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Sterostreins F–O, illudalanes and norilludalanes from cultures of the Basidiomycete Stereum ostrea BCC 22955
Phytochemistry 79 (2012) 116–120
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Sterostreins F–O, illudalanes and norilludalanes from cultures of the Basidiomycete Stereum ostrea BCC 22955 Masahiko Isaka ⇑, Urarat Srisanoh, Malipan Sappan, Sumalee Supothina, Thitiya Boonpratuang National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
a r t i c l e
i n f o
Article history: Received 7 December 2011 Received in revised form 17 February 2012 Available online 15 May 2012
a b s t r a c t Sterostreins F–O (1–10), 10 illudalanes and norilludalanes, were isolated from cultures of the Basidiomycete Stereum ostrea BCC 22955. Their structures were elucidated by analyses of the NMR spectroscopic and mass spectrometry data. Sterostreins M (8), N (9), and O (10) are pyridine-containing illudalanes. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Stereum ostrea Illudalane Norilludalane Basidiomycete
1. Introduction Woody mushrooms belonging to the genus Stereum have been the source of bioactive compounds. One of the major metabolite groups of this fungal genus are sesquiterpenoids, such as hirsutanes (Yun et al., 2002; Yoo et al., 2006; Liermann et al., 2010), sterpuranes (Xie et al., 1992), cadinanes (Li et al., 2006, 2008), stereumanes (Li et al., 2011), and drimanes (Kim et al., 2006), respectively. As part of our research program on biologically active fungal metabolites, dimeric and monomeric illudalane-type sesquiterpenes, sterostreins A–E, were recently isolated from cultures of Stereum ostrea BCC 22955 (Isaka et al., 2011). Further studies on this fungal strain resulted in the isolation of additional 10 new illudalanes and norilludalanes, sterostreins F–O (1–10) (Fig. 1). 2. Results and discussion In the proceeding study (Isaka et al., 2011), very slow mycelial growth of the producing fungus was observed under static fermentation. The use of a bioreactor accelerated the mycelial growth, but the conditions were not suitable for production of the targeted terpenoids. A new static fermentation was performed in threefold larger scale (250 ml 60 in 1 l Erlenmeyer flasks, and additional for time course study) than the preceding batch. A flask was harvested, extracted, and analyzed by 1H NMR every 10 or 15 day of incubation, which suggested that sterostreins started to be produced from 100 days of final fermentation. The cultures were harvested at day 130, which was the same duration as the previous ⇑ Corresponding author. Tel.: +66 25646700x3554; fax: +66 25646707. E-mail address: [email protected] (M. Isaka). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.04.009
fermentation. Extracts from the new batch contained 10 new analogs 1–10, together with the major constituents, sterostreins D (11) and E (12), and a small amount of a dimeric analog, sterostrein B. Sterostrein F (1) was isolated as a pale yellow gum, and the molecular formula was determined by HRESIMS as C15H20O5, which had one more oxygen atom than the major metabolites sterostreins D (11) and E (12). The 1H and 13C NMR spectroscopic data (Tables 1 and 2) were similar to those of 11. The difference was the absence of a methine, replaced by an oxygenated quaternary carbon (dC 83.1). The location of the tertiary alcohol was assigned to C-9 position on the basis of the HMBC correlations from Ha-1, Hb-1, Hb-3, and H-4 to this carbon. Sterostrein G (2) possessed the same molecular formula as 1, C15H20O5 (HRESIMS). The 1H and 13C NMR resonance patterns more resembled those of 12 rather than 11. Detailed analysis of 2D NMR data, including the HMBC correlations from Ha-1, Hb-1, and Hb-3 to C-9 (dC 84.3), established that it was also a 9-hydroxy analog of 11/12. The relative configurations of 1 and 2 were assigned on the basis of NOESY correlations as shown in Fig. 2. NOESY correlation of Ha-1/Ha-3 for 1 suggested the cis ring junction, while the correlation of Hb-1/H-4 for 2 indicated the trans ring junction. Examinations of the conformations of both isomers 9b-OH and 9a-OH isomers with a molecular model further supported this conclusion. The unusual downfield shift of Ha-3 (dH 2.06) for 2, when compared with 1 (dH 0.99), 11 (dH 0.98), and 12 (dH 1.48), can be explained by the deshielding of this proton by the 9a-OH group. The molecular formula of sterostrein H (3) was determined by HRESIMS as C15H18O4. The 1H and 13C NMR spectroscopic data and DEPT135 indicated the presence of a trisubstituted olefin, replacing a methine and a methylene of 11/12. This compound was identified as the 1,9-dehydro derivative of 11/12 on the basis
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15
7
1
8
6 13
4
5
14
11 2
9
10
3 12
15 7 14 6
13
1 8
11
2
9 4
5
10
3 12
3
15
14
7
6
13
8
1 9
5
11
2 4 3
10
12
Fig. 1. Structures of compounds 1–12.
Table 1 13 C NMR spectroscopic data (CDCl3, 125 MHz) for sterostreins F–O (1–10). No.
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a b,c
50.3 35.3 43.2 58.0 68.7 125.6 121.0 195.4 83.1 30.9 30.6 25.7 151.5 56.1 144.7
2
3
49.0 35.6 37.3 56.7 70.3 129.8 123.3 192.6 84.3 32.6 31.9 26.2 151.2 56.9 144.1
4 b
151.1 45.9 38.8 56.4 71.9 130.0 125.6 181.6 139.1 28.7 27.6 25.3 149.8 57.0 143.3
43.67 37.9 43.72b 50.2 70.7 154.2 132.7 201.5 47.3 30.6 30.3 27.5 31.8 61.7 12.1
5
6
7
8
9
10
42.6 37.5 43.9 51.9 70.3 160.4 127.5 201.5 47.6 31.2a 31.2a 26.4 35.6 63.0
40.2 36.4 41.4 54.8 73.8 168.6 127.3 200.4 51.7 31.8 31.6 19.5 32.8 63.1
148.2 45.4 39.5 54.8 74.4 171.6 127.6 186.5 137.5 28.6 27.6 22.8 33.7 62.9
40.0 36.6 41.2 53.7 73.0 160.3 126.2 198.2 52.2 31.7 31.5 22.8 120.6 153.7 148.8
53.3 38.7 45.0 58.0 70.9 153.8 126.8 200.7 83.3 29.3 27.9 23.9 119.5 154.7 148.6
44.4 37.8 47.4 164.2 68.4 156.6 125.2 181.8 136.3 29.33c 29.30c 29.2 120.5 152.6 148.6
The carbon resonances were superimposed. The assignment of carbons can be interchanged.
