Phytochemistry xxx (2014) xxx–xxx
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Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum Xiao-Yan Yang a,b, Tao Feng a, Gang-Qiang Wang c, Jian-Hai Ding a,b, Zheng-Hui Li a, Yan Li a, Shuang-Hui He d, Ji-Kai Liu a,⇑ a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China University of Chinese Academy of Sciences, Beijing 100049, China College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China d Institute of Microbiology, Beijing Forestry University, Beijing 100083, China b c
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 26 November 2013 Available online xxxx Keywords: Basidiomycete Trichaptum pargamenum Cadinane-type sesquiterpenes 13-Carbon c-lactones
a b s t r a c t Four cadinane-type sesquiterpenes and four 13-carbon c-lactones, together with three known compounds, were isolated from cultures of the basidiomycete Trichaptum pargamenum. Their structures were elucidated on the basis of extensive spectroscopic methods. The absolute configurations of two of the cadinene type sesquiterpenes 1 and 3 were confirmed by single crystal X-ray diffractions. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The genus Trichaptum belongs to order Hymenochaetales. Species of the genus causes a similar fragile, lacy white, pocket rot on both angiosperm and gymnosperm wood. Most species of Trichaptum occur in temperate and boreal forests, and 13 species in the genus were found in China (Dai et al., 2009a; Dai 2012). The mushroom T. pargamenum (Fr.) G. Cunn. (=T. biforme (Fr.) Ryvarden) has a wide distribution in China (Cui et al., 2008; Dai et al., 2009a), and has been used as a medicinal fungus for treating cancer, fungal, and bacterial diseases in areas of China (Dai et al., 2009b; Yang et al., 2005). The secondary metabolites produced by this fungus have not been reported previously. In the course of ongoing search for novel secondary metabolites, a chemical investigation of cultures of T. pargamenum, led to isolation of four new cadinane-type sesquiterpenes (Fig. 1), (+)-(1R,3R,6S,7S,11R)-3,12-dihydroxy-a-muurolene (1), (+)-(1R,3R, 6S,7S,11S)-3,12-dihydroxy-a-muurolene (2), 3a-hydroxyartemisinic acid (3), (+)-(1R,3R,6S,7S,8R,11S)-3,8,12-trihydroxy-a-muurolene (4), and four new 13-carbon c-lactones (Fig. 1), (6Z, 11S)-3,4-trans-11-oxo-3-methyldodec-cis-6-en-4-olide (5), (6Z,11S)-3,4-trans-11-hydroxy-3-methyldodec-cis-6-en-4-olide (6), (6Z,11S)-3,4-trans-9-hydroxy-3-methyldodec-cis-6-en-4-olide (7), ⇑ Corresponding author. Tel.: +86 871 5216327; fax: +86 871 5219934. E-mail address: [email protected] (J.-K. Liu).
(6Z,11S)-3,4-trans-9-oxo-3-methyldodec-cis-6-en-4-olide (8), along with three known compounds, (+)-3b-hydroxy-a-muurolene (9) (Affeld et al., 2009), ent-T-muurolol (10) (Nagashima et al., 1994), and 2-methyl-6-methylene-oct-7-en-2,3-diol (11) (Barrero et al., 1992; Bohlmann et al., 1983). The structures of new compounds were elucidated by means of spectroscopic methods, while the known compounds were identified by comparison with data reported in the literature. According to the medicinal use of this fungus, all isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines. 2. Results and discussion Compound 1 was obtained as colorless cubic crystals (MeOH). Its molecular formula C15H24O2 was determined by its HREIMS at m/z 236.1782 [M]+ (calcd. for 236.1776), corresponding to four degrees of unsaturation. The IR data at 3300 cm1 corresponded to hydroxy groups. The 13C NMR spectroscopic data suggested 15 carbon resonances, which were ascribed to three methyls, three methylenes (including one oxygenated), seven methines (including one oxygenated and two olefinic), and two sp2 quaternary carbons, respectively. In the 1H NMR spectrum (Table 1), signals at dH 1.80 and 1.69 (each 3H, s) were readily identified as methyl groups connected to double bonds. Meanwhile, the 1H–1H COSY spectrum (Fig. 2) had correlations of H-11 with H2-12 and H3-13. These data suggested that compound 1 should be a cadinane-type sesquiter-
http://dx.doi.org/10.1016/j.phytochem.2014.04.015 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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X.-Y. Yang et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 2. Key 2D NMR correlations of compounds 1 and 5.
Fig. 1. Structures of compounds 1–8.
pene, with similarities to (+)-3b-hydroxy-a-muurolene (Affeld et al., 2009). The key difference between them was that the methyl group at C-12 in (+)-3b-hydroxy-a-muurolene was replaced by a hydroxymethylene (dC 66.9, C-12) moiety in compound 1, as supported by the HMBC correlations (Fig. 2) from dH 3.39 (2H, m, H12) to dC 36.0 (d, C-11) and 10.7 (q, C-13). Detailed analysis of the 2D NMR spectroscopic data indicated that the other parts of 1 were the same to those of (+)-3b-hydroxy-a-muurolene (Affeld et al., 2009). Due to free rotation of bond of C-7/C-11, the stereochemistry of C-11 in 1 could not be established readily. Fortunately, a single crystal X-ray diffraction identified the absolute configuration of 1 as 1R,3R,6S,7S,11R (Fig. 3). In addition, comparison of the optical rotation data of 1 (+122.2) with those reported in the literature (Affeld et al., 2009; Ohloff and Pawlak, 1970) also supported the absolute structure of 1. Hence, structure 1 was deduced as (+)-(1R,3R,6S,7S,11R)-3,12-dihydroxy-a-muurolene. Compound 2 gave the same molecular formula C15H24O2 as that of 1 by HREIMS. Comparison of 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 2) with those of 1 suggested that compound 2 exhibited a similar structure to that of 1. Preliminary analysis of HSQC, 1H–1H COSY and HMBC data suggested that compound 2
Fig. 3. ORTEP drawing of crystal structure of 1.
had the same overall structure to that of 1; analysis of the ROESY correlations also suggested that the configurations at C-1, C-3, C6 and C-7 in 2 were in agreement with those of 1. However, signif-
Table 1 H NMR spectroscopic data for 1–4 (d in ppm; J in Hz).
1
a b c
No.
1a
1 2a 2b 3 5 6 7 8a 8b 9 11 12a 12b 13 14 15
2.31, 1.93, 1.61, 3.88, 5.68, 2.09, 1.79, 1.80,
2a m m m t (3.4) br d (4.2) m overlap 2H, overlap
5.41, br s 1.99, m 3.39, 2H, overlap 0.86, d (7.0) 1.80, s 1.69, s
2.24, 1.89, 1.69, 3.95, 5.67, 2.19, 1.54, 1.98, 1.79, 5.40, 1.94, 3.74, 3.46, 0.99, 1.81, 1.68,
3b m m m t (3.4) br d (3.3) m m m overlap br s m dd (10.5, 4.5) dd (10.5, 8.6) d (6.9) s s
2.31, 1.93, 1.71, 3.98, 5.56, 2.10, 2.06, 1.91, 1.85, 5.39, 2.77,
4c m m m t (3.4) br d (3.1) m m m m br s m
1.11, d (7.0) 1.82, s 1.70, s
2.23, 1.86, 1.52, 3.97, 5.83, 2.14, 2.04, 4.19,
m m m t (3.4) br d (4.2) m m br s
5.63, 2.58, 3.83, 3.52, 1.14, 1.83, 1.79,
br s m t (7.8) t (7.8) d (7.2) s s
Measured at 400 MHz. Measured at 500 MHz. Measured at 600 MHz.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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X.-Y. Yang et al. / Phytochemistry xxx (2014) xxx–xxx Table 2 C NMR spectroscopic data of 1–8 (d in ppm; J in Hz).
13
a b c
No.
1a
2a
3b
4c
5a
6a
7a
8a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
35.1, d 35.0, t 68.6, d 136.4, s 128.8, d 37.6, d 35.5, d 25.4, t 122.4, d 137.0, s 36.0, d 66.9, t 10.7, q 21.6, q 21.7, q
34.0, d 33.7, t 68.0, d 135.3, s 128.4, d 36.6, d 37.8, d 26.5, t 121.7, d 135.7, s 35.5, d 65.0, t 15.6, q 20.9, q 21.4, q
33.9, d 33.8, t 68.0, d 136.6, s 126.9, d 36.5, d 36.6, d 25.7, t 121.0, d 135.8, s 39.4, d 181.2, s 10.5, q 20.9, q 21.3, q
34.1, d 33.1, t 67.9, d 134.5, s 129.9, d 31.3, d 41.6, d 76.7, d 121.0, d 141.2, s 37.4, d 72.7, t 15.2, q 21.3, q 21.8, q
176.1, s 37.0, t 35.2, d 86.6, d 31.2, t 123.8, d 132.6, d 26.6, t 23.2, t 42.7, t 208.9, s 29.9, q 17.5, q
176.4, s 36.9, t 35.1, d 86.7, d 31.1, t 123.0, d 133.3, d 27.2, t 25.4, t 38.7, t 67.7, d 23.4, q 17.5, q
176.2, s 37.0, t 35.2, d 86.5, d 31.3, t 126.0, d 129.3, d 35.4, t 71.0, d 39.1, t 18.9,t 14.0, q 17.5, q
175.7, s 36.9, t 35.2, d 86.2, d 31.5, t 126.6, d 124.9, d 41.5, t 208.3, s 44.5, t 17.2, t 13.7, q 17.5, q
Measured at 400 MHz. Measured at 500 MHz. Measured at 600 MHz.
