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Drimane sesquiterpenoids from cultures of the basidiomycete Gymnopilus sp. BCC 19384
Phytochemistry Letters 35 (2020) 141–146
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Drimane sesquiterpenoids from cultures of the basidiomycete Gymnopilus sp. BCC 19384
T
Masahiko Isakaa,*, Malipan Sappana, Rapheephat Suvannakada, Thitiya Boonpratuangb, Tuksaporn Thummarukcharoenb a b
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand National Biobank of Thailand, 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
ARTICLE INFO
ABSTRACT
Keywords: Gymnopilus Drimane sesquiterpene Hymenogastraceae
Seven previously undescribed drimane sesquiterpenoids, gymnodrimanes A–G (1–7), were isolated from cultures of the basidiomycete Gymnopilus sp. BCC 19384. The structures were elucidated by spectroscopic analysis. The absolute configurations of 1 and 2 were determined by application of the modified Mosher’s method.
1. Introduction Gymnopilus is a genus of gilled mushroom within the fungal family Hymenogastraceae. Although over 250 species have been described, extensive chemical investigations have been done only for the biglaughter mushroom, Gymnopilus spectabilis (currently named as G. junonius): polyisoprenepolyols such as gymnoprenols and gymnopilins (Aoyagi et al., 1983; Nozoe et al., 1983a,b; Tanaka et al., 1993), and styryl 2-pyranones (Hatfield and Brady, 1969; Lee et al., 2008). There are many other chemically unexplored or rarely investigated species. As part of our continuing search for novel bioactive compounds from basidiomycetes (Isaka et al., 2017, 2019), a strain of Gymnopilus sp. (BCC 19384) was chemically investigated. Extracts from cultures of this strain displayed a unique 1H NMR spectroscopic profile, suggesting the presence of several terpenoid metabolites. Herein, we report the isolation and structure elucidation of seven new drimane sesquiterpenoids, named gymnodrimanes A–G (1–7) (Fig. 1). 2. Results and discussion Gymnodrimane A (1) was isolated as a colorless solid, and its molecular formula was determined to be C15H22O5 by HRESIMS. The 1H and 13C NMR spectroscopic data of 1 suggested that it is a ca. 2:1 mixture (in acetone-d6) of interchangeable isomers. The ratios of the isomers were ca. 2:1 in CDCl3 (poor solubility) and ca. 2:1 in DMSO-d6. Since the 1H and 13C NMR spectra in acetone-d6 displayed good peak dispersions, both isomers were successfully identified by interpretation of the 2D NMR data (Table 1). The NMR analysis and assignments of
⁎
protons and carbons were performed first for the major isomer. The DEPT-135 and HSQC data supported the presence of a carbonyl carbon (δC 168.5), a trisubstituted olefin, an acetal methine carbon (δC 102.0; δH 5.39, s), an oxymethine (δC 78.1; δH 3.28, m), an oxymethylene (δC 71.1; δH 4.19 and 3.64), two quaternary carbons, two methines, three methylenes, and two methyls. A drimane-type carbon skeleton was deduced from the COSY and HMBC data (Fig. 2). Thus, the presence of geminal methyl groups and the location of the secondary alcohol at C-3 were elucidated by the HMBC correlations from both singlet methyl protons, H3-13 (δH 1.04) and H3-14 (δH 0.94), to C-3, C-4, and C-5, and the cross correlations from H3-13 to C-14 and from H3-14 to C-13. The presence of a tetrahydrofuran ring, containing a hemiacetal, was revealed by the HMBC correlations from the hemiacetal proton (H-15) to C-1 and the oxymethylene carbon (C-11), and from the oxymethylene protons (H2-11) to C-8, C-9, C-10, and C-15. Location of the carboxyl group at C-12 was evident from the HMBC correlation from the olefinic proton (H-7) to the carbonyl carbon. The planar structure of the minor isomer was also elucidated similarly by interpretation of the 2D NMR spectra to be the same as the major isomer. Consequently, compound 1 should be a mixture of C-15 epimers. The relative configurations of these isomers were established on the basis of the NOESY data (Fig. 3). NOESY correlations between Hα-1/H-3, Hα-1/H-5, Hα-1/H-9, and H-3/ H-5 of the major isomer suggested axial- and α-orientation of these protons. The major isomer displayed NOESY cross-peaks of the acetal methine proton (H-15) with Hβ-1 and Hβ-2, which indicated an α-orientation of H-15. On the other hand, intense NOESY cross-peaks of H15 with H3-14 and Hβ-6 of the minor isomer demonstrated β-orientation of H-15.