Table 2 1 H (500 MHz, CDCl3) NMR spectroscopic data for sterostreins F–H (1–3). No. 1 3 4 10 11 12 14 15
1
2
a 2.18, d (13.5)
a 1.68, d (14.7)
b 1.64, d (13.5) a 0.99, t (13.1) b 1.68, dd (12.9, 7.4) 2.73, dd (13.3, 7.4) 0.87, s 1.16, s 1.64, s 4.71, d (13.7) 4.65, d (13.7) 7.94, s
b 1.97, d (14.7) a 2.06, dd (13.3, 11.8) b 1.75, dd (11.8, 6.7) 2.52, dd (13.3, 6.7) 1.26, s 1.11, s 1.73, s 4.87, d (13.9) 4.69, d (13.9) 7.92, s
3
1 4
6.72, d (2.4)
a 1.87, dd (13.1, 8.4) b 2.01, dd (13.1, 8.4) 3.59, dt (2.4, 8.4) 1.24, s 1.15, s 1.39, s 4.82, d (13.7) 4.72, d (13.7) 7.89, s
of HMBC correlations from H-1 (dH 6.72) to C-2, C-3, C-8, and C-9, and the correlations from H-4 to the olefinic carbons C-1 (dC 151.1)
3
12
10 11
3
3
3
3
3
3
Fig. 2. Key NOESY correlations for 1 and 2.
and C-9 (dC 139.1). The upfield shift of the ketone C-8 (dC 181.8) when compared with 11 (dC 198.4) and 12 (dC 196.0) was consistent with the enone conjugation. The molecular formula of sterostrein I (4) was determined as C15H24O3 by HRESIMS. The 1H and 13C NMR spectroscopic data
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suggested the absence of the furan ring and the structural difference at this moiety. The resonance patterns for the cyclopentane and the central six-membered ketone were similar to those of 11 (Tables 1 and 3). Instead of the hydroxymethyl-furan (C-6, C-7, C-13, C-14, and C-15) in 11, sterostrein I (4) possessed a tetrasubstituted olefin (dC 132.7 and 154.2), an allylic methyl group at dC 12.1 (dH 1.82, s), and a 2-hydroxyethyl group. Its structure was established by analysis of HMBC correlations: from H3-15 to C-6, C-7, C-8; from H-4 and H3-12 to C-6; from H2-13 to C-5, C-6, and C-7; and from H2-14 to C-6. The cis ring junction was confirmed by the NOESY correlation of H-4/H-9 and the correlations from these protons to H3-11. This compound can also be assigned as an illudalane-type sesquiterpene. The molecular formula of sterostrein J (5), C14H22O3 (HRESIMS), suggested that it was a norilludalane. The 1H and 13C NMR spectroscopic data were similar to those of 4. The only difference was the absence of the allylic methyl group CH3-15 in 5, and a quaternary sp2 carbon (C-7) of 4 was replaced by an sp2 methine at dC 127.3 (dH 5.88, H-7). The location of the trisubstituted olefin (C-6/C-7) was confirmed by the HMBC correlations from H-7 to C-5, C-9, and C-13, and the correlations from H-4, H3-12, H2-13, and H2-14 to C-6. Sterostrein K (6) possessed the same molecular formula as 5 (C14H22O3, HRESIMS). The 1H and 13C NMR spectroscopic data for the cyclopentane moiety were similar to those of 12. It was identified as the C-9 epimer of 5, on the basis of the NOESY correlations H-4/Hb-1, H3-12/H-9, and H3-12/Ha-3. Sterostrein L (7), C14H20O3 (HRESIMS), was identified as the 1,9-dehydro derivative of 5/6. The location of the olefinic methine proton (H-1) was confirmed by the HMBC correlations from this proton to C-2, C-3, C4, C-8, C-9, and C-10. The relative configuration was suggested by the NOESY correlation of H3-12/Ha-3. The molecular formula of sterostrein M (8) was determined by HRESIMS as C15H19NO2, which indicated the incorporation of one nitrogen atom in the molecule. Analysis of the 1H and 13C NMR, DEPT135, HMQC and COSY spectroscopic data demonstrated the trans fused C-1–C-9 bicyclic ring moiety similar to 12. The NOESY correlations of H-9/Ha-3 and H-9/H3-12 indicated the relative configuration of C-9/C-4/C-5. Instead of the hydroxymethyl-furan C5 unit (C-6, C-7, C-13, C-14, and C-15), compound 8 contained three sp2 methines and two sp2 quaternary carbons. The pyridine-fused structure was addressed on the basis of the HMBC correlations (Fig. 3) and the chemical shifts of the heteroaromatic protons (H13, H-14, and H-15) and carbons (C-6, C-7, C-13, C-14, and C-15) (Tables 1 and 4). This compound is also assignable as an illudalane-type sesquiterpene. The 1H and 13C NMR spectroscopic data for sterostrein N (9) suggested that it was also a pyridine-containing illudalane. The molecular formula was determined by HRESIMS as C15H19NO3,
3 3 3
Fig. 3. Selected HMBC correlations for 8.
Table 4 1 H (500 MHz, CDCl3) NMR spectroscopic data for sterostreins M–O (8–10). No. 1 3 4 9 10 11 12 13 14 15
8
9
10
a 1.91, dd (13.3, 8.1)
a 1.59, d (13.9)
a 2.59, d (16.3)
b 1.77, dd (13.3, 10.3) a 1.60, t (12.3) b 1.81, dd (12.3, 6.7) 2.50, m 2.77, m 1.16, s 1.09, s 1.50, s 7.68, d (5.2) 8.66, d (5.2) 9.04, s
b 1.64, dd (13.9, 2.3) a 0.77, t (12.6) b 1.78, ddd (12.6, 7.6, 2.3) 2.96, dd (12.6, 7.6) 0.86, 1.22, 1.65, 7.36, 8.75, 8.92,
s s s d (5.1) d (5.1) s
b 2.53, d (16.3)
a 2.62, d (18.3) b 2.76, d (18.3)
1.19, 1.17, 1.60, 7.67, 8.64, 9.15,
s s s d (5.2) d (5.2) s
which had one oxygen atom more than 8. HMBC correlations from Ha-1, Hb-1, Hb-3, and H-4 to the tertiary alcohol carbon (dC 83.3) revealed that it was a 9-hydroxy analog. The cis ring junction was suggested by the close resemblance of the 1H and 13C NMR data for this region to those of 1. This relative configuration was strongly supported by the NOESY correlations of Ha-1/Ha-3 and H3-12/Ha-3, and the observation of a W-type 1H–1H coupling (J = 2.3 Hz) for Hb-1/Hb-3. The molecular formula of sterostrein O (10) was determined as C15H17NO2 by HRESIMS. It was identified as a 4,9-dehydro derivative by following key HMBC correlations: from H2-1 to C-4, C-8, and C-9; from H2-3 to C-4, C-5, and C-9; and from H3-12 to C-4, C-5, and C-6. The chemical shifts of C-8 (dC 181.8) and C-4 (dC 164.2) were consistent with the enone conjugation. The absolute configuration of the most abundant metabolite, sterostrein D (11) was previously determined by its conversion to the 8b-hydroxy secondary alcohol derivative (LiAlH4/THF) followed by application of the modified Mosher’s method. Application of the same strategy to the absolute configuration of sterostrein F (4) was unsuccessful, as LiAlH4 reduction of 4 gave a mixture of more than three products. Attempts at recrystallization of several of the new compounds and acylated derivatives of 4 for X-ray crystallographic analysis were also unsuccessful; therefore the absolute configurations of 4–10 remain unassigned. Although not