Fig. 4. ORTEP drawing of crystal structure of 3.
icant downfield chemical shifts of the methyl group at C-13 (dH 0.99, dC 15.6) in 2 suggested an opposite absolute configuration at C-11 in compound 2. Hence, the structure of 2 was deduced as (+)-(1R,3R,6S,7S,11S)-3,12-dihydroxy-a-muurolene. Compound 3 possessed a molecular formula C15H22O3 as assigned by the HREIMS, indicating five degrees of unsaturation. The 13C NMR spectrum showed 15 carbon signals, which were assigned to one carboxyl, two double bands, one oxymethine, two methylenes, four sp3 methines, and three methyls by the HSQC spectrum (Table 2). These data indicated that compound 3 possessed similar patterns to that of 1, except that the hydroxymethylene group in 1 was replaced by a carboxyl group (dC 181.2) in 3, as supported by HMBC correlations from dH 2.77 (1H, m, H-11) and 1.11 (3H, d, H-13) to dC 181.2 (s, C-12). Detailed analysis of other 2D NMR data suggested that the other parts of 3 were the same to those of 1. In addition, a single crystal X-ray diffraction confirmed the structure as elucidated above, which also determined the absolute configuration of 3 to be 1R,3R,6S,7S,11R (Fig. 4). Therefore, the structure of 3 was determined as 3a-hydroxyartemisinic acid. Compound 4 had the molecular formula C15H24O3 indicated by the HREIMS. The 1H and 13C NMR spectra of 4 (Tables 1 and 2) were very similar to those of 2, suggesting similar overall structures. The only difference was one more hydroxy group at C-8 in 4, which resulted in downfield shifts of CH-8 (dH 4.19; dC 76.7), as deduced by HMBC correlations from dH 4.19 (1H, br s, H-8) to dC 121.0
(d, C-9), and dC 141.2 (s, C-10), as well as 1H–1H COSY correlation between dH 2.04 (1H, m, H-7) and H-8. The ROESY correlation of H-7/H-8 indicated that H-8 and H-7 were on the same side, which suggested that the absolute configurations of C-7 and C-8 should be S and R, respectively. Hence, structure 4 was deduced as (+)-(1R,3R,6S,7S,8R,11R)-3,8,12-trihydroxy-a-muurolene. Compound 5 was isolated as a colorless oil. Its molecular formula (C13H20O3) was established by HREIMS, indicating four degrees of unsaturation. The IR spectrum revealed a lactone carbonyl group (1777 cm1), a vinyl group (1629 cm1), and a ketone group (1710 cm1). The 13C NMR spectroscopic data showed 13 carbon signals (Table 2), which could be classified into for two methyls, five methylenes, four methines (one oxygenated and two olefinic), and two carbonyl carbons, respectively, by DEPT and HSQC spectra. Analyses of 1H–1H COSY correlations established a fragment as shown in Fig. 2. The HMBC correlations (Fig. 2) from dH 4.06 (1H, m, H-4) to dC 176.1 (s, C-1), 37.0 (t, C-2), and 17.5 (q, C-13) suggested that a five-membered lactone ring was formed according to C-4–O–C-1. Furthermore, the methyl signal at dH 2.14 (3H, s, H-12) and dH 2.45 (2H, t, J = 7.0 Hz, H-10) showed key HMBC correlations to dC 208.9 (s, C-11), which suggested a ketone group at C-11. The trans relationship of the two substitutes on the c-lactone ring was deduced by ROESY correlations (Fig. 2) of H-4 with H3-13 (Findlay et al., 2003). The double bond was assigned as having a cis geometry based on the ROESY correlation (Fig. 2) of H2-5 with H2-8. Therefore, compound 5 was established as (6Z,11S)-3,4-trans-11-oxo-3-methyldodeccis-6-en-4-olide. Compound 6, a colorless oil, had a molecular formula of C13H22O3, as established by HREIMS at m/z 226.1566 (calcd. for 226.1569). The IR spectrum showed absorptions for hydroxy (3441 cm1) and lactone carbonyl (1778 cm1) groups. Detailed comparison of the 13C NMR spectroscopic data (Table 2) of 6 with those of 5 showed that they were similar in structure. The key difference between them was that the carbonyl group in 5 was replaced by a hydroxy group (Tables 2 and 3, dH 3.76, dC 67.7) in 6, which was confirmed by the 1H–1H COSY correlations of dH 3.76 (H-11) with dH 1.15 (H3-12) and dH 1.42 (H2-10), and the HMBC correlations from H-12, H-10, and H-9 to dC 67.7 (C-11). The ROESY correlation of H-4/H-13 suggested a trans relationship between H-3 and H-4, while the cis geometry of the double bond between C-6 and C-7 was determined by the ROESY correlation of H2-5 with H2-8. The modified Mosher method was applied to determine the absolute configuration at C-11 in compound 6 (Dale and Mosher, 1973; Ohtani et al., 1991; Sullivan et al.,
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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X.-Y. Yang et al. / Phytochemistry xxx (2014) xxx–xxx
Table 3 H NMR (400 MHz) spectroscopic data of 5–8 (d in ppm; J in Hz).
1
No.
5
6
2 2b 3 4 5a 5b 6 7 8 9 10 11a 11b 12 13
2.68, dd (16.8, 7.6) 2.19, (overlap) 2.27, m 4.06, m 2.46 m 2.40 m 5.45, m 5.53, m 2.05, 2H, m 1.65, 2H, m 2.45, 2H, t (7.0)
2.65, 2.16, 2.25, 4.04, 2.40,
dd (16.8, 7.8) dd (16.8, 9.3) m m 2H, m
5.39, 5.54, 2.03, 1.41, 1.42, 3.76,
m m 2H, m 2H, overlap 2H, overlap m
2.14, s 1.14, d (6.4)
7 2.66, 2.17, 2.27, 4.07, 2.50, 2.42, 5.59, 5.62, 2.22, 3.65, 1.44, 1.45, 1.35, 0.92, 1.13,
1.15, d (6.2) 1.11, d (6.6)
8 dd (16.8, 7.7) (overlap) m m m m m m 2H, m m 2H, m m m t (6.8) d (6.6)
2.68, 2.20, 2.27, 4.07, 2.48, 2.41, 5.65, 5.77, 3.19,
dd (17.0, 7.8) (overlap) m m m m m m 2H, m
2.43, m 1.60, 2H, m 0.92, t (7.2) 1.14, d (6.6)
3. Conclusion
Fig. 5. Dd Values (dS dR) in ppm of the two MPTA esters derived from 6 and 7.
1973). From the values of Dd (dS dR) (Fig. 5), the absolute configuration at C-11 in 6 was determined as S. On the basis of these data, compound 6 was assigned as (6Z,11S)-3,4-trans-11-hydroxy-3methyldodec-cis-6-en-4-olide. The molecular formula of compound 7 was assigned as C13H22O3 by HREIMS at m/z 226.1576 (calcd. for 226.1569). Its IR spectrum showed the presence of hydroxy (3441 cm1) and lactone (1777 cm1) groups. Its 13C NMR spectroscopic data (Table 2) were similar to those of compound 6, which suggested that compound 7 possessed the same patterns to those of 6. Detailed analysis of the 2D NMR data suggested that the hydroxy group should be placed at C-9 in 7 rather than that at C-11 in 6, as supported by the HMBC correlations from dH 3.65 (1H, m, H-9) to dC 129.3 (d, C7) and dC 18.9 (t, C-11). The stereochemistry of the double bond and lactone ring substituents were resolved by the ROESY data as in case of 6. In addition, the absolute configuration of C-9 in 7 was determined as S by the modified Mosher method (Fig. 5). Therefore, compound 7 was elucidated as (6Z,11S)-3,4-trans9-hydroxy-3-methyldodec-cis-6-en-4-olide. Compound 8 possessed a molecular formula C13H20O3 as determined by the HREIMS. Its 13C NMR spectroscopic data (Table 2) were similar to those of compound 7, except for the hydroxy group in 7 being replaced by a carbonyl group (dC 208.3) in 8, as supported by HMBC correlations from dH 3.19 (2H, m, H-8) and dH 2.43 (2H, m, H-10) to dC 208.3 (s, C-9). The cis geometry of the double bond between C-6 and C-7 was determined by the ROESY correlation of H-5 with H-8, and the relative configuration at C-4 and C-3 were determined by the ROESY correlation of H-4 with Me-13. Hence, compound 8 was elucidated as (6Z,11S)-3,4-trans-9-oxo3-methyldodec-cis-6-en-4-olide. Since the fungus T. pargamenum is used for treating cancer in areas of China, all compounds were evaluated for their cytotoxicity against five human cancer cell lines, SK-BR-3 breast cancer, SMMC7721 hepatocellular carcinoma, HL-60 myeloid leukemia, PANC-1 pancreatic cancer, and A-549 lung cancer, using the MTT method as reported previously (Mosmann, 1983). Unfortunately, none of them had significant activity (IC50 > 10 lmol).