Corresponding author. E-mail address: [email protected] (M. Isaka).
https://doi.org/10.1016/j.phytol.2019.11.016 Received 26 September 2019; Received in revised form 15 November 2019; Accepted 19 November 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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presence of an aldehyde (δC 205.2; δH 9.76) and an acetal (δC 105.1; δH 5.05). Detailed analysis of 2D NMR spectroscopic data revealed a drimane-type carbon skeleton (Fig. 2). Location of the formyl group (C-15) was indicated by the HMBC correlations from the formyl proton (H-15) to C-1 and C-10, and from H2-1, H-5, and H-9 to C-15. The presence of a methyl acetal, locating at C-11 and forming a γ-lactone, was revealed by the COSY correlation of H-9/H-11 and HMBC correlations from the acetal proton (H-11) to C-9, C-10, and the carbonyl carbon (δC 166.3, C12), and from the methoxy protons to C-11. The relative configuration of 3 was determined on the basis of NOESY correlations (Fig. 3). Correlations between Hα-1/H-5, Hα-1/H-9, and Hα-3/H-5 indicated α-orientation of these protons. Key NOESY correlations on the β-face were H-15/H3-14, H-15/Hβ-6, H-15/H-11, and Hβ-1/H-11. The molecular formula of gymnodrimane D (4) was determined to be C16H22O4 by HRESIMS. Its NMR spectra demonstrated the presence of two acetal carbons at δC 111.7 (δH 4.83, s) and δC 102.8 (δH 5.95, d, J = 5.5 Hz) (Table 2). The former acetal carbon (C-15) is bonded to a methoxy group, which was confirmed by the HMBC correlation from the methoxy protons to C-15. The other acetal carbon (C-11) links two five-membered rings, which was revealed by the HMBC correlations from the acetal proton (H-11) to C-8, C-15, and a carbonyl carbon at δC 169.6 (C-12) (Fig. 2). The relative configuration of 4 was elucidated by analysis of NOESY data (Fig. 3). The correlations between Hα-1/H-9 and H-5/H-9 demonstrated axial- and α-orientation of these protons. Intense cross-peak between H-9 and H-11 indicated a cis ring junction of the five-membered rings. The NOESY correlations between H-15/Hβ1, H-15/Hβ-2, and H3-14/15-OCH3, and the absence of the correlation between H3-14 and H-15 revealed β-orientation of 15-OCH3 (15S configuration). A cross-peak of Hβ-1 and H-11 further supported the proposed relative configuration. The molecular formula of gymnodrimane E (5) was determined to be C17H26O5 by HRESIMS. Interpretation of the 2D NMR spectroscopic data revealed the presence of two methyl acetal groups at δC 107.8 (C15) and δC 106.5 (C-11). A tetrahydrofuran ring, involving these acetal carbons, C-9, and C-10, was elucidated by the HMBC correlations from H-15 to C-1, C-5, and C-11, from H-11 to C-8, C-10, and C-15, and from H-9 to C-1, C-7, C-8, C-10, and C-11. The relative configuration of C-5/ C-9/C-10/C-11/C-15 was suggested by the NOESY correlations between H-15/H3-14, H-15/Hβ-6, H-15/H-11, and Hα-1/H-9. The small vicinal coupling constant for H-9 and H-11 (H-15 resonated as a singlet) in the 1 H NMR spectrum, and the very low cross-peak intensity of H-9/H-11 in the NOESY spectrum were consistent with the trans relation of these protons. Presence of an allylic coupling of H-7/H-9 (J =3.0 Hz) was confirmed by their COSY correlation. The molecular formula of gymnodrimane F (6), determined by HRESIMS, was the same as 5. Its 1H and 13C NMR spectroscopic data were similar to those of 5. Analysis of the 2D NMR data (COSY and HMBC) revealed