Table 3 1 H (500 MHz, CDCl3) NMR spectroscopic data for sterostreins I–L (4–7). No. 1 3 4 7 9 10 11 12 13 14 15
4
5
6
7
a 1.91, dd (13.3, 4.7)
a 2.13, dd (13.5, 2.5)
a 1.81, dd (13.2, 7.9)
6.51, d (2.6)
b 1.73, dd (13.3, 8.6) a 1.19, t (12.6) b 1.55, dd (12.6, 7.2) 2.64, m
b 1.67, dd (13.5, 8.8) a 1.15, t (12.7) b 1.53, dd (12.7, 6.6) 2.64, dt (12.7, 6.6) 5.88, s 2.92, m 0.97, s 1.04, s 1.39, s 2.69, ddd (14.4, 9.9, 3.9) 2.38, dt (14.4, 3.9) 4.02, m 3.76, dt (3.9, 9.9)
b 1.60, dd (13.2, 10.7) a 1.48, t (12.2) b 1.72, dd (12.2, 6.6) 2.32, ddd (13.7, 12.2, 6.6) 5.82, d (1.0) 2.52, ddd (13.7, 10.7, 7.9) 1.11, s 1.07, s 1.33, s 2.76, ddd (14.1, 9.8, 5.2) 2.44, ddt (14.1, 1.0, 4.2) 3.99, m 3.76, dt (4.2, 9.8)
2.97, dt (4.7, 8.1) 0.96, s 1.02, s 1.35, s 2.68–2.65, m 3.95, dt (9.8, 4.4) 3.72, m 1.82, s
a 1.82, dd (13.2, 8.2) b 1.96, dd (13.2, 8.2) 3.44, dt (2.6, 8.2) 5.89, s 1.20, 1.10, 1.17, 2.83, 2.44, 3.99, 3.77,
s s s ddd (13.9, 9.9, 5.0) dt (13.9, 4.0) m dt (4.0, 9.9)
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certain, the co-occurrence suggested that the new furan-containing analogs 1–3 also possess the same sense of absolute configuration of C-4 and C-5 as sterostrein D (11). 3. Conclusions Although many terpenoids have been isolated from fruiting bodies or cell cultures of Basidiomycetes, illudalane-type sesquiterpenes are relatively rare (Clericuzio et al., 1997; Suzuki et al., 2005; Pettit et al., 2010; Fushimi et al., 2010) when compared with biogenetically related illudanes. Most of the known illudalanes, from plants and fungi, possess benzene ring fused with a fivemembered ring. Sterostreins D–H (11, 12, 1–3) and M–O (8–10) are novel furan- and pyridine-containing tricyclic illudalanes, respectively. On the other hand, sterostreins J–L (5–7) are new norilludalanes. These compounds were produced after unusually long fermentation, which may be one of the reasons why they have been missed. Taking together the proceeding report of the dimeric analogs, the fungus S. ostrea BCC 22955 has probed to be a unique source of secondary metabolites. 4. Experimental 4.1. General procedures Melting points were measured with an Electrothermal IA9100 digital melting point apparatus. Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded on a GBC Cintra 404 spectrophotometer. FTIR spectra were taken on Bruker VECTOR 22 and ALPHA spectrometers. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. 4.2. Fungal material The mushroom S. ostrea was collected on bark of a dead hardwood tree in Khao Yai National Park, Nakhon Nayok Province, Thailand, by one of the authors (T.B.). The natural mushroom speciment was deposited in the BIOTEC Bangkok Herbarium as BBH 17035, and the living culture was deposited in the BIOTEC Culture Collection as BCC 22955 on August 21, 2006. 4.3. Fermentation and isolation Fermentation was performed in threefold larger scale than the previous study (Isaka et al., 2011), but under the similar conditions. The fungus BCC 22955 was maintained on potato dextrose agar at 25 °C. The agar was cut into small plugs and inoculated into 6 250 ml Erlenmeyer flasks containing 25 ml of potato dextrose broth (PDB; potato starch 4.0 g/l, dextrose 20.0 g/l). After incubation at 25 °C for 7 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1 l Erlenmeyer flask containing 250 ml of the same liquid medium (PDB), and incubated at 25 °C for 7 days on a rotary shaker (200 rpm). These secondary cultures were pooled and each 25 ml portion was transferred into 60 1 l Erlenmeyer flasks containing 250 ml of malt extract broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g/l, maltose 1.8 g/l, dextrose 6.0 g/l), and the final fermentation was carried out at 25 °C for 130 days under static conditions. The cultures were filtered to separate broth (filtrate) and mycelia (residue). The EtOAc extract from the culture filtrate (3.00 g) was subjected to CC on silica gel (5.0 18 cm, MeOH/CH2Cl2, step gradient elution from 0:100 to 15:85) to obtain nine pooled fractions. Fraction 5 (357 mg) was purified by preparative HPLC (SunFire Prep C18 OBD,
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19 150 mm, 5 lm; mobile phase MeCN/H2O, 20:80, flow rate 10 ml/min) to furnish 3 (4.6 mg), 12 (60 mg), 10 (8.0 mg), and 8 (15 mg). Fraction 6 (858 mg) was further fractionated by CC on silica gel (4.0 16 cm, MeOH/CH2Cl2, step gradient elution from 0:100 to 15:85), and the sub-fractions were further purified by preparative HPLC (SunFire Prep C18 OBD; MeCN/H2O, 20:80 or 25:75) to obtain 12 (15 mg), 4 (7.5 mg), sterostrein B (6.1 mg), 7 (28 mg), 6 (1.8 mg), 9 (78 mg), 5 (21 mg), and 11 (20 mg). Fraction 7 (350 mg) was subjected to preparative HPLC (SunFire Prep C18 OBD; MeCN/H2O, 30:70) to furnish 2 (3.8 mg), 7 (3.0 mg), 5 (9.0 mg), and 11 (178 mg). Fraction 8 (150 mg) was also purified by preparative HPLC (SunFire Prep C18 OBD; MeCN/H2O, 20:80) to afford 1 (9.1 mg) and 11 (21 mg). The mycelial extract (278 mg) contained much less quantities of the terpenoids, and no additional new compound was found by chromatographic fractionation. 