Four new cadinane-type sesquiterpenes (1–4) and four new 13-carbon c-lactones (5–8) were isolated from the culture of T. pargamenum. Compounds 1–4 represented a structure characteristic of hydroxymethylene or carboxyl groups substituted at C-11. The absolute stereochemistries of 1 and 3 were determined by X-ray crystal diffraction. The absolute stereochemistry of 6 and 7 were determined using a modified Mosher’s method.
4. Experimental 4.1. General experimental procedures Melting points were measured on X-4 microscopic melting point meter (Yuhua instrument company, Gongyi, China). Optical rotations were obtained using a Horiba SEPA-300 polarimeter, whereas UV spectra were acquired using a Shimadzu UV-2401A spectrometer. IR spectra were determined using a Bruker Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR experiments were performed on a Bruker AM-400, DRX-500 spectrometer with TMS as the internal standard. Chemical shifts (d) were expressed in ppm with reference to the solvent signals. Mass spectra (MS) were recorded on a VG Auto Spec-3000 or an APIQSTAR time-of-flight spectrometer. Silica gel (200–300 mesh; Qingdao Marine Chemical Ltd., China), MPLC was performed on a Büchi Sepacore system (Büchi Labortechnik AG, Switzerland), reversed-phase C18 (RP18) gel (40–75 lm, Fuji Silysia Chemical Ltd., Japan), and Sephadex LH-20 (Amersham Biosciences, Sweden). Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd. Qingdao), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH.
4.2. Fungal material and cultivation condition The fruiting bodies of fungus T. pargamenum was collected from the Shennongjia Forest Region in Hubei Province, China (latitude: 31°, 310 , 700 ; longitude: 110°, 190 , 4700 ), in October 2004, and authenticated by Prof. Dai Yucheng, Beijing Forestry University. A voucher isolate (No. HF20040812Y) was deposited in the Herbarium of Kunming Institute of Botany, CAS. Culture medium: glucose 5%, peptone 0.15%, yeast powder 0.5%, KH2PO4 0.05% and MgSO4 0.05%. Fermentation was carried out on a shaker at 24 °C and 150 r/min for 23 days.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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4.3. Extraction and isolation The whole culture broth of T. pargamenum (20 L) was initially filtered, and the filtrate was extracted three times with EtOAc. The organic layer was concentrated under reduced pressure to give an oily residue (8.2 g). The residue was subjected to column chromatography (CC) over silica gel (200–300 mesh) eluting with CHCl3/MeOH gradient (from 1:0 to 1:1) to afford fractions A–D. Fraction A was further separated by Sephadex LH-20 (CHCl3/ MeOH, 1:1) and silica gel CC (petroleum ether/Me2CO, 20:1), followed by MPLC (MeOH/H2O, eluting from 62:38 to 68:32 for 16 mins with a flow of 20 mL/min) to afford 9 (10.2 mg) and 10 (2.3 mg). Fraction B was separated by repeated reversed-phase C18 HPLC or (MeOH/H2O, from 3:7 to 5:5) to obtain four subfractions (Fr B1–B4). Fr B1 was further separated by silica gel (petroleum ether/EtOAc, 4:1) and Sephadex LH-20 (Me2CO) to give 1 (22.1 mg), 6 (156.4 mg), 8 (3.8 mg), and 11 (2.1 mg), respectively. Fr B2 was separated by silica gel CC (petroleum ether/EtOAc, 5:1) to yield 7 (83.9 mg). Fr B3 was further purified by repeated silica gel CC and Sephadex LH-20 (Me2CO) to give 2 (5.1 mg), 3 (1.9 mg), 4 (3.8 mg) and 5 (48.5 mg), respectively. 4.3.1. (+)-(1R,3R,6S,7S,11R)-3,12-Dihydroxy-a-muurolene (1) Colorless cubic crystals (MeOH); mp 189–191 °C; [a]26 D +122.2 (c 0.29, MeOH); IR (KBr) mmax 3300, 2956, 2920, 1449, 1435, 1383, 1345, 1042, 1028, 968, 893 cm1; for 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (positive) m/z 259 [M+Na]+; HREIMS (positive) m/z 236.1782 (calcd for C15H24O2, 236.1776). 4.3.2. (+)-(1R,3R,6S,7S,11S)-3,12-Dihydroxy-a-muurolene (2) Colorless crystals; [a]24 D +95.0 (c 0.28, MeOH); IR (KBr) mmax 3440, 2960, 2919, 1631, 1439, 1383, 1030, 972 cm1; for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (positive) m/z 259 [M+Na]+; HREIMS (positive) m/z 236.1776 (calcd for C15H24O2, 236.1776). 4.3.3. 3a-Hydroxyartemisinic acid (3) Colorless crystals; mp 170–172 °C; [a]25 D +56.0 (c 0.14, MeOH); IR (KBr) mmax 3440, 1688, 1629, 1384, 1102 cm1; for 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (negative) m/z 249 [MH]+; HREIMS (positive) m/z 250.1568 (calcd for C15H22O3, 250.1569). 4.3.4. (+)-(1R,3R,6S,7S,8R,11S)-3,8,12-Trihydroxy-a-muurolene (4) Colorless oil; [a]25 D +91.2 (c 0.08, MeOH); IR (KBr) mmax 3430, 2960, 2923, 1639, 1451, 1383, 1261, 1031, 802 cm1; for 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (positive) m/z 253 [M+H]+; HRESIMS (positive) m/z 253.1802 [M+H]+ (calcd for C15H25O3, 253.1803). 4.3.5. (6Z,11S)-3,4-Trans-11-oxo-3-methyldodec-cis-6-en-4-olide (5) Colorless oil; [a]24 D +19.9 (c 0.23, MeOH); IR (KBr) mmax 2957, 2923, 1777, 1710, 1629, 1423, 1383, 1210, 1163, 1028 cm1; for 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 247 [M+Na]+; HREIMS (positive) m/z 224.1401 (calcd for C13H20O3, 224.1412). 4.3.6. (6Z,11S)-3,4-Trans-11-hydroxy-3-methyldodec-cis-6-en-4-olide (6) Colorless oil; [a]24 D +36.5 (c 2.08, MeOH); IR (KBr) mmax 3441, 3011, 2964, 2932, 1778, 1631, 1458, 1210, 1157, 1007, 941 cm1; for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 249 [M+Na]+; HREIMS (positive) m/z 226.1566 (calcd for C13H22O3, 226.1569).
