) (d) (s) (s) (d) (s) (s) (q) (d)
6.09 (d, J ¼ 2.5 Hz) 3.38 (d, J ¼ 2.3 Hz) 3.18 – 3.20 (m) 1.64 – 1.66 (m) 1.25 – 1.27 (m) 0.84 (t, J ¼ 7.0 Hz) 3.77 (s)
6
O
4
10
11 10
7
OHC
9
8
O
2
O
3
6
9
O
7
1
OH
O
12 4
11
1
8
O 2
1O
3 12
(s) (d) (s) (d) (s) (d) (d) (t) (t) (q) (q)
Pestalotiopsis acaciae except for the presence of formyl group and the absence of hydroxymethyl group in 1 [11], and this suggested that a hydroxymethyl group is replaced by a formyl group supported by the following key HMBC correlations (Figure 2): from H-5 to C-4, C-7 and C9, from H-8 to C-7 and C-12, from H-12 to C-6 and C-8, from H-11 to C-2, C-3 and C4. From the above data, compound 1 was elucidated as 4,6-dihydroxy-7-formyl-3methylcoumarin (Figure 1). Compound 2 was isolated as an optically active white powder with ½a21:8 D 2 98.7 (c 0.6, MeOH). The molecular
OH 5
163.9 88.0 171.5 99.6 167.2 54.7 61.5 33.9 19.2 14.5 56.8
5.49 (d, J ¼ 2.5 Hz)
7.01 (1H, s) displayed a distinct NOE correlation with one formyl proton at d 10.33 (1H, s), which further confirmed the presence of a 1,2,4,5-tetrasubstituted phenyl ring. The interpretation of its 13C NMR and HSQC spectroscopic data displayed 11 carbon signals accounting for 1 ester carbonyl carbon, 1 formyl group, 1 1,2,4,5-tetrasubstituted phenyl ring, 1 tetrasubstituted double bond, and 1 methyl group (Table 1). Detailed analysis of its NMR data exhibited similarities to the known compound 4,6-dihydroxy-7-hydroxymethyl-3-methylcoumarin previously isolated from another endophytic fungi
HO
dC
5
OCH3
OCH3 OH
2
3
O O
O
O O O
OCH3
O
O
O
O OCH3
OH
OCH3
OH
HO 4
Figure 1. The structures of compounds 1 – 6.
5
6
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3
O
OH HO
O
OHC
O
OCH3
O O
1
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Figure 2.
1
2
H, 1H COSY and selected key HMBC correlations of compounds 1 and 2.
formula of 2 was established as C11H14O4 on the basis of positive HR-TOF-MS analysis at m/z 211.0973 [M þ H]þ. In 1H NMR spectrum (Table 1), the signals at dH 5.49 (1H, d, J ¼ 2.5 Hz) and 6.09 (1H, d, J ¼ 2.5 Hz) due to olefinic protons, and at dH 3.77 (3H, s) due to ZOCH3, along with IR absorption bands at 1738, 1654, 1576, 1235 cm21 and a UV maximum at 282 nm, revealed the presence of a 4-methoxy-6substituted a-pyrone moiety in 2 [12]. The cross-peaks between H-7 and H-8/H-9/H10/H-11 were observed in the 1H, 1H COSY spectrum (Figure 2), which allowed us to establish one spin system from C-7 to C-11. Careful analysis of its NMR data revealed features very closely resembled those of a coexisting known compound 3 except for 18 mass less than 3 [13], indicating that compound 3 lost water molecule to form epoxide 2. Finally, the planar structure of 2 was constructed as shown in Figure 1. Comparing the structure of 2 with that of one known compound, 6-[20 -propyloxiran-1-yl]-2Hpyran-2-one isolated from Trichoderma viride UCA 06 [14], we found that both compounds have the same 2-propyloxiran1-yl moiety attached to C-6 position. Significantly, the absolute configuration of 2-propyloxiran-1-yl moiety in 6-[20 propyloxiran-1-yl]-2H-pyran-2-one has been determined to be 1S, 2R through chemical synthesis of its stereoisomers. Thus, the absolute configuration of 2 was finally determined to be 7S and 8R by comparing its optical rotation (½a21:8 D 2 98.78 (c 0.6, MeOH)) and NMR data with those of 6-[(10 S, 20 R)-20 -propyloxiran-
1-yl]-2H-pyran-2-one (½a25 D 2 908 (c 0.45, CHCI3)), which is identical with that of coisolated known compound 3 in which its absolute configuration was also determined to be 7S and 8R by chemical synthesis [13]. Compounds 3 –6 were identified as LL-P880g (3) [13], scirpyrone A (4) [15], scirpyrone B (5) [15], and 6-pentyl-4methoxy-pyran-2-one (6) [16] by comparison of their NMR and MS data with those reported. Compounds 1 – 6 were tested for antifungal activities against three fungal strains including Fusarium solani, Ustilago maydis, and Candida albicans. However, all compounds were proved to be devoid of significant antifungal activity in the bioassays used.