4.3.1. Sterostrein F (1) Pale yellow gum; [a]27D 77 (c 0.10, MeOH); UV (MeOH) kmax (log e) 205 (4.11), 262 (3.37) nm; IR (ATR) mmax 3352, 2951, 1685, 1619, 1549, 1367, 1141, 1025, 899 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 2; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 303.1208 [M+Na]+ (calcd for C15H20O5Na, 303.1203). 4.3.2. Sterostrein G (2) Pale yellow gum; [a]24D 8 (c 0.21, MeOH); UV (MeOH) kmax (log e) 208 (3.98) nm; IR (ATR) mmax 3353, 2951, 1685, 1544, 1366, 1031 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 2; 13 C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 303.1216 [M+Na]+ (calcd for C15H20O5Na, 303.1203). 4.3.3. Sterostrein H (3) Pale yellow amorphous solid; [a]24D 48 (c 0.14, MeOH); UV (MeOH) kmax (log e) 209 (3.96), 274 (3.86) nm; IR (ATR) mmax 3316, 2955, 1654, 1610, 1559, 1145, 1121 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 2; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 263.1281 [M+H]+ (calcd for C15H19O4, 263.1278). 4.3.4. Sterostrein I (4) Colorless amorphous solid; [a]26D +20 (c 0.11, MeOH); UV (MeOH) kmax (log e) 252 (4.02), 287 (3.90) nm; IR (ATR) mmax 3352, 2951, 1647, 1366, 1041, 901 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 275.1607 [M+Na]+ (calcd for C15H24O3Na, 275.1618). 4.3.5. Sterostrein J (5) Colorless gum; [a]27D +59 (c 0.075, MeOH); UV (MeOH) kmax (log e) 234 (3.95) nm; IR (ATR) mmax 3350, 2950, 1649, 1366, 1044, 909 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 239.1640 [M+H]+ (calcd for C14H23O3, 239.1642). 4.3.6. Sterostrein K (6) Pale yellow amorphous solid; [a]27D 18 (c 0.09, MeOH); UV (MeOH) kmax (log e) 234 (3.90) nm; IR (ATR) mmax 3397, 3337, 2949, 1656, 1141, 1029 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 236.1639 [M+H]+ (calcd for C14H23O3, 239.1642). 4.3.7. Sterostrein L (7) Colorless solid; mp 142–144 °C; [a]26D 119 (c 0.105, MeOH); UV (MeOH) kmax (log e) 253 (3.89), 282 sh (3.79) nm; IR (ATR) mmax 3359, 3255, 2954, 1619, 1599, 1303, 1032, 836, 689 cm 1; 1H NMR
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(500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 237.1481 [M+H]+ (calcd for C14H21O3, 237.1485). 4.3.8. Sterostrein M (8) Pale yellow solid; mp 152–154 °C; [a]26D +82 (c 0.095, MeOH); UV (MeOH) kmax (log e) 234 (3.89), 268 (3.29) nm; IR (ATR) mmax 3197, 2955, 1701, 1594, 854, 589 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 4; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 264.1489 [M+H]+ (calcd for C15H20NO2, 264.1489). 4.3.9. Sterostrein N (9) Pale yellow gum; [a]26D +48 (c 0.05, MeOH); UV (MeOH) kmax (log e) 233 (3.89), 264 sh (3.49) nm; IR (ATR) mmax 3277, 2949, 1692, 1594, 1368, 1029, 845 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 4; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 262.1431 [M+H]+ (calcd for C15H20NO3, 262.1438). 4.3.10. Sterostrein O (10) Pale brown solid; mp 163–164 °C; [a]23D 13 (c 0.10, MeOH); UV (MeOH) kmax (log e) 241 (3.82), 282 (3.77) nm; IR (ATR) mmax 3134, 1655, 1592, 1292, 1045, 605 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 4; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 244.1340 [M+H]+ (calcd for C15H18NO2, 244.1332). Acknowledgment Financial support from the Bioresources Research Network, National Center for Genetic Engineering and Biotechnology (BIOTEC), is gratefully acknowledged. 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.2012. 04.009.
References Clericuzio, M., Pan, F., Han, F., Pang, Z., Sterner, O., 1997. Stearoyldelicone, an unstable protoilludane sesquiterpenoid from intact fruit bodies of Russula delica. Tetrahedron Lett. 38, 8237–8240. Fushimi, K., Horikawa, M., Suzuki, K., Sekiya, A., Kanno, S., Shimura, S., Kawagishi, H., 2010. Applanatines A–E from the culture broth of Ganoderma applanatum. Tetrahedron 66, 9332–9335. Isaka, M., Srisanoh, U., Choowong, W., Boonpratuang, T., 2011. Sterostreins A–E, new terpenoids from cultures of the Basidiomycete Stereum ostrea BCC 22955. Org. Lett. 13, 4886–4889. Kim, Y.-H., Yun, B.-S., Ryoo, I.-J., Kim, J.-P., Koshino, H., Yoo, I.-D., 2006. Methoxylaricinolic acid, a new sesquiterpene from the fruiting bodies of Stereum ostrea. J. Antibiot. 59, 432–434. Li, G.-H., Li, L., Duan, M., Zhang, K.-Q., 2006. The chemical constituents of the fungus Stereum sp. Chem. Biodiv. 3, 210–216. Li, G.-H., Duan, M., Yu, Z.-F., Li, L., Dong, J.-Y., Wang, X.-B., Guo, J.-W., Huang, R., Wang, M., Zhang, K.-Q., 2008. Stereumin A–E, sesquiterpenoids from the fungus Stereum sp. CCTCC AF 207024. Phytochemistry 69, 1439–1445. Li, G., Liu, F., Shen, L., Zhu, H., Zhang, K., 2011. Stereumins H–J, stereumane-type sesquiterpenes from the fungus Stereum sp. J. Nat. Prod. 74, 296–299. Liermann, J.C., Schüffler, A., Wollinsky, B., Birnbacher, J., Kolshorn, H., Anke, T., Opatz, T., 2010. Hirsutane-type sesquiterpenes with uncommon modifications from three Basidiomycetes. J. Org. Chem. 75, 2955–2961. Pettit, G.R., Meng, Y., Pettit, R.K., Herald, D.L., Cichacz, Z.A., Doubek, D.L., Richert, L., 2010. Antineoplastic agents: 556. Isolation and structure of coprinastatin 1 from Coprinus cinereus. J. Nat. Prod. 73, 388–392. Suzuki, S., Murayama, T., Shiono, Y., 2005. Illudalane sesquiterpenoids, echinolactones A and B, from a mycelial culture of Echinodontium japonicum. Phytochemistry 66, 2329–2333. Xie, J.-L., Li, L.-P., Dai, Z.-Q., 1992. Isolation and identification of two new metabolites from silver leaf fungus Stereum purpureum. J. Org. Chem. 57, 2313–2316. Yoo, N.-H., Kim, J.-P., Yun, B.-S., Ryoo, I.-J., Lee, I.-K., Yoon, E.-S., Koshino, H., Yoo, I.D., 2006. Hirsutenols D, E, and F, new sesquiterpenes from the culture broth of Stereum hirsutum. J. Antibiot. 59, 110–113. Yun, B.-S., Lee, I.-K., Cho, Y., Cho, S.-M., Yoo, I.-D., 2002. New tricyclic triterpenes from the fermentation broth of Stereum hirsutum. J. Nat. Prod. 65, 786–788.