the same planar structure as 5. NOESY correlations between Hα-1/H-5, Hα-1/H-9, and H-5/H-9 demonstrated the same relative configuration of C-5/C-9/C-10 as 5. A β-orientation of 15-OCH3 was deduced from the NOESY correlations between 15-OCH3/H3-14, H15/Hβ-1, and H-15/Hβ-2. Similarly to 5, a trans relation of H-9 and H11 (α-orientation of 11-OCH3) was suggested by the small coupling constant value for H-9 and H-11 (singlet), and their very low NOESY cross-peak intensity. NOESY correlations between H-11/15-OCH3 and H-15/11-OCH3 further supported the trans relation of the methoxy groups. Consequently, gymnodrimane F (6) was identified as the C-15 epimer of gymnodrimane E (5). The molecular formula of gymnodrimane G (7) was determined by HRESIMS as C15H20O4. The NMR spectroscopic data of 7 demonstrated the presence of two carbonyl carbons. Location of one of them at C-12 (δC 175.2), forming a γ-lactone, was deduced from the HMBC correlations from H-8 and H2-11 to C-12 (Fig. 2). Location of the other carbonyl group C-15 (δC 174.5), bridging C-7 and C-10 to form a δ-lactone, was revealed by the HMBC correlations from Hα-1, H-5, H-7, and H-9 to C-15. The relative configuration was determined on the basis of NOESY
Fig. 1. Structures of compounds 1–8, 9a, and 9b.
Gymnodrimane B (2) was assigned the molecular formula C16H24O5 by HRESIMS. The 1H and 13C NMR spectra displayed similarity to those of the major isomer (15β-OH) of 1. The difference was the presence of a methoxy group (δC 54.1; δH 3.07), replacing 15-OH of 1 (15β-OH). Location of the methoxy group was confirmed by the HMBC correlation from the methoxy protons to the acetal carbon (δC 109.6, C-15). The relative configuration of 2 was elucidated by analysis of NOESY data to be the same as 1 (15β-OH isomer). Thus, NOESY correlations between Hα-1/H-3, Hα-1/H-5, Hα-1/H-9, and H-3/H-5 demonstrated α-orientation of these protons. Intense NOESY correlation of H-15/Hβ-2, weaker correlations of H-15/H3-14 and H-15/Hβ-1, and the absence of the H15/Hβ-6 cross-peak indicated α-orientation of H-15. The structural relation between 1 and 2 was confirmed by a chemical correlation, and the absolute configuration of the secondary alcohol carbon (C-3) was determined by application of the modified Mosher’s method (Ohtani et al., 1991). Treatment of 1 with pTsOH·H2O in dry MeOH gave a mixture of 2 (major) and 15-epi-2 (minor), which was subsequently converted to the methyl esters 8 (major) and 15-epi-8 (minor) and separated. The methyl ester 8 was converted to Mosher ester derivatives 9a and 9b (Fig. 4). The Δδ-values indicated 3S-configuration of 8. Consequently, the absolute configuration of 1 was determined to be 3S,5S,9R,10R, and the absolute configuration of 2 was assigned to be 3S,5S,9R,10R,15S. The Mosher esters 9a and 9b were produced each as a single diastereomer, which indicated that the substrate 1 was not a nonequivalent mixture of enantiomers (enantiomerically pure). Therefore, it is not unreasonable to propose that other cometabolites, gymnodrimanes C–G (3–7), whose structures discussed below, should share the same absolute configuration of the drimane core (C-5/C-9/C-10) with 1 and 2 as shown in Fig. 1. The molecular formula of gymnodrimane C (3) was determined by HRESIMS to be C16H22O4. The NMR spectroscopic data indicated the 142
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Table 1 1 H and 13C NMR spectroscopic data for compounds 1 (acetone-d6), 2 (acetone-d6), and 3 (CDCl3). 1 : 15β-OH