5
4.3.7. (6Z,11S)-3,4-Trans-9-hydroxy-3-methyldodec-cis-6-en-4-olide (7) Colorless oil; [a]24 D +18.2 (c 0.20, MeOH); IR (KBr) mmax 3441, 3019, 2959, 1777, 1630, 1459, 1384, 1211, 1158, 1008 cm1; for 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 249 [M+Na]+; HREIMS (positive) m/z 226.1576 (calcd for C13H22O3, 226.1569). 4.3.8. (6Z,11S)-3,4-trans-9-oxo-3-methyldodec-cis-6-en-4-olide (8) Colorless oil; [a]25 D +68.4 (c 0.07, MeOH); IR (KBr) mmax 2964, 2934, 2877, 1778, 1713, 1630, 1459, 1420, 1382, 1210, 1157, 1006 cm1; for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 247 [M+Na]+; HREIMS (positive) m/z 224.1416 (calcd for C13H20O3, 224.1412). 4.3.9. MTPA esters of 6 and 7 The methyl esters by reaction with TMSCHN2 were prepared from 6 and 7 by a procedure published in the literature (Hashimoto et al., 1981). A mixture of 6 (30.0 mg), (S)-MTPA (93.0 mg), 4-(dimethylamino) pyridine (DMAP, 16.0 mg), and 1,3-dicyclohexylcarbodiimide (DCC, 96.0 mg) was dissolved in dry CH2Cl2 (3 mL) and stirred at room temperature for 4 h. The reaction mixture was filtered, and the concentrated filtrate was applied to a silica gel column (eluted with petroleum ether/EtOAc, 12:1) to yield the purified Mosher ester of 6 (42.4 mg). Other MTPA esters were prepared in the same manner for 7 and characterized by measurement of their 1H spectroscopic data in CDCl3. 4.3.10. Bis [(S)-MTPA] ester of 6 1 H NMR (CDCl3) d 5.51 (1H, m, H-7), 5.44(1H, m, H-6), 5.14 (1H, m, H-11), 4.05 (1H, m, H-4), 2.66 (1H, dd, J = 16.9, 7.8 Hz, H-2a), 2.41 (2H, m, H-5), 2.25 (1H, m, H-3), 2.17 (1H, dd, J = 16.9, 9.3 Hz, H-2b), 2.06 (2H, m, H-8), 1.70 (1H, m, H-10a), 1.58 (1H, m, H-10b), 1.42 (2H, m, H-9), 1.26 (3H, d, J = 6.3 Hz, H-12), 1.13 (3H, d, J = 7.2 Hz, H-13). 4.3.11. Bis [(R)-MTPA] ester of 6 1 H NMR (CDCl3) d 5.44 (1H, m, H-7), 5.41(1H, m, H-6), 5.15 (1H, m, H-11), 4.03 (1H, m, H-4), 2.66 (1H, dd, J = 16.9, 7.7 Hz, H-2a), 2.39 (2H, m, H-5), 2.24 (1H, m, H-3), 2.17 (1H, dd, J = 16.9, 9.3 Hz, H-2b), 1.98 (2H, m, H-8), 1.61 (1H, m, H-10a), 1.52 (1H, m, H-10b), 1.28 (2H, m, H-9), 1.33 (3H, d, J = 6.2 Hz, H-12), 1.12 (3H, d, J = 7.0 Hz, H-13). 4.3.12. Bis [(S)-MTPA] ester of 7 1 H NMR (CDCl3) d 5.58 (1H, m, H-7), 5.57 (1H, m, H-6), 5.11 (1H, m, H-9), 4.01 (1H, m, H-4), 2.66 (1H, m, H-2a), 2.41 (2H, m, H-5), 2.21 (1H, m, H-3), 2.25 (2H, m, H-8), 2.17 (1H, m, H-2b), 1.64 (2H, m, H-10), 1.56 (1H, m, H-11a), 1.24 (1H, m, H-11b), 1.11 (3H, d, J = 6.4 Hz, H-13), 0.86 (3H, t, J = 7.3 Hz, H-12). 4.3.13. Bis [(R)-MTPA] ester of 7 1 H NMR (CDCl3) d 5.47 (1H, m, H-7), 5.45 (1H, m, H-6), 5.11 (1H, m, H-9), 3.96 (1H, m, H-4), 2.65 (1H, m, H-2a), 2.36 (2H, m, H-5), 2.20 (1H, m, H-3), 2.22 (2H, m, H-8), 2.16 (1H, m, H-2b), 1.67 (2H, m, H-10), 1.62 (1H, m, H-11a), 1.34 (1H, m, H-11b), 1.11 (3H, d, J = 6.3 Hz, H-13), 0.92 (3H, t, J = 7.4 Hz, H-12). 4.4. X-ray crystallography The crystals of 1 and 3 were used for measurement on a Bruker APEX DUO with a graphite monochromater, Cu Ka radiation. The crystal structures of 1 and 3 were solved using the program
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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SHELXS-97 (Sheldrick 2008) and refined anisotropically by fullmatrix least-squares on F2 using SHELXL-97 (Sheldrick 2008). The absolute configurations were determined by refinement of the Flack (Flack 1983) parameter based on resonant scattering of the light atoms and computation of the Hooft parameter (Hooft et al., 2008), in all cases yielding a probability of 1.000 that the reported configuration is correct. Crystal data for 1: 2(C15H24O2), MW = 472.68; monoclinic, space group P21; a = 9.7826 (2), b = 14.4461 (3), c = 9.7855 (2) Å, a = c = 90°, b = 90.0170 (10)°, V = 1382.89 (5) Å3, Z = 2, d = 1.35 g/cm3, crystal dimensions 0.23 0.32 0.80 mm3. The total number of reflections measured was 5766, of which 3323 were observed, I > 2r (I). Final indices: R1 = 0.0887, wR2 = 0.2066. Flack parameter = 0.3 (3). The Hooft parameter is 0.11 (9) for 974 Bijvoet pairs. Crystal data for 3: C15H22O3, MW = 250.33; monoclinic, space group P21; a = 6.0112 (2) Å, b = 12.9540 (4) Å, c = 18.1285 (6) Å, a = 90.00°, b = 99.478 (2)°, c = 90.00°, V = 1392.38 (8) Å3, T = 100 (2) K, Z = 4, d = 1.94 mg/m3, crystal dimensions 0.30 0.08 0.04 mm3. The total number of reflections measured was 10492, of which 4475 were observed, I > 2r (I). The final R1 values were 0.1225 (I > 2r (I)). The final wR (F2) values were 0.2794 (I > 2r (I)). The final R1 values were 0.1327 (all data). The final wR (F2) values were 0.3018 (all data). The goodness of fit on F2 was 1.326. Flack parameter = 0.2 (4). The Hooft parameter is 0.03 (18) for 1836 Bijvoet pairs. Crystallographic data for 1 (deposition no. CCDC 913634) and 3 (CCDC 935182) have been deposited with the Cambridge Crystallographic Data Centre. Copies of these data can be obtained free of charge via www.ccdc.cam.ac.uk.
4.6. Cytotoxicity assay Five human cancer cell lines: SK-BR-3 breast cancer, SMMC7721 hepatocellular carcinoma, HL-60 myeloid leukemia, PANC-1 pancreatic cancer and A-549 lung cancer. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO2 at 37 °C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates. Briefly, 100 lL adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with initial density of 1 105 cells/mL. Each tumor cell line was exposed to the test compound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 lmol in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control (IC50: SK-BR-3, 13.4 lmol; SMMC-7721, 11.2 lmol; HL-60, 2.5 lmol; PANC-1, 18.6 lmol; A-549, 17.6 lmol). After compound treatment, cell viability was detected and cell growth curve was graphed.
Acknowledgments This project was supported by the National Basic Research Program of China (973 Program, 2009CB522300), the National Natural Sciences Foundation of China (U1132607), and Youth Innovation Promotion Association CAS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 04.015. References Affeld, S., Kehraus, S., Wägele, H., König, G.M., 2009. Dietary derived sesquiterpenes from Phyllodesmium lizardensis. J. Nat. Prod. 72, 298–300. Barrero, A.F., Sanchez, J.F., Altarejos, J., Zafra, M.J., 1992. Homoditerpenes from the essential oil of Tanacetum annuum. Phytochemistry 31, 1727–1730. Bohlmann, F., Ahmed, M., King, R.M., Robinson, H., 1983. Polyacetylenic compounds. Part 262. Acetylenic compounds from Bidens graveolens. Phytochemistry 22, 1281–1283. Cui, B.K., Yuan, H.S., Dai, Y.C., 2008. Wood-rotting fungi in eastern China 1. Polypores from Wuyi Mountains. Fujian Province. Sydowia 60, 25–40. Dai, Y.C., Cui, B.K., Yuan, H.S., 2009a. Trichaptum (Basidiomycota, Hymenochaetales) from China with a description of three new species. Mycol. Prog., 281–287. Dai, Y.C., Yang, Z.L., Cui, B.K., Yu, C.J., Zhou, L.W., 2009b. Species diversity and utilization of medicinal mushrooms and fungi in China (Review). Int. J. Med. Mushrooms 11, 287–302. Dai, Y.C., 2012. Polypore diversity in China with an annotated checklist of Chinese polypores. Mycoscience 53, 49–80. Dale, J.A., Mosher, H.S., 1973. Nuclear magnetic resonance enantiomer regents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and a-methoxy-atrifluoromethylphenylacetate (MTPA) esters. J. Am. Chem. Soc. 95, 512–519. Findlay, J.A., Li, G.Q., Miller, D., Womiloju, T.O., 2003. Insect toxins from spruce endophytes. Can. J. Chem. 81, 284–292. Flack, H.D., 1983. On enantiomorph-polarity estimation. Acta Cryst. A39, 876–881. Hashimoto, N., Aoyama, T., Shioiri, T., 1981. New methods and reagents in organic synthesis. A simple efficient preparation of methyl esters with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chem. Pharm. Bull. 29, 1475–1478. Hooft, R.W.W., Stravera, L.H., Spek, A.L., 2008. Determination of absolute structure using Bayesian statistics on Bijvoet differences. J. Appl. Cryst. 41, 96–103. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Nagashima, F., Suda, K., Asakawa, Y., 1994. Cadinane-type sesquiterpenoids from the liverwort Scapania undulata. Phytochemistry 37, 1323–1325. Ohloff, G., Pawlak, M., 1970. Acid-catalyzed isomerization of ()-a-copaeneepoxide. Helv. Chim. Acta 53, 245–250. Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids. J. Am. Chem. Soc. 113, 4092–4096. Sheldrick, G.M., 2008. A short history of SHELX. Acta Cryst. A64, 112–122. Sullivan, G.R., Dale, J.A., Mosher, H.S., 1973. Correlation of configuration and 19F chemical shifts of a-methoxy-a-trifluoromethylphenylacetate derivatives. J. Org. Chem. 38, 2143–2147. Yang, X.P., Li, F.Q., Han, S.L., 2005. Study on the resources of medicinal macrofungi in the Dabie mountain of Henan. Wuhan Bot. Res. 23, 393–397.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum Xiao-Yan Yang a,b, Tao Feng a, Gang-Qiang Wang c, Jian-Hai Ding a,b, Zheng-Hui Li a, Yan Li a, Shuang-Hui He d, Ji-Kai Liu a,⇑ a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China University of Chinese Academy of Sciences, Beijing 100049, China College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China d Institute of Microbiology, Beijing Forestry University, Beijing 100083, China b c
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 26 November 2013 Available online xxxx Keywords: Basidiomycete Trichaptum pargamenum Cadinane-type sesquiterpenes 13-Carbon c-lactones
a b s t r a c t Four cadinane-type sesquiterpenes and four 13-carbon c-lactones, together with three known compounds, were isolated from cultures of the basidiomycete Trichaptum pargamenum. Their structures were elucidated on the basis of extensive spectroscopic methods. The absolute configurations of two of the cadinene type sesquiterpenes 1 and 3 were confirmed by single crystal X-ray diffractions. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The genus Trichaptum belongs to order Hymenochaetales. Species of the genus causes a similar fragile, lacy white, pocket rot on both angiosperm and gymnosperm wood. Most species of Trichaptum occur in temperate and boreal forests, and 13 species in the genus were found in China (Dai et al., 2009a; Dai 2012). The mushroom T. pargamenum (Fr.) G. Cunn. (=T. biforme (Fr.) Ryvarden) has a wide distribution in China (Cui et al., 2008; Dai et al., 2009a), and has been used as a medicinal fungus for treating cancer, fungal, and bacterial diseases in areas of China (Dai et al., 2009b; Yang et al., 2005). The secondary metabolites produced by this fungus have not been reported previously. In the course of ongoing search for novel secondary metabolites, a chemical investigation of cultures of T. pargamenum, led to isolation of four new cadinane-type sesquiterpenes (Fig. 1), (+)-(1R,3R,6S,7S,11R)-3,12-dihydroxy-a-muurolene (1), (+)-(1R,3R, 6S,7S,11S)-3,12-dihydroxy-a-muurolene (2), 3a-hydroxyartemisinic acid (3), (+)-(1R,3R,6S,7S,8R,11S)-3,8,12-trihydroxy-a-muurolene (4), and four new 13-carbon c-lactones (Fig. 1), (6Z, 11S)-3,4-trans-11-oxo-3-methyldodec-cis-6-en-4-olide (5), (6Z,11S)-3,4-trans-11-hydroxy-3-methyldodec-cis-6-en-4-olide (6), (6Z,11S)-3,4-trans-9-hydroxy-3-methyldodec-cis-6-en-4-olide (7), ⇑ Corresponding author. Tel.: +86 871 5216327; fax: +86 871 5219934. E-mail address: [email protected] (J.-K. Liu).