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Sterostreins F–O, illudalanes and norilludalanes from cultures of the Basidiomycete Stereum ostrea BCC 22955 Masahiko Isaka ⇑, Urarat Srisanoh, Malipan Sappan, Sumalee Supothina, Thitiya Boonpratuang National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
a r t i c l e
i n f o
Article history: Received 7 December 2011 Received in revised form 17 February 2012 Available online 15 May 2012
a b s t r a c t Sterostreins F–O (1–10), 10 illudalanes and norilludalanes, were isolated from cultures of the Basidiomycete Stereum ostrea BCC 22955. Their structures were elucidated by analyses of the NMR spectroscopic and mass spectrometry data. Sterostreins M (8), N (9), and O (10) are pyridine-containing illudalanes. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Stereum ostrea Illudalane Norilludalane Basidiomycete
1. Introduction Woody mushrooms belonging to the genus Stereum have been the source of bioactive compounds. One of the major metabolite groups of this fungal genus are sesquiterpenoids, such as hirsutanes (Yun et al., 2002; Yoo et al., 2006; Liermann et al., 2010), sterpuranes (Xie et al., 1992), cadinanes (Li et al., 2006, 2008), stereumanes (Li et al., 2011), and drimanes (Kim et al., 2006), respectively. As part of our research program on biologically active fungal metabolites, dimeric and monomeric illudalane-type sesquiterpenes, sterostreins A–E, were recently isolated from cultures of Stereum ostrea BCC 22955 (Isaka et al., 2011). Further studies on this fungal strain resulted in the isolation of additional 10 new illudalanes and norilludalanes, sterostreins F–O (1–10) (Fig. 1). 2. Results and discussion In the proceeding study (Isaka et al., 2011), very slow mycelial growth of the producing fungus was observed under static fermentation. The use of a bioreactor accelerated the mycelial growth, but the conditions were not suitable for production of the targeted terpenoids. A new static fermentation was performed in threefold larger scale (250 ml 60 in 1 l Erlenmeyer flasks, and additional for time course study) than the preceding batch. A flask was harvested, extracted, and analyzed by 1H NMR every 10 or 15 day of incubation, which suggested that sterostreins started to be produced from 100 days of final fermentation. The cultures were harvested at day 130, which was the same duration as the previous ⇑ Corresponding author. Tel.: +66 25646700x3554; fax: +66 25646707. E-mail address: [email protected] (M. Isaka). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.04.009
fermentation. Extracts from the new batch contained 10 new analogs 1–10, together with the major constituents, sterostreins D (11) and E (12), and a small amount of a dimeric analog, sterostrein B. Sterostrein F (1) was isolated as a pale yellow gum, and the molecular formula was determined by HRESIMS as C15H20O5, which had one more oxygen atom than the major metabolites sterostreins D (11) and E (12). The 1H and 13C NMR spectroscopic data (Tables 1 and 2) were similar to those of 11. The difference was the absence of a methine, replaced by an oxygenated quaternary carbon (dC 83.1). The location of the tertiary alcohol was assigned to C-9 position on the basis of the HMBC correlations from Ha-1, Hb-1, Hb-3, and H-4 to this carbon. Sterostrein G (2) possessed the same molecular formula as 1, C15H20O5 (HRESIMS). The 1H and 13C NMR resonance patterns more resembled those of 12 rather than 11. Detailed analysis of 2D NMR data, including the HMBC correlations from Ha-1, Hb-1, and Hb-3 to C-9 (dC 84.3), established that it was also a 9-hydroxy analog of 11/12. The relative configurations of 1 and 2 were assigned on the basis of NOESY correlations as shown in Fig. 2. NOESY correlation of Ha-1/Ha-3 for 1 suggested the cis ring junction, while the correlation of Hb-1/H-4 for 2 indicated the trans ring junction. Examinations of the conformations of both isomers 9b-OH and 9a-OH isomers with a molecular model further supported this conclusion. The unusual downfield shift of Ha-3 (dH 2.06) for 2, when compared with 1 (dH 0.99), 11 (dH 0.98), and 12 (dH 1.48), can be explained by the deshielding of this proton by the 9a-OH group. The molecular formula of sterostrein H (3) was determined by HRESIMS as C15H18O4. The 1H and 13C NMR spectroscopic data and DEPT135 indicated the presence of a trisubstituted olefin, replacing a methine and a methylene of 11/12. This compound was identified as the 1,9-dehydro derivative of 11/12 on the basis
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15
7
1
8
6 13
4
5
14
11 2
9
10
3 12
15 7 14 6
13
1 8
11
2
9 4
5
10
3 12
3
15
14
7
6
13
8
1 9
5
11
2 4 3
10
12
Fig. 1. Structures of compounds 1–12.
Table 1 13 C NMR spectroscopic data (CDCl3, 125 MHz) for sterostreins F–O (1–10). No.
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a b,c
50.3 35.3 43.2 58.0 68.7 125.6 121.0 195.4 83.1 30.9 30.6 25.7 151.5 56.1 144.7
2
3
49.0 35.6 37.3 56.7 70.3 129.8 123.3 192.6 84.3 32.6 31.9 26.2 151.2 56.9 144.1
4 b
151.1 45.9 38.8 56.4 71.9 130.0 125.6 181.6 139.1 28.7 27.6 25.3 149.8 57.0 143.3
43.67 37.9 43.72b 50.2 70.7 154.2 132.7 201.5 47.3 30.6 30.3 27.5 31.8 61.7 12.1
5
6
7
8
9
10
42.6 37.5 43.9 51.9 70.3 160.4 127.5 201.5 47.6 31.2a 31.2a 26.4 35.6 63.0
40.2 36.4 41.4 54.8 73.8 168.6 127.3 200.4 51.7 31.8 31.6 19.5 32.8 63.1
148.2 45.4 39.5 54.8 74.4 171.6 127.6 186.5 137.5 28.6 27.6 22.8 33.7 62.9
40.0 36.6 41.2 53.7 73.0 160.3 126.2 198.2 52.2 31.7 31.5 22.8 120.6 153.7 148.8
53.3 38.7 45.0 58.0 70.9 153.8 126.8 200.7 83.3 29.3 27.9 23.9 119.5 154.7 148.6
44.4 37.8 47.4 164.2 68.4 156.6 125.2 181.8 136.3 29.33c 29.30c 29.2 120.5 152.6 148.6
The carbon resonances were superimposed. The assignment of carbons can be interchanged.