1 : 15α-OH
2
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
1
38.2
α 1.21, m; β 1.83, dt (13.4, 3.4)
33.2
α 1.30, m; β 2.44, dt (13.2, 3.4)
38.7
α 1.23, m; β 1.82, m
32.1
2 3
29.6 78.1
α 1.64, m; β 1.64, m 3.28, m
30.3 78.0
α 1.57, m; β 2.04, m 3.24, m
30.0 78.5
α 1.65, m; β 1.67, m 3.29, dd (9.4, 6.7)
18.3 41.5
4 5 6 7 8 9 10 11
39.8 46.8 25.5 142.4 132.7 47.2 48.1 71.1
α 1.04, dt (4.8, 12.4); β 2.38, m α 1.51, m; β 1.48, m α 1.30, dt (6.3, 13.7); β 1.50, m
12 13 14 15 11-OCH3 15-OCH3
168.5 27.3 15.9 102.0
1.54, dd (12.4, 4.9) α 2.16, m; β 2.82, m 7.10, d (6.2) 2.59, m α 4.19, t (8.9); β 3.64, dd (8.9, 5.0) 1.04, s 0.94, s 5.39, s
39.6 47.5 22.6 142.0 132.2 53.7 48.0 67.1 168.2 27.2 14.2 98.4
1.37, dd (12.0, 4.0) α 2.33, m; β 2.18, m 7.16, d (6.4) 2.61, m α 4.01, t (8,9); β 3.64, dd (8.9, 5.9) 1.03, s 0.92, s 5.34, s
40.1 47.2 25.7 141.8 133.0 47.5 48.6 71.7 168.7 27.4 15.7 109.6 54.1
3
1.52, dd (12.2, 4.8) α 2.15, m; β 2.65, m 7.07, d (6.3) 2.60, m α 4.20, t (9.0); β 3.47, dd (9.0, 5.0) 1.03, s 0.84, s 4.83, s 3.07, s
33.4 49.2 24.8 135.6 126.8 53.6 47.6 105.1 166.3 31.2 20.3 205.2 58.0
1.82, dd (11.7, 6.3) α 2.72, m; β 2.54, m 6.92, m 2.74, m 5.05, d (6.1) 0.98, 0.79, 9.76, 3.55,
s s d (1.2) s
3. Experimental 3.1. General procedures Optical rotations were determined using a JASCO P-1030 digital polarimeter. UV spectra were recorded on an Analytik-jena SPEKOL 1200 spectrophotometer. FTIR spectra were acquired using a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured using a Bruker micrOTOF mass spectrometer. Preparative HPLC was performed on a Waters 600 System equipped with a Waters 2296 photodiode array detector (Waters, Milford, USA). Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and Silica gel 60H (particle size, 90 % < 45 μM; Merck KGaA, Darmstadt, Germany) were used for column chromatography. 3.2. Fungal material The mushroom specimens, growing on soil, were collected in Khao Phanoen Thung trail, Prachin Buri Province, Thailand, on December 1, 2005. The living culture is preserved in the BIOTEC Culture Collection as BCC 19384. On the basis of the ITS rDNA sequence data (GenBank accession number: MN220537) and phylogenetic algorithm used Maximum Likelihood (ML) to infer the evolutionary relationship of all taxa related under this genus, including this strain (BCC 19384) was identified as the genus Gymnopilus (Hymenogastraceae), but it was not assignable to the species level.
Fig. 2. COSY and HMBC correlations for 1, 3, 4, and 7.
3.3. Fermentation, extraction, and isolation
correlations (Fig. 3). Correlations between H-9/Hα-1, H-9/H-5, and H9/H-8 indicated α-orientation of these protons. The present study demonstrates that Gymnopilus is also a source of drimane-type sesquiterpenoids. Since the methyl acetal derivatives 2–6 were isolated through non-MeOH extraction/isolation procedures (see Experimental), it can be concluded that they were not isolation artifacts. The isolated compounds were tested for antibacterial (Bacillus cereus and Enterococcus faecium), antimycobacterial (Mycobacterium tuberculosis H37Ra), and antiplasmodial (Plasmodium falciparum K1) activities, and cytotoxicity to cancer cell-lines (MCF-7 and NCI-H187). However, all tested compounds were inactive in these biological assays at a concentration of 50 μg/ml (only for the antiplasmodial assay, the tested sample concentration was 10 μg/ml).