(6Z,11S)-3,4-trans-9-oxo-3-methyldodec-cis-6-en-4-olide (8), along with three known compounds, (+)-3b-hydroxy-a-muurolene (9) (Affeld et al., 2009), ent-T-muurolol (10) (Nagashima et al., 1994), and 2-methyl-6-methylene-oct-7-en-2,3-diol (11) (Barrero et al., 1992; Bohlmann et al., 1983). The structures of new compounds were elucidated by means of spectroscopic methods, while the known compounds were identified by comparison with data reported in the literature. According to the medicinal use of this fungus, all isolated compounds were evaluated for their cytotoxicities against five human cancer cell lines. 2. Results and discussion Compound 1 was obtained as colorless cubic crystals (MeOH). Its molecular formula C15H24O2 was determined by its HREIMS at m/z 236.1782 [M]+ (calcd. for 236.1776), corresponding to four degrees of unsaturation. The IR data at 3300 cm1 corresponded to hydroxy groups. The 13C NMR spectroscopic data suggested 15 carbon resonances, which were ascribed to three methyls, three methylenes (including one oxygenated), seven methines (including one oxygenated and two olefinic), and two sp2 quaternary carbons, respectively. In the 1H NMR spectrum (Table 1), signals at dH 1.80 and 1.69 (each 3H, s) were readily identified as methyl groups connected to double bonds. Meanwhile, the 1H–1H COSY spectrum (Fig. 2) had correlations of H-11 with H2-12 and H3-13. These data suggested that compound 1 should be a cadinane-type sesquiter-
http://dx.doi.org/10.1016/j.phytochem.2014.04.015 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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X.-Y. Yang et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 2. Key 2D NMR correlations of compounds 1 and 5.
Fig. 1. Structures of compounds 1–8.
pene, with similarities to (+)-3b-hydroxy-a-muurolene (Affeld et al., 2009). The key difference between them was that the methyl group at C-12 in (+)-3b-hydroxy-a-muurolene was replaced by a hydroxymethylene (dC 66.9, C-12) moiety in compound 1, as supported by the HMBC correlations (Fig. 2) from dH 3.39 (2H, m, H12) to dC 36.0 (d, C-11) and 10.7 (q, C-13). Detailed analysis of the 2D NMR spectroscopic data indicated that the other parts of 1 were the same to those of (+)-3b-hydroxy-a-muurolene (Affeld et al., 2009). Due to free rotation of bond of C-7/C-11, the stereochemistry of C-11 in 1 could not be established readily. Fortunately, a single crystal X-ray diffraction identified the absolute configuration of 1 as 1R,3R,6S,7S,11R (Fig. 3). In addition, comparison of the optical rotation data of 1 (+122.2) with those reported in the literature (Affeld et al., 2009; Ohloff and Pawlak, 1970) also supported the absolute structure of 1. Hence, structure 1 was deduced as (+)-(1R,3R,6S,7S,11R)-3,12-dihydroxy-a-muurolene. Compound 2 gave the same molecular formula C15H24O2 as that of 1 by HREIMS. Comparison of 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 2) with those of 1 suggested that compound 2 exhibited a similar structure to that of 1. Preliminary analysis of HSQC, 1H–1H COSY and HMBC data suggested that compound 2
Fig. 3. ORTEP drawing of crystal structure of 1.
had the same overall structure to that of 1; analysis of the ROESY correlations also suggested that the configurations at C-1, C-3, C6 and C-7 in 2 were in agreement with those of 1. However, signif-
Table 1 H NMR spectroscopic data for 1–4 (d in ppm; J in Hz).
1
a b c
No.
1a
1 2a 2b 3 5 6 7 8a 8b 9 11 12a 12b 13 14 15
2.31, 1.93, 1.61, 3.88, 5.68, 2.09, 1.79, 1.80,
2a m m m t (3.4) br d (4.2) m overlap 2H, overlap
5.41, br s 1.99, m 3.39, 2H, overlap 0.86, d (7.0) 1.80, s 1.69, s
2.24, 1.89, 1.69, 3.95, 5.67, 2.19, 1.54, 1.98, 1.79, 5.40, 1.94, 3.74, 3.46, 0.99, 1.81, 1.68,
3b m m m t (3.4) br d (3.3) m m m overlap br s m dd (10.5, 4.5) dd (10.5, 8.6) d (6.9) s s
2.31, 1.93, 1.71, 3.98, 5.56, 2.10, 2.06, 1.91, 1.85, 5.39, 2.77,
4c m m m t (3.4) br d (3.1) m m m m br s m
1.11, d (7.0) 1.82, s 1.70, s
2.23, 1.86, 1.52, 3.97, 5.83, 2.14, 2.04, 4.19,
m m m t (3.4) br d (4.2) m m br s
5.63, 2.58, 3.83, 3.52, 1.14, 1.83, 1.79,
br s m t (7.8) t (7.8) d (7.2) s s
Measured at 400 MHz. Measured at 500 MHz. Measured at 600 MHz.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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X.-Y. Yang et al. / Phytochemistry xxx (2014) xxx–xxx Table 2 C NMR spectroscopic data of 1–8 (d in ppm; J in Hz).
13
a b c
No.
1a
2a
3b
4c
5a
6a
7a
8a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
35.1, d 35.0, t 68.6, d 136.4, s 128.8, d 37.6, d 35.5, d 25.4, t 122.4, d 137.0, s 36.0, d 66.9, t 10.7, q 21.6, q 21.7, q
34.0, d 33.7, t 68.0, d 135.3, s 128.4, d 36.6, d 37.8, d 26.5, t 121.7, d 135.7, s 35.5, d 65.0, t 15.6, q 20.9, q 21.4, q
33.9, d 33.8, t 68.0, d 136.6, s 126.9, d 36.5, d 36.6, d 25.7, t 121.0, d 135.8, s 39.4, d 181.2, s 10.5, q 20.9, q 21.3, q
34.1, d 33.1, t 67.9, d 134.5, s 129.9, d 31.3, d 41.6, d 76.7, d 121.0, d 141.2, s 37.4, d 72.7, t 15.2, q 21.3, q 21.8, q
176.1, s 37.0, t 35.2, d 86.6, d 31.2, t 123.8, d 132.6, d 26.6, t 23.2, t 42.7, t 208.9, s 29.9, q 17.5, q
176.4, s 36.9, t 35.1, d 86.7, d 31.1, t 123.0, d 133.3, d 27.2, t 25.4, t 38.7, t 67.7, d 23.4, q 17.5, q
176.2, s 37.0, t 35.2, d 86.5, d 31.3, t 126.0, d 129.3, d 35.4, t 71.0, d 39.1, t 18.9,t 14.0, q 17.5, q
175.7, s 36.9, t 35.2, d 86.2, d 31.5, t 126.6, d 124.9, d 41.5, t 208.3, s 44.5, t 17.2, t 13.7, q 17.5, q
Measured at 400 MHz. Measured at 500 MHz. Measured at 600 MHz.