Table 2 1 H (500 MHz, CDCl3) NMR spectroscopic data for sterostreins F–H (1–3). No. 1 3 4 10 11 12 14 15
1
2
a 2.18, d (13.5)
a 1.68, d (14.7)
b 1.64, d (13.5) a 0.99, t (13.1) b 1.68, dd (12.9, 7.4) 2.73, dd (13.3, 7.4) 0.87, s 1.16, s 1.64, s 4.71, d (13.7) 4.65, d (13.7) 7.94, s
b 1.97, d (14.7) a 2.06, dd (13.3, 11.8) b 1.75, dd (11.8, 6.7) 2.52, dd (13.3, 6.7) 1.26, s 1.11, s 1.73, s 4.87, d (13.9) 4.69, d (13.9) 7.92, s
3
1 4
6.72, d (2.4)
a 1.87, dd (13.1, 8.4) b 2.01, dd (13.1, 8.4) 3.59, dt (2.4, 8.4) 1.24, s 1.15, s 1.39, s 4.82, d (13.7) 4.72, d (13.7) 7.89, s
of HMBC correlations from H-1 (dH 6.72) to C-2, C-3, C-8, and C-9, and the correlations from H-4 to the olefinic carbons C-1 (dC 151.1)
3
12
10 11
3
3
3
3
3
3
Fig. 2. Key NOESY correlations for 1 and 2.
and C-9 (dC 139.1). The upfield shift of the ketone C-8 (dC 181.8) when compared with 11 (dC 198.4) and 12 (dC 196.0) was consistent with the enone conjugation. The molecular formula of sterostrein I (4) was determined as C15H24O3 by HRESIMS. The 1H and 13C NMR spectroscopic data
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suggested the absence of the furan ring and the structural difference at this moiety. The resonance patterns for the cyclopentane and the central six-membered ketone were similar to those of 11 (Tables 1 and 3). Instead of the hydroxymethyl-furan (C-6, C-7, C-13, C-14, and C-15) in 11, sterostrein I (4) possessed a tetrasubstituted olefin (dC 132.7 and 154.2), an allylic methyl group at dC 12.1 (dH 1.82, s), and a 2-hydroxyethyl group. Its structure was established by analysis of HMBC correlations: from H3-15 to C-6, C-7, C-8; from H-4 and H3-12 to C-6; from H2-13 to C-5, C-6, and C-7; and from H2-14 to C-6. The cis ring junction was confirmed by the NOESY correlation of H-4/H-9 and the correlations from these protons to H3-11. This compound can also be assigned as an illudalane-type sesquiterpene. The molecular formula of sterostrein J (5), C14H22O3 (HRESIMS), suggested that it was a norilludalane. The 1H and 13C NMR spectroscopic data were similar to those of 4. The only difference was the absence of the allylic methyl group CH3-15 in 5, and a quaternary sp2 carbon (C-7) of 4 was replaced by an sp2 methine at dC 127.3 (dH 5.88, H-7). The location of the trisubstituted olefin (C-6/C-7) was confirmed by the HMBC correlations from H-7 to C-5, C-9, and C-13, and the correlations from H-4, H3-12, H2-13, and H2-14 to C-6. Sterostrein K (6) possessed the same molecular formula as 5 (C14H22O3, HRESIMS). The 1H and 13C NMR spectroscopic data for the cyclopentane moiety were similar to those of 12. It was identified as the C-9 epimer of 5, on the basis of the NOESY correlations H-4/Hb-1, H3-12/H-9, and H3-12/Ha-3. Sterostrein L (7), C14H20O3 (HRESIMS), was identified as the 1,9-dehydro derivative of 5/6. The location of the olefinic methine proton (H-1) was confirmed by the HMBC correlations from this proton to C-2, C-3, C4, C-8, C-9, and C-10. The relative configuration was suggested by the NOESY correlation of H3-12/Ha-3. The molecular formula of sterostrein M (8) was determined by HRESIMS as C15H19NO2, which indicated the incorporation of one nitrogen atom in the molecule. Analysis of the 1H and 13C NMR, DEPT135, HMQC and COSY spectroscopic data demonstrated the trans fused C-1–C-9 bicyclic ring moiety similar to 12. The NOESY correlations of H-9/Ha-3 and H-9/H3-12 indicated the relative configuration of C-9/C-4/C-5. Instead of the hydroxymethyl-furan C5 unit (C-6, C-7, C-13, C-14, and C-15), compound 8 contained three sp2 methines and two sp2 quaternary carbons. The pyridine-fused structure was addressed on the basis of the HMBC correlations (Fig. 3) and the chemical shifts of the heteroaromatic protons (H13, H-14, and H-15) and carbons (C-6, C-7, C-13, C-14, and C-15) (Tables 1 and 4). This compound is also assignable as an illudalane-type sesquiterpene. The 1H and 13C NMR spectroscopic data for sterostrein N (9) suggested that it was also a pyridine-containing illudalane. The molecular formula was determined by HRESIMS as C15H19NO3,
3 3 3
Fig. 3. Selected HMBC correlations for 8.
Table 4 1 H (500 MHz, CDCl3) NMR spectroscopic data for sterostreins M–O (8–10). No. 1 3 4 9 10 11 12 13 14 15
8
9
10
a 1.91, dd (13.3, 8.1)
a 1.59, d (13.9)
a 2.59, d (16.3)
b 1.77, dd (13.3, 10.3) a 1.60, t (12.3) b 1.81, dd (12.3, 6.7) 2.50, m 2.77, m 1.16, s 1.09, s 1.50, s 7.68, d (5.2) 8.66, d (5.2) 9.04, s
b 1.64, dd (13.9, 2.3) a 0.77, t (12.6) b 1.78, ddd (12.6, 7.6, 2.3) 2.96, dd (12.6, 7.6) 0.86, 1.22, 1.65, 7.36, 8.75, 8.92,
s s s d (5.1) d (5.1) s
b 2.53, d (16.3)
a 2.62, d (18.3) b 2.76, d (18.3)
1.19, 1.17, 1.60, 7.67, 8.64, 9.15,
s s s d (5.2) d (5.2) s
which had one oxygen atom more than 8. HMBC correlations from Ha-1, Hb-1, Hb-3, and H-4 to the tertiary alcohol carbon (dC 83.3) revealed that it was a 9-hydroxy analog. The cis ring junction was suggested by the close resemblance of the 1H and 13C NMR data for this region to those of 1. This relative configuration was strongly supported by the NOESY correlations of Ha-1/Ha-3 and H3-12/Ha-3, and the observation of a W-type 1H–1H coupling (J = 2.3 Hz) for Hb-1/Hb-3. The molecular formula of sterostrein O (10) was determined as C15H17NO2 by HRESIMS. It was identified as a 4,9-dehydro derivative by following key HMBC correlations: from H2-1 to C-4, C-8, and C-9; from H2-3 to C-4, C-5, and C-9; and from H3-12 to C-4, C-5, and C-6. The chemical shifts of C-8 (dC 181.8) and C-4 (dC 164.2) were consistent with the enone conjugation. The absolute configuration of the most abundant metabolite, sterostrein D (11) was previously determined by its conversion to the 8b-hydroxy secondary alcohol derivative (LiAlH4/THF) followed by application of the modified Mosher’s method. Application of the same strategy to the absolute configuration of sterostrein F (4) was unsuccessful, as LiAlH4 reduction of 4 gave a mixture of more than three products. Attempts at recrystallization of several of the new compounds and acylated derivatives of 4 for X-ray crystallographic analysis were also unsuccessful; therefore the absolute configurations of 4–10 remain unassigned. Although not