3. Experimental 3.1 General experimental procedures Optical rotation spectra were recorded on JASCO P-1020 spectropolarimeter (Shimadzu, Tokyo, Japan). IR spectra were measured on a Perkin-Elmer 577 spectrometer (Perkin-Elmer, Waltham, MA, USA). UV spectra were measured on Shimadzu UV-2401PC spectrometer (Shimadzu, Kyoto, Japan), lmax (log 1) in nm. 1D and 2D NMR spectra were recorded on a Bruker AM-600 spectrometer (Bruker, Faellanden, Switzerland) with TMS as an internal standard, d in ppm, J in Hz. The HR-TOF-MS data were measured on a Bruker apex-ultra 7.0 T spectrometer (Bruker).
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X.-L. Yang et al.
3.2 Fungal material and cultivation conditions Pestalotiopsis versicolor NBRC 6319 was obtained from NITE Biological Resource Center. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 288C for 7 days and then inoculated into 200 ml of potato dextrose broth (PDB) in 500 ml Erlenmeyer flasks under shaking conditions at 308C for 14 days. 3.3
Extraction and isolation
The culture broths (4.0 l) were extracted three times with ethyl acetate. After evaporation in vacuo, the crude extract (1.3 g) was subjected to ODS column chromatography (Cosmosil 75C18-PREP, Nacalai Tesque, Kyoto, Japan) eluted with a stepwise gradient system from water to methanol (0%, 25%, 50%, 75%, and 100% methanol (v/v)) to afford five fractions 1 – 5. The major peaks of fractions 1 and 2 are similar, and thus we combined them and further separated by preparative HPLC (Shimadzu, LC-20AD and SPD-20A Prominence Diode Array Detector) using Cosmosil 5C18-PAQ Waters (10 mm £ 250 mm, Nacalai Tesque) at a flow rate of 2.5 ml/min with a mixture of H2O and CH3CN, both containing 0.05% trifluoroacetic acid (TFA). Separation was performed with a linear gradient from solvent B/solvent A of 10:90 to 100:0 for 30 min, 100:0 for an additional 20 min, 100:0 to 10:90 for 5 min, 10:90 for an additional 5 min, which resulted in the isolation of 1 (1.5 mg, tR 22.3 min), 2 (2.0 mg, tR 16.1 min), 3 (6.8 mg, tR 15.5 min), 4 (37.0 mg, tR 24.2 min), 5 (7.0 mg, tR 21.3 min), and 6 (2.0 mg, tR 23.1 min). 3.3.1 4,6-Dihydroxy-7-formyl-3methylcoumarin (1) White powder; UV (MeOH) lmax (log 1): 354 (3.78), 281 (4.18), 254 (3.17) nm. IR (KBr) vmax: 3389, 1743, 1726, 1651, 1546,
1215 cm21. 1H and 13C NMR spectral data, see Table 1. HR-TOF-MS (positive): m/z 221.0447 [M þ H] þ (calcd for C11H9O5, 221.0444).
3.3.2 6-[(7S,8R)-8-propyloxiran-1-yl]4-methoxy-pyran-2-one (2) White powder, ½a21:8 2 98.7 (c 0.6, D MeOH). UV (MeOH) lmax (log 1): 226 (3.58), 282 (3.97) nm. IR (KBr) vmax: 3084, 2973, 1738, 1654, 1576, 1235 cm21. 1 H and 13C NMR spectral data, see Table 1. HR-TOF-MS (positive): m/z 211.0973 [M þ H] þ (calcd for C11 H15O 4, 211.0965).
3.4
Antifungal assays
Antifungal bioassays were conducted as described previously in the literature [17]. The fungal strains, F. solani, U. maydis, and C. albicans, were obtained from NITE Biological Resource Center. The strains were grown on PDA. Targeted microbes (3 – 4 colonies) were prepared from broth culture (288C for 72 h), and the final spore suspensions of fungal (in PDB) were 104 mycelial fragments/ml. Test samples (1 mg/ml as stock solution in DMSO and serial dilutions) were transferred to 96well clear plate in triplicate, and the suspension of the test organisms was added to each well, achieving a final volume of 120 ml (ketoconazole was used as the positive control). After incubation, the minimum inhibitory concentration was defined as the lowest test concentration that completely inhibited the growth of the test organisms.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21202033), the Natural Science Foundation of Hebei Province (C2012201047) and Foundation of Hebei University (179).
Journal of Asian Natural Products Research
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