Fig. 4. ORTEP drawing of crystal structure of 3.
icant downfield chemical shifts of the methyl group at C-13 (dH 0.99, dC 15.6) in 2 suggested an opposite absolute configuration at C-11 in compound 2. Hence, the structure of 2 was deduced as (+)-(1R,3R,6S,7S,11S)-3,12-dihydroxy-a-muurolene. Compound 3 possessed a molecular formula C15H22O3 as assigned by the HREIMS, indicating five degrees of unsaturation. The 13C NMR spectrum showed 15 carbon signals, which were assigned to one carboxyl, two double bands, one oxymethine, two methylenes, four sp3 methines, and three methyls by the HSQC spectrum (Table 2). These data indicated that compound 3 possessed similar patterns to that of 1, except that the hydroxymethylene group in 1 was replaced by a carboxyl group (dC 181.2) in 3, as supported by HMBC correlations from dH 2.77 (1H, m, H-11) and 1.11 (3H, d, H-13) to dC 181.2 (s, C-12). Detailed analysis of other 2D NMR data suggested that the other parts of 3 were the same to those of 1. In addition, a single crystal X-ray diffraction confirmed the structure as elucidated above, which also determined the absolute configuration of 3 to be 1R,3R,6S,7S,11R (Fig. 4). Therefore, the structure of 3 was determined as 3a-hydroxyartemisinic acid. Compound 4 had the molecular formula C15H24O3 indicated by the HREIMS. The 1H and 13C NMR spectra of 4 (Tables 1 and 2) were very similar to those of 2, suggesting similar overall structures. The only difference was one more hydroxy group at C-8 in 4, which resulted in downfield shifts of CH-8 (dH 4.19; dC 76.7), as deduced by HMBC correlations from dH 4.19 (1H, br s, H-8) to dC 121.0
(d, C-9), and dC 141.2 (s, C-10), as well as 1H–1H COSY correlation between dH 2.04 (1H, m, H-7) and H-8. The ROESY correlation of H-7/H-8 indicated that H-8 and H-7 were on the same side, which suggested that the absolute configurations of C-7 and C-8 should be S and R, respectively. Hence, structure 4 was deduced as (+)-(1R,3R,6S,7S,8R,11R)-3,8,12-trihydroxy-a-muurolene. Compound 5 was isolated as a colorless oil. Its molecular formula (C13H20O3) was established by HREIMS, indicating four degrees of unsaturation. The IR spectrum revealed a lactone carbonyl group (1777 cm1), a vinyl group (1629 cm1), and a ketone group (1710 cm1). The 13C NMR spectroscopic data showed 13 carbon signals (Table 2), which could be classified into for two methyls, five methylenes, four methines (one oxygenated and two olefinic), and two carbonyl carbons, respectively, by DEPT and HSQC spectra. Analyses of 1H–1H COSY correlations established a fragment as shown in Fig. 2. The HMBC correlations (Fig. 2) from dH 4.06 (1H, m, H-4) to dC 176.1 (s, C-1), 37.0 (t, C-2), and 17.5 (q, C-13) suggested that a five-membered lactone ring was formed according to C-4–O–C-1. Furthermore, the methyl signal at dH 2.14 (3H, s, H-12) and dH 2.45 (2H, t, J = 7.0 Hz, H-10) showed key HMBC correlations to dC 208.9 (s, C-11), which suggested a ketone group at C-11. The trans relationship of the two substitutes on the c-lactone ring was deduced by ROESY correlations (Fig. 2) of H-4 with H3-13 (Findlay et al., 2003). The double bond was assigned as having a cis geometry based on the ROESY correlation (Fig. 2) of H2-5 with H2-8. Therefore, compound 5 was established as (6Z,11S)-3,4-trans-11-oxo-3-methyldodeccis-6-en-4-olide. Compound 6, a colorless oil, had a molecular formula of C13H22O3, as established by HREIMS at m/z 226.1566 (calcd. for 226.1569). The IR spectrum showed absorptions for hydroxy (3441 cm1) and lactone carbonyl (1778 cm1) groups. Detailed comparison of the 13C NMR spectroscopic data (Table 2) of 6 with those of 5 showed that they were similar in structure. The key difference between them was that the carbonyl group in 5 was replaced by a hydroxy group (Tables 2 and 3, dH 3.76, dC 67.7) in 6, which was confirmed by the 1H–1H COSY correlations of dH 3.76 (H-11) with dH 1.15 (H3-12) and dH 1.42 (H2-10), and the HMBC correlations from H-12, H-10, and H-9 to dC 67.7 (C-11). The ROESY correlation of H-4/H-13 suggested a trans relationship between H-3 and H-4, while the cis geometry of the double bond between C-6 and C-7 was determined by the ROESY correlation of H2-5 with H2-8. The modified Mosher method was applied to determine the absolute configuration at C-11 in compound 6 (Dale and Mosher, 1973; Ohtani et al., 1991; Sullivan et al.,
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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Table 3 H NMR (400 MHz) spectroscopic data of 5–8 (d in ppm; J in Hz).
1
No.
5
6
2 2b 3 4 5a 5b 6 7 8 9 10 11a 11b 12 13
2.68, dd (16.8, 7.6) 2.19, (overlap) 2.27, m 4.06, m 2.46 m 2.40 m 5.45, m 5.53, m 2.05, 2H, m 1.65, 2H, m 2.45, 2H, t (7.0)
2.65, 2.16, 2.25, 4.04, 2.40,
dd (16.8, 7.8) dd (16.8, 9.3) m m 2H, m
5.39, 5.54, 2.03, 1.41, 1.42, 3.76,
m m 2H, m 2H, overlap 2H, overlap m
2.14, s 1.14, d (6.4)
7 2.66, 2.17, 2.27, 4.07, 2.50, 2.42, 5.59, 5.62, 2.22, 3.65, 1.44, 1.45, 1.35, 0.92, 1.13,
1.15, d (6.2) 1.11, d (6.6)
8 dd (16.8, 7.7) (overlap) m m m m m m 2H, m m 2H, m m m t (6.8) d (6.6)
2.68, 2.20, 2.27, 4.07, 2.48, 2.41, 5.65, 5.77, 3.19,
dd (17.0, 7.8) (overlap) m m m m m m 2H, m
2.43, m 1.60, 2H, m 0.92, t (7.2) 1.14, d (6.6)
3. Conclusion
Fig. 5. Dd Values (dS dR) in ppm of the two MPTA esters derived from 6 and 7.
1973). From the values of Dd (dS dR) (Fig. 5), the absolute configuration at C-11 in 6 was determined as S. On the basis of these data, compound 6 was assigned as (6Z,11S)-3,4-trans-11-hydroxy-3methyldodec-cis-6-en-4-olide. The molecular formula of compound 7 was assigned as C13H22O3 by HREIMS at m/z 226.1576 (calcd. for 226.1569). Its IR spectrum showed the presence of hydroxy (3441 cm1) and lactone (1777 cm1) groups. Its 13C NMR spectroscopic data (Table 2) were similar to those of compound 6, which suggested that compound 7 possessed the same patterns to those of 6. Detailed analysis of the 2D NMR data suggested that the hydroxy group should be placed at C-9 in 7 rather than that at C-11 in 6, as supported by the HMBC correlations from dH 3.65 (1H, m, H-9) to dC 129.3 (d, C7) and dC 18.9 (t, C-11). The stereochemistry of the double bond and lactone ring substituents were resolved by the ROESY data as in case of 6. In addition, the absolute configuration of C-9 in 7 was determined as S by the modified Mosher method (Fig. 5). Therefore, compound 7 was elucidated as (6Z,11S)-3,4-trans9-hydroxy-3-methyldodec-cis-6-en-4-olide. Compound 8 possessed a molecular formula C13H20O3 as determined by the HREIMS. Its 13C NMR spectroscopic data (Table 2) were similar to those of compound 7, except for the hydroxy group in 7 being replaced by a carbonyl group (dC 208.3) in 8, as supported by HMBC correlations from dH 3.19 (2H, m, H-8) and dH 2.43 (2H, m, H-10) to dC 208.3 (s, C-9). The cis geometry of the double bond between C-6 and C-7 was determined by the ROESY correlation of H-5 with H-8, and the relative configuration at C-4 and C-3 were determined by the ROESY correlation of H-4 with Me-13. Hence, compound 8 was elucidated as (6Z,11S)-3,4-trans-9-oxo3-methyldodec-cis-6-en-4-olide. Since the fungus T. pargamenum is used for treating cancer in areas of China, all compounds were evaluated for their cytotoxicity against five human cancer cell lines, SK-BR-3 breast cancer, SMMC7721 hepatocellular carcinoma, HL-60 myeloid leukemia, PANC-1 pancreatic cancer, and A-549 lung cancer, using the MTT method as reported previously (Mosmann, 1983). Unfortunately, none of them had significant activity (IC50 > 10 lmol).