Table 3 1 H (500 MHz, CDCl3) NMR spectroscopic data for sterostreins I–L (4–7). No. 1 3 4 7 9 10 11 12 13 14 15
4
5
6
7
a 1.91, dd (13.3, 4.7)
a 2.13, dd (13.5, 2.5)
a 1.81, dd (13.2, 7.9)
6.51, d (2.6)
b 1.73, dd (13.3, 8.6) a 1.19, t (12.6) b 1.55, dd (12.6, 7.2) 2.64, m
b 1.67, dd (13.5, 8.8) a 1.15, t (12.7) b 1.53, dd (12.7, 6.6) 2.64, dt (12.7, 6.6) 5.88, s 2.92, m 0.97, s 1.04, s 1.39, s 2.69, ddd (14.4, 9.9, 3.9) 2.38, dt (14.4, 3.9) 4.02, m 3.76, dt (3.9, 9.9)
b 1.60, dd (13.2, 10.7) a 1.48, t (12.2) b 1.72, dd (12.2, 6.6) 2.32, ddd (13.7, 12.2, 6.6) 5.82, d (1.0) 2.52, ddd (13.7, 10.7, 7.9) 1.11, s 1.07, s 1.33, s 2.76, ddd (14.1, 9.8, 5.2) 2.44, ddt (14.1, 1.0, 4.2) 3.99, m 3.76, dt (4.2, 9.8)
2.97, dt (4.7, 8.1) 0.96, s 1.02, s 1.35, s 2.68–2.65, m 3.95, dt (9.8, 4.4) 3.72, m 1.82, s
a 1.82, dd (13.2, 8.2) b 1.96, dd (13.2, 8.2) 3.44, dt (2.6, 8.2) 5.89, s 1.20, 1.10, 1.17, 2.83, 2.44, 3.99, 3.77,
s s s ddd (13.9, 9.9, 5.0) dt (13.9, 4.0) m dt (4.0, 9.9)
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certain, the co-occurrence suggested that the new furan-containing analogs 1–3 also possess the same sense of absolute configuration of C-4 and C-5 as sterostrein D (11). 3. Conclusions Although many terpenoids have been isolated from fruiting bodies or cell cultures of Basidiomycetes, illudalane-type sesquiterpenes are relatively rare (Clericuzio et al., 1997; Suzuki et al., 2005; Pettit et al., 2010; Fushimi et al., 2010) when compared with biogenetically related illudanes. Most of the known illudalanes, from plants and fungi, possess benzene ring fused with a fivemembered ring. Sterostreins D–H (11, 12, 1–3) and M–O (8–10) are novel furan- and pyridine-containing tricyclic illudalanes, respectively. On the other hand, sterostreins J–L (5–7) are new norilludalanes. These compounds were produced after unusually long fermentation, which may be one of the reasons why they have been missed. Taking together the proceeding report of the dimeric analogs, the fungus S. ostrea BCC 22955 has probed to be a unique source of secondary metabolites. 4. Experimental 4.1. General procedures Melting points were measured with an Electrothermal IA9100 digital melting point apparatus. Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded on a GBC Cintra 404 spectrophotometer. FTIR spectra were taken on Bruker VECTOR 22 and ALPHA spectrometers. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. 4.2. Fungal material The mushroom S. ostrea was collected on bark of a dead hardwood tree in Khao Yai National Park, Nakhon Nayok Province, Thailand, by one of the authors (T.B.). The natural mushroom speciment was deposited in the BIOTEC Bangkok Herbarium as BBH 17035, and the living culture was deposited in the BIOTEC Culture Collection as BCC 22955 on August 21, 2006. 4.3. Fermentation and isolation Fermentation was performed in threefold larger scale than the previous study (Isaka et al., 2011), but under the similar conditions. The fungus BCC 22955 was maintained on potato dextrose agar at 25 °C. The agar was cut into small plugs and inoculated into 6 250 ml Erlenmeyer flasks containing 25 ml of potato dextrose broth (PDB; potato starch 4.0 g/l, dextrose 20.0 g/l). After incubation at 25 °C for 7 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1 l Erlenmeyer flask containing 250 ml of the same liquid medium (PDB), and incubated at 25 °C for 7 days on a rotary shaker (200 rpm). These secondary cultures were pooled and each 25 ml portion was transferred into 60 1 l Erlenmeyer flasks containing 250 ml of malt extract broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g/l, maltose 1.8 g/l, dextrose 6.0 g/l), and the final fermentation was carried out at 25 °C for 130 days under static conditions. The cultures were filtered to separate broth (filtrate) and mycelia (residue). The EtOAc extract from the culture filtrate (3.00 g) was subjected to CC on silica gel (5.0 18 cm, MeOH/CH2Cl2, step gradient elution from 0:100 to 15:85) to obtain nine pooled fractions. Fraction 5 (357 mg) was purified by preparative HPLC (SunFire Prep C18 OBD,
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19 150 mm, 5 lm; mobile phase MeCN/H2O, 20:80, flow rate 10 ml/min) to furnish 3 (4.6 mg), 12 (60 mg), 10 (8.0 mg), and 8 (15 mg). Fraction 6 (858 mg) was further fractionated by CC on silica gel (4.0 16 cm, MeOH/CH2Cl2, step gradient elution from 0:100 to 15:85), and the sub-fractions were further purified by preparative HPLC (SunFire Prep C18 OBD; MeCN/H2O, 20:80 or 25:75) to obtain 12 (15 mg), 4 (7.5 mg), sterostrein B (6.1 mg), 7 (28 mg), 6 (1.8 mg), 9 (78 mg), 5 (21 mg), and 11 (20 mg). Fraction 7 (350 mg) was subjected to preparative HPLC (SunFire Prep C18 OBD; MeCN/H2O, 30:70) to furnish 2 (3.8 mg), 7 (3.0 mg), 5 (9.0 mg), and 11 (178 mg). Fraction 8 (150 mg) was also purified by preparative HPLC (SunFire Prep C18 OBD; MeCN/H2O, 20:80) to afford 1 (9.1 mg) and 11 (21 mg). The mycelial extract (278 mg) contained much less quantities of the terpenoids, and no additional new compound was found by chromatographic fractionation. 4.3.1. Sterostrein F (1) Pale yellow gum; [a]27D 77 (c 0.10, MeOH); UV (MeOH) kmax (log e) 205 (4.11), 262 (3.37) nm; IR (ATR) mmax 3352, 2951, 1685, 1619, 1549, 1367, 1141, 1025, 899 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 2; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 303.1208 [M+Na]+ (calcd for C15H20O5Na, 303.1203). 