The fungus BCC 19384 was maintained on potato dextrose agar at 25 °C. The agar was cut into small plugs and inoculated into four 250-ml Erlenmeyer flasks containing 25 ml of malt extract broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g/l, maltose 1.8 g/l, and dextrose 6.0 g/ l). After incubation at 25 °C for 7 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1000-ml Erlenmeyer flask containing 250 ml of the same liquid medium (MEB), and incubated at 25 °C for 7 days on a rotary shaker (200 rpm). The secondary cultures were pooled, and each 25 ml portion was transferred into one of 40 1000-ml Erlenmeyer flasks containing 250 ml of MEB. The final fermentation was carried out at 25 °C for 95 days under static conditions. The cultures were filtered to separate broth and mycelia. The filtrate 143
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Fig. 3. Key NOESY correlations for 1, 3, 4, and 7.
Fraction 2 (3.32 g) contained terpenoid metabolites. This fraction was further separated by silica gel CC (3.5 × 15 cm, EtOAc/CH2Cl2, step gradient elution from 0:100 to 60:40) to obtain eleven fractions: fractions 2-1–2-11. Fraction 2-2 (479 mg) was purified by preparative HPLC using a reversed-phase column (Phenomenex Luna C18 100A, 21.2 × 250 mm, 10 μm; mobile phase MeCN/H2O, 70.30; flow rate 8 ml/min) to yield 3 (285 mg). Fraction 2–4 (15 mg) was further separated by silica gel CC (1.5 × 15 cm, acetone/CH2Cl2, step gradient elution from 0:100 to 10:90) to yield 7 (3.0 mg). Fraction 2–7 (370 mg) was purified by preparative HPLC (Phenomenex Luna; MeCN/H2O, 70.30) to yield 5 (11 mg). Considering a possibility that the methyl acetal derivatives (3 and 5) were formed during the chromatographic separation processes using MeOH, another half portion of the broth extract (5.0 g) was subjected to non-MeOH isolation procedures. The extract was fractionated by silica gel CC (3.5 × 15 cm, EtOAc/CH2Cl2, step gradient elution from 0:100 to 60:40) to obtain ten fractions: fractions 1–10. Fraction 2 (228 mg) was further separated by preparative HPLC (Phenomenex Luna; MeCN/ H2O, 70.30) to yield 3 (12 mg) and 4 (159 mg). Fraction 5 (538 mg) was purified by preparative HPLC (Merck LiChroCART 250-10, 21.2 × 250 mm, 10 μm; MeCN/H2O, 52:48; flow rate 4 ml/min) to
Fig. 4. Δδ-Values (δS–δR) of the Mosher esters 9a and 9b.
(broth) was extracted with EtOAc (2 × 8 l), and the combined organic layer was concentrated under reduced pressure to obtain a brown gum (10.2 g). A half portion of this extract (5.0 g) was subjected to fractionation by column chromatography (CC) on Sephadex LH-20 (4.0 × 60 cm, MeOH) to obtain four pooled fractions: fractions 1–4. Table 2 1 H and 13C NMR spectroscopic data for compounds 4–7 (CDCl3). 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 11-OCH3 15-OCH3
5
6
7
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
36.5 19.2 42.1 33.5 45.9 27.1 142.9 125.0 51.9 46.0 102.8 169.6 31.1 20.8 111.7
α 1.18, m; β 1.80, m 1.62–1.58 (2 H), m α 1.34, m;β 1.54, m
α 1.18, m; β 2.71, m α 1.38, m; β 1.93, m α 1.21, m; β 1.40, m
29.8 18.3 40.7 34.1 46.0 27.3 74.9 43.6 44.5 43.3 68.1 175.2 30.7 19.3 174.5