Four new cadinane-type sesquiterpenes (1–4) and four new 13-carbon c-lactones (5–8) were isolated from the culture of T. pargamenum. Compounds 1–4 represented a structure characteristic of hydroxymethylene or carboxyl groups substituted at C-11. The absolute stereochemistries of 1 and 3 were determined by X-ray crystal diffraction. The absolute stereochemistry of 6 and 7 were determined using a modified Mosher’s method.
4. Experimental 4.1. General experimental procedures Melting points were measured on X-4 microscopic melting point meter (Yuhua instrument company, Gongyi, China). Optical rotations were obtained using a Horiba SEPA-300 polarimeter, whereas UV spectra were acquired using a Shimadzu UV-2401A spectrometer. IR spectra were determined using a Bruker Tensor 27 spectrometer with KBr pellets. 1D and 2D NMR experiments were performed on a Bruker AM-400, DRX-500 spectrometer with TMS as the internal standard. Chemical shifts (d) were expressed in ppm with reference to the solvent signals. Mass spectra (MS) were recorded on a VG Auto Spec-3000 or an APIQSTAR time-of-flight spectrometer. Silica gel (200–300 mesh; Qingdao Marine Chemical Ltd., China), MPLC was performed on a Büchi Sepacore system (Büchi Labortechnik AG, Switzerland), reversed-phase C18 (RP18) gel (40–75 lm, Fuji Silysia Chemical Ltd., Japan), and Sephadex LH-20 (Amersham Biosciences, Sweden). Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd. Qingdao), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH.
4.2. Fungal material and cultivation condition The fruiting bodies of fungus T. pargamenum was collected from the Shennongjia Forest Region in Hubei Province, China (latitude: 31°, 310 , 700 ; longitude: 110°, 190 , 4700 ), in October 2004, and authenticated by Prof. Dai Yucheng, Beijing Forestry University. A voucher isolate (No. HF20040812Y) was deposited in the Herbarium of Kunming Institute of Botany, CAS. Culture medium: glucose 5%, peptone 0.15%, yeast powder 0.5%, KH2PO4 0.05% and MgSO4 0.05%. Fermentation was carried out on a shaker at 24 °C and 150 r/min for 23 days.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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4.3. Extraction and isolation The whole culture broth of T. pargamenum (20 L) was initially filtered, and the filtrate was extracted three times with EtOAc. The organic layer was concentrated under reduced pressure to give an oily residue (8.2 g). The residue was subjected to column chromatography (CC) over silica gel (200–300 mesh) eluting with CHCl3/MeOH gradient (from 1:0 to 1:1) to afford fractions A–D. Fraction A was further separated by Sephadex LH-20 (CHCl3/ MeOH, 1:1) and silica gel CC (petroleum ether/Me2CO, 20:1), followed by MPLC (MeOH/H2O, eluting from 62:38 to 68:32 for 16 mins with a flow of 20 mL/min) to afford 9 (10.2 mg) and 10 (2.3 mg). Fraction B was separated by repeated reversed-phase C18 HPLC or (MeOH/H2O, from 3:7 to 5:5) to obtain four subfractions (Fr B1–B4). Fr B1 was further separated by silica gel (petroleum ether/EtOAc, 4:1) and Sephadex LH-20 (Me2CO) to give 1 (22.1 mg), 6 (156.4 mg), 8 (3.8 mg), and 11 (2.1 mg), respectively. Fr B2 was separated by silica gel CC (petroleum ether/EtOAc, 5:1) to yield 7 (83.9 mg). Fr B3 was further purified by repeated silica gel CC and Sephadex LH-20 (Me2CO) to give 2 (5.1 mg), 3 (1.9 mg), 4 (3.8 mg) and 5 (48.5 mg), respectively. 4.3.1. (+)-(1R,3R,6S,7S,11R)-3,12-Dihydroxy-a-muurolene (1) Colorless cubic crystals (MeOH); mp 189–191 °C; [a]26 D +122.2 (c 0.29, MeOH); IR (KBr) mmax 3300, 2956, 2920, 1449, 1435, 1383, 1345, 1042, 1028, 968, 893 cm1; for 1H NMR (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (positive) m/z 259 [M+Na]+; HREIMS (positive) m/z 236.1782 (calcd for C15H24O2, 236.1776). 4.3.2. (+)-(1R,3R,6S,7S,11S)-3,12-Dihydroxy-a-muurolene (2) Colorless crystals; [a]24 D +95.0 (c 0.28, MeOH); IR (KBr) mmax 3440, 2960, 2919, 1631, 1439, 1383, 1030, 972 cm1; for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (positive) m/z 259 [M+Na]+; HREIMS (positive) m/z 236.1776 (calcd for C15H24O2, 236.1776). 4.3.3. 3a-Hydroxyartemisinic acid (3) Colorless crystals; mp 170–172 °C; [a]25 D +56.0 (c 0.14, MeOH); IR (KBr) mmax 3440, 1688, 1629, 1384, 1102 cm1; for 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (negative) m/z 249 [MH]+; HREIMS (positive) m/z 250.1568 (calcd for C15H22O3, 250.1569). 4.3.4. (+)-(1R,3R,6S,7S,8R,11S)-3,8,12-Trihydroxy-a-muurolene (4) Colorless oil; [a]25 D +91.2 (c 0.08, MeOH); IR (KBr) mmax 3430, 2960, 2923, 1639, 1451, 1383, 1261, 1031, 802 cm1; for 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) spectroscopic data, see Tables 1 and 2; ESIMS (positive) m/z 253 [M+H]+; HRESIMS (positive) m/z 253.1802 [M+H]+ (calcd for C15H25O3, 253.1803). 4.3.5. (6Z,11S)-3,4-Trans-11-oxo-3-methyldodec-cis-6-en-4-olide (5) Colorless oil; [a]24 D +19.9 (c 0.23, MeOH); IR (KBr) mmax 2957, 2923, 1777, 1710, 1629, 1423, 1383, 1210, 1163, 1028 cm1; for 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 247 [M+Na]+; HREIMS (positive) m/z 224.1401 (calcd for C13H20O3, 224.1412). 4.3.6. (6Z,11S)-3,4-Trans-11-hydroxy-3-methyldodec-cis-6-en-4-olide (6) Colorless oil; [a]24 D +36.5 (c 2.08, MeOH); IR (KBr) mmax 3441, 3011, 2964, 2932, 1778, 1631, 1458, 1210, 1157, 1007, 941 cm1; for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 249 [M+Na]+; HREIMS (positive) m/z 226.1566 (calcd for C13H22O3, 226.1569).