4.3.2. Sterostrein G (2) Pale yellow gum; [a]24D 8 (c 0.21, MeOH); UV (MeOH) kmax (log e) 208 (3.98) nm; IR (ATR) mmax 3353, 2951, 1685, 1544, 1366, 1031 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 2; 13 C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 303.1216 [M+Na]+ (calcd for C15H20O5Na, 303.1203). 4.3.3. Sterostrein H (3) Pale yellow amorphous solid; [a]24D 48 (c 0.14, MeOH); UV (MeOH) kmax (log e) 209 (3.96), 274 (3.86) nm; IR (ATR) mmax 3316, 2955, 1654, 1610, 1559, 1145, 1121 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 2; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 263.1281 [M+H]+ (calcd for C15H19O4, 263.1278). 4.3.4. Sterostrein I (4) Colorless amorphous solid; [a]26D +20 (c 0.11, MeOH); UV (MeOH) kmax (log e) 252 (4.02), 287 (3.90) nm; IR (ATR) mmax 3352, 2951, 1647, 1366, 1041, 901 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 275.1607 [M+Na]+ (calcd for C15H24O3Na, 275.1618). 4.3.5. Sterostrein J (5) Colorless gum; [a]27D +59 (c 0.075, MeOH); UV (MeOH) kmax (log e) 234 (3.95) nm; IR (ATR) mmax 3350, 2950, 1649, 1366, 1044, 909 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 239.1640 [M+H]+ (calcd for C14H23O3, 239.1642). 4.3.6. Sterostrein K (6) Pale yellow amorphous solid; [a]27D 18 (c 0.09, MeOH); UV (MeOH) kmax (log e) 234 (3.90) nm; IR (ATR) mmax 3397, 3337, 2949, 1656, 1141, 1029 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 236.1639 [M+H]+ (calcd for C14H23O3, 239.1642). 4.3.7. Sterostrein L (7) Colorless solid; mp 142–144 °C; [a]26D 119 (c 0.105, MeOH); UV (MeOH) kmax (log e) 253 (3.89), 282 sh (3.79) nm; IR (ATR) mmax 3359, 3255, 2954, 1619, 1599, 1303, 1032, 836, 689 cm 1; 1H NMR
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(500 MHz, CDCl3) data, see Table 3; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 237.1481 [M+H]+ (calcd for C14H21O3, 237.1485). 4.3.8. Sterostrein M (8) Pale yellow solid; mp 152–154 °C; [a]26D +82 (c 0.095, MeOH); UV (MeOH) kmax (log e) 234 (3.89), 268 (3.29) nm; IR (ATR) mmax 3197, 2955, 1701, 1594, 854, 589 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 4; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 264.1489 [M+H]+ (calcd for C15H20NO2, 264.1489). 4.3.9. Sterostrein N (9) Pale yellow gum; [a]26D +48 (c 0.05, MeOH); UV (MeOH) kmax (log e) 233 (3.89), 264 sh (3.49) nm; IR (ATR) mmax 3277, 2949, 1692, 1594, 1368, 1029, 845 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 4; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 262.1431 [M+H]+ (calcd for C15H20NO3, 262.1438). 4.3.10. Sterostrein O (10) Pale brown solid; mp 163–164 °C; [a]23D 13 (c 0.10, MeOH); UV (MeOH) kmax (log e) 241 (3.82), 282 (3.77) nm; IR (ATR) mmax 3134, 1655, 1592, 1292, 1045, 605 cm 1; 1H NMR (500 MHz, CDCl3) data, see Table 4; 13C NMR (125 MHz, CDCl3) data, see Table 1; HRESIMS m/z 244.1340 [M+H]+ (calcd for C15H18NO2, 244.1332). Acknowledgment Financial support from the Bioresources Research Network, National Center for Genetic Engineering and Biotechnology (BIOTEC), is gratefully acknowledged. 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.2012. 04.009.
References Clericuzio, M., Pan, F., Han, F., Pang, Z., Sterner, O., 1997. Stearoyldelicone, an unstable protoilludane sesquiterpenoid from intact fruit bodies of Russula delica. Tetrahedron Lett. 38, 8237–8240. Fushimi, K., Horikawa, M., Suzuki, K., Sekiya, A., Kanno, S., Shimura, S., Kawagishi, H., 2010. Applanatines A–E from the culture broth of Ganoderma applanatum. Tetrahedron 66, 9332–9335. Isaka, M., Srisanoh, U., Choowong, W., Boonpratuang, T., 2011. Sterostreins A–E, new terpenoids from cultures of the Basidiomycete Stereum ostrea BCC 22955. Org. Lett. 13, 4886–4889. Kim, Y.-H., Yun, B.-S., Ryoo, I.-J., Kim, J.-P., Koshino, H., Yoo, I.-D., 2006. Methoxylaricinolic acid, a new sesquiterpene from the fruiting bodies of Stereum ostrea. J. Antibiot. 59, 432–434. Li, G.-H., Li, L., Duan, M., Zhang, K.-Q., 2006. The chemical constituents of the fungus Stereum sp. Chem. Biodiv. 3, 210–216. Li, G.-H., Duan, M., Yu, Z.-F., Li, L., Dong, J.-Y., Wang, X.-B., Guo, J.-W., Huang, R., Wang, M., Zhang, K.-Q., 2008. Stereumin A–E, sesquiterpenoids from the fungus Stereum sp. CCTCC AF 207024. Phytochemistry 69, 1439–1445. Li, G., Liu, F., Shen, L., Zhu, H., Zhang, K., 2011. Stereumins H–J, stereumane-type sesquiterpenes from the fungus Stereum sp. J. Nat. Prod. 74, 296–299. Liermann, J.C., Schüffler, A., Wollinsky, B., Birnbacher, J., Kolshorn, H., Anke, T., Opatz, T., 2010. Hirsutane-type sesquiterpenes with uncommon modifications from three Basidiomycetes. J. Org. Chem. 75, 2955–2961. Pettit, G.R., Meng, Y., Pettit, R.K., Herald, D.L., Cichacz, Z.A., Doubek, D.L., Richert, L., 2010. Antineoplastic agents: 556. Isolation and structure of coprinastatin 1 from Coprinus cinereus. J. Nat. Prod. 73, 388–392. Suzuki, S., Murayama, T., Shiono, Y., 2005. Illudalane sesquiterpenoids, echinolactones A and B, from a mycelial culture of Echinodontium japonicum. Phytochemistry 66, 2329–2333. Xie, J.-L., Li, L.-P., Dai, Z.-Q., 1992. Isolation and identification of two new metabolites from silver leaf fungus Stereum purpureum. J. Org. Chem. 57, 2313–2316. Yoo, N.-H., Kim, J.-P., Yun, B.-S., Ryoo, I.-J., Lee, I.-K., Yoon, E.-S., Koshino, H., Yoo, I.D., 2006. Hirsutenols D, E, and F, new sesquiterpenes from the culture broth of Stereum hirsutum. J. Antibiot. 59, 110–113. Yun, B.-S., Lee, I.-K., Cho, Y., Cho, S.-M., Yoo, I.-D., 2002. New tricyclic triterpenes from the fermentation broth of Stereum hirsutum. J. Nat. Prod. 65, 786–788.