α 0.96, dt (4.4, 13.4); β 2.28, m α 1.55, m; β 1.84, m α 1.17, dt (3.7, 12.9); β 1.54, m
3.19, s
38.9 20.1 42.1 33.3 46.4 25.4 146.4 127.7 54.0 46.0 109.2 171.6 31.8 21.4 110.1 56.4 54.7
α 1.12, m; β 2.20, m α 1.52, m; β 1.62, m α 1.28, dt (4.1, 13.3); β 1.47, m
55.9
36.7 20.6 41.9 33.6 47.9 22.7 146.4 129.0 57.9 46.8 106.5 171.8 32.4 19.5 107.8 55.1 56.8
1.87, dd (10.4, 7.7) α 2.41, m; β 2.35, m 6.94, dt (4.1, 3.0) 2.81, m 5.95, d (5.5) 0.96, s 0.80, s 4.83, s
1.32, dd (12.5, 3.4) α 2.36, m; β 2.08, m 7.38, d (6.6) 2.91, br s 4.61, s 0.92, 0.83, 4.86, 3.38, 3.46,
s s s s s
1.51, m α 2.17, m; β 2.61, m 7.30, br d (4.1) 2.58, br s 4.87, s 0.91, 0.83, 4.95, 3.44, 3.22,
144
s s s s s
1.59, dd (10.4, 6.8) α 1.90, m; β 2.01, ddd (14.6, 6.8, 4.0) 5.08, dt (4.0, 1.9) 2.87, dd (11.0, 1.1) 2.72, ddd (11.0, 9.7, 4.9) α 4.40, dd (10.4, 9.7); β 4.02, dd (10.4, 4.9) 0.88, s 0.81, s
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yield 6 (32 mg). Fraction 6 (380 mg) was subjected to preparative HPLC (Phenomenex Luna; MeCN/H2O, 70:30) to yield 5 (25 mg). Fraction 7 (826 mg) was purified by preparative HPLC (Merck LiChroCART; MeCN/H2O, 55:45) to furnish 2 (11 mg). Fraction 8 (826 mg) was also purified by preparative HPLC (Phenomenex Luna; MeCN/H2O, 70:30) to yield 1 (596 mg).
CH2Cl2–MeOH (1:1, 1.0 ml) was added a solution of (trimethylsilyl) diazomethane in hexane (0.6 M, 0.40 ml) and the mixture was stirred at room temperature for 1 h. The reaction was quenched by addition of AcOH (150 μl), then 2 drops of ammonia solution was added, and the mixture was concentrated under reduced pressure to leave a pale yellow gum, which was purified by preparative HPLC (MeCN/H2O) to furnish the methyl ester derivatives 8 (5.3 mg) and 15-epi-8 (1.2 mg).
3.3.1. Gymnodrimane A (1) MeOH Colorless powder; [α]24 (nm) D ‒82 (c 0.10, MeOH); UV λmax (log ε): 230 (3.41); IR νmax ATR (cm−1): 3391, 1686, 1650, 1260, 1037; 1 H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6) data, Table 1; HRESIMS (m/z): 305.1364 [M+Na]+ (calc. for C15H22O5Na, 305.1359).
3.4.1. Gymnodrimane B methyl ester 8 Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.05 (1H, d, J = 6.3 Hz, H-7), 4.84 (1H, s, H-15), 4.22 (1H, t, J = 9.0 Hz, Hα-11), 3.66 (3H, s, –CO2CH3), 3.62 (1H, d, J = 5.3 Hz, 3-OH), 3.42 (1H, dd, J = 9.0, 5.0 Hz, Hβ-11), 3.28 (1H, m, H-3), 3.06 (3H, s, 15-OCH3), 2.64 (1H, m, Hβ-6), 2.59 (1H, m, H-9), 2.16 (1H, m, Hα-6), 1.82 (1H, m, Hβ1), 1.69-1.62 (2H, m, Hα-2 and Hβ-2), 1.52 (1H, dd, J = 12.4, 4.8 Hz, H5), 1.24 (1H, m, Hα-1), 1.03 (3H, s, H-13), 0.83 (3H, s, H-14); HRESIMS (m/z): 333.1670 [M+Na]+ (calc. for C17H26NaO5, 333.1672).
3.3.2. Gymnodrimane B (2) MeOH Colorless powder; [α]24 (nm) D ‒25 (c 0.10, MeOH); UV λmax −1 (log ε): 231 (3.43); IR νmax ATR (cm ): 3410, 1703, 1651, 1104, 1043; 1 H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6) data, Table 1; HRESIMS (m/z): 319.1523 [M+Na]+ (calc. for C16H24O5Na, 319.1516).
3.4.2. 15-Epi-8 Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.14 (1H, d, J = 6.5 Hz, H-7), 4.77 (1H, s, H-15), 3.88 (1H, dd, J = 8.8, 4.6 Hz, Hα11), 3.68 (3H, s, –CO2CH3), 3.46 (1H, d, J = 5.3 Hz, 3-OH), 3.40 (d, J = 8.8 Hz, Hβ-11), 