5
4.3.7. (6Z,11S)-3,4-Trans-9-hydroxy-3-methyldodec-cis-6-en-4-olide (7) Colorless oil; [a]24 D +18.2 (c 0.20, MeOH); IR (KBr) mmax 3441, 3019, 2959, 1777, 1630, 1459, 1384, 1211, 1158, 1008 cm1; for 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 249 [M+Na]+; HREIMS (positive) m/z 226.1576 (calcd for C13H22O3, 226.1569). 4.3.8. (6Z,11S)-3,4-trans-9-oxo-3-methyldodec-cis-6-en-4-olide (8) Colorless oil; [a]25 D +68.4 (c 0.07, MeOH); IR (KBr) mmax 2964, 2934, 2877, 1778, 1713, 1630, 1459, 1420, 1382, 1210, 1157, 1006 cm1; for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 2 and 3; ESIMS (positive) m/z 247 [M+Na]+; HREIMS (positive) m/z 224.1416 (calcd for C13H20O3, 224.1412). 4.3.9. MTPA esters of 6 and 7 The methyl esters by reaction with TMSCHN2 were prepared from 6 and 7 by a procedure published in the literature (Hashimoto et al., 1981). A mixture of 6 (30.0 mg), (S)-MTPA (93.0 mg), 4-(dimethylamino) pyridine (DMAP, 16.0 mg), and 1,3-dicyclohexylcarbodiimide (DCC, 96.0 mg) was dissolved in dry CH2Cl2 (3 mL) and stirred at room temperature for 4 h. The reaction mixture was filtered, and the concentrated filtrate was applied to a silica gel column (eluted with petroleum ether/EtOAc, 12:1) to yield the purified Mosher ester of 6 (42.4 mg). Other MTPA esters were prepared in the same manner for 7 and characterized by measurement of their 1H spectroscopic data in CDCl3. 4.3.10. Bis [(S)-MTPA] ester of 6 1 H NMR (CDCl3) d 5.51 (1H, m, H-7), 5.44(1H, m, H-6), 5.14 (1H, m, H-11), 4.05 (1H, m, H-4), 2.66 (1H, dd, J = 16.9, 7.8 Hz, H-2a), 2.41 (2H, m, H-5), 2.25 (1H, m, H-3), 2.17 (1H, dd, J = 16.9, 9.3 Hz, H-2b), 2.06 (2H, m, H-8), 1.70 (1H, m, H-10a), 1.58 (1H, m, H-10b), 1.42 (2H, m, H-9), 1.26 (3H, d, J = 6.3 Hz, H-12), 1.13 (3H, d, J = 7.2 Hz, H-13). 4.3.11. Bis [(R)-MTPA] ester of 6 1 H NMR (CDCl3) d 5.44 (1H, m, H-7), 5.41(1H, m, H-6), 5.15 (1H, m, H-11), 4.03 (1H, m, H-4), 2.66 (1H, dd, J = 16.9, 7.7 Hz, H-2a), 2.39 (2H, m, H-5), 2.24 (1H, m, H-3), 2.17 (1H, dd, J = 16.9, 9.3 Hz, H-2b), 1.98 (2H, m, H-8), 1.61 (1H, m, H-10a), 1.52 (1H, m, H-10b), 1.28 (2H, m, H-9), 1.33 (3H, d, J = 6.2 Hz, H-12), 1.12 (3H, d, J = 7.0 Hz, H-13). 4.3.12. Bis [(S)-MTPA] ester of 7 1 H NMR (CDCl3) d 5.58 (1H, m, H-7), 5.57 (1H, m, H-6), 5.11 (1H, m, H-9), 4.01 (1H, m, H-4), 2.66 (1H, m, H-2a), 2.41 (2H, m, H-5), 2.21 (1H, m, H-3), 2.25 (2H, m, H-8), 2.17 (1H, m, H-2b), 1.64 (2H, m, H-10), 1.56 (1H, m, H-11a), 1.24 (1H, m, H-11b), 1.11 (3H, d, J = 6.4 Hz, H-13), 0.86 (3H, t, J = 7.3 Hz, H-12). 4.3.13. Bis [(R)-MTPA] ester of 7 1 H NMR (CDCl3) d 5.47 (1H, m, H-7), 5.45 (1H, m, H-6), 5.11 (1H, m, H-9), 3.96 (1H, m, H-4), 2.65 (1H, m, H-2a), 2.36 (2H, m, H-5), 2.20 (1H, m, H-3), 2.22 (2H, m, H-8), 2.16 (1H, m, H-2b), 1.67 (2H, m, H-10), 1.62 (1H, m, H-11a), 1.34 (1H, m, H-11b), 1.11 (3H, d, J = 6.3 Hz, H-13), 0.92 (3H, t, J = 7.4 Hz, H-12). 4.4. X-ray crystallography The crystals of 1 and 3 were used for measurement on a Bruker APEX DUO with a graphite monochromater, Cu Ka radiation. The crystal structures of 1 and 3 were solved using the program
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
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X.-Y. Yang et al. / Phytochemistry xxx (2014) xxx–xxx
SHELXS-97 (Sheldrick 2008) and refined anisotropically by fullmatrix least-squares on F2 using SHELXL-97 (Sheldrick 2008). The absolute configurations were determined by refinement of the Flack (Flack 1983) parameter based on resonant scattering of the light atoms and computation of the Hooft parameter (Hooft et al., 2008), in all cases yielding a probability of 1.000 that the reported configuration is correct. Crystal data for 1: 2(C15H24O2), MW = 472.68; monoclinic, space group P21; a = 9.7826 (2), b = 14.4461 (3), c = 9.7855 (2) Å, a = c = 90°, b = 90.0170 (10)°, V = 1382.89 (5) Å3, Z = 2, d = 1.35 g/cm3, crystal dimensions 0.23 0.32 0.80 mm3. The total number of reflections measured was 5766, of which 3323 were observed, I > 2r (I). Final indices: R1 = 0.0887, wR2 = 0.2066. Flack parameter = 0.3 (3). The Hooft parameter is 0.11 (9) for 974 Bijvoet pairs. Crystal data for 3: C15H22O3, MW = 250.33; monoclinic, space group P21; a = 6.0112 (2) Å, b = 12.9540 (4) Å, c = 18.1285 (6) Å, a = 90.00°, b = 99.478 (2)°, c = 90.00°, V = 1392.38 (8) Å3, T = 100 (2) K, Z = 4, d = 1.94 mg/m3, crystal dimensions 0.30 0.08 0.04 mm3. The total number of reflections measured was 10492, of which 4475 were observed, I > 2r (I). The final R1 values were 0.1225 (I > 2r (I)). The final wR (F2) values were 0.2794 (I > 2r (I)). The final R1 values were 0.1327 (all data). The final wR (F2) values were 0.3018 (all data). The goodness of fit on F2 was 1.326. Flack parameter = 0.2 (4). The Hooft parameter is 0.03 (18) for 1836 Bijvoet pairs. Crystallographic data for 1 (deposition no. CCDC 913634) and 3 (CCDC 935182) have been deposited with the Cambridge Crystallographic Data Centre. Copies of these data can be obtained free of charge via www.ccdc.cam.ac.uk.
4.6. Cytotoxicity assay Five human cancer cell lines: SK-BR-3 breast cancer, SMMC7721 hepatocellular carcinoma, HL-60 myeloid leukemia, PANC-1 pancreatic cancer and A-549 lung cancer. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO2 at 37 °C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates. Briefly, 100 lL adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with initial density of 1 105 cells/mL. Each tumor cell line was exposed to the test compound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 lmol in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control (IC50: SK-BR-3, 13.4 lmol; SMMC-7721, 11.2 lmol; HL-60, 2.5 lmol; PANC-1, 18.6 lmol; A-549, 17.6 lmol). After compound treatment, cell viability was detected and cell growth curve was graphed.
Acknowledgments This project was supported by the National Basic Research Program of China (973 Program, 2009CB522300), the National Natural Sciences Foundation of China (U1132607), and Youth Innovation Promotion Association CAS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 04.015. References Affeld, S., Kehraus, S., Wägele, H., König, G.M., 2009. Dietary derived sesquiterpenes from Phyllodesmium lizardensis. J. Nat. Prod. 72, 298–300. Barrero, A.F., Sanchez, J.F., Altarejos, J., Zafra, M.J., 1992. Homoditerpenes from the essential oil of Tanacetum annuum. Phytochemistry 31, 1727–1730. Bohlmann, F., Ahmed, M., King, R.M., Robinson, H., 1983. Polyacetylenic compounds. Part 262. Acetylenic compounds from Bidens graveolens. Phytochemistry 22, 1281–1283. Cui, B.K., Yuan, H.S., Dai, Y.C., 2008. Wood-rotting fungi in eastern China 1. Polypores from Wuyi Mountains. Fujian Province. Sydowia 60, 25–40. Dai, Y.C., Cui, B.K., Yuan, H.S., 2009a. Trichaptum (Basidiomycota, Hymenochaetales) from China with a description of three new species. Mycol. Prog., 281–287. Dai, Y.C., Yang, Z.L., Cui, B.K., Yu, C.J., Zhou, L.W., 2009b. Species diversity and utilization of medicinal mushrooms and fungi in China (Review). Int. J. Med. Mushrooms 11, 287–302. Dai, Y.C., 2012. Polypore diversity in China with an annotated checklist of Chinese polypores. Mycoscience 53, 49–80. Dale, J.A., Mosher, H.S., 1973. Nuclear magnetic resonance enantiomer regents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and a-methoxy-atrifluoromethylphenylacetate (MTPA) esters. J. Am. Chem. Soc. 95, 512–519. Findlay, J.A., Li, G.Q., Miller, D., Womiloju, T.O., 2003. Insect toxins from spruce endophytes. Can. J. Chem. 81, 284–292. Flack, H.D., 1983. On enantiomorph-polarity estimation. Acta Cryst. A39, 876–881. Hashimoto, N., Aoyama, T., Shioiri, T., 1981. New methods and reagents in organic synthesis. A simple efficient preparation of methyl esters with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chem. Pharm. Bull. 29, 1475–1478. Hooft, R.W.W., Stravera, L.H., Spek, A.L., 2008. Determination of absolute structure using Bayesian statistics on Bijvoet differences. J. Appl. Cryst. 41, 96–103. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Nagashima, F., Suda, K., Asakawa, Y., 1994. Cadinane-type sesquiterpenoids from the liverwort Scapania undulata. Phytochemistry 37, 1323–1325. Ohloff, G., Pawlak, M., 1970. Acid-catalyzed isomerization of ()-a-copaeneepoxide. Helv. Chim. Acta 53, 245–250. Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids. J. Am. Chem. Soc. 113, 4092–4096. Sheldrick, G.M., 2008. A short history of SHELX. Acta Cryst. A64, 112–122. Sullivan, G.R., Dale, J.A., Mosher, H.S., 1973. Correlation of configuration and 19F chemical shifts of a-methoxy-a-trifluoromethylphenylacetate derivatives. J. Org. Chem. 38, 2143–2147. Yang, X.P., Li, F.Q., Han, S.L., 2005. Study on the resources of medicinal macrofungi in the Dabie mountain of Henan. Wuhan Bot. Res. 23, 393–397.
Please cite this article in press as: Yang, X.-Y., et al. Chemical constituents from cultures of the basidiomycete Trichaptum pargamenum. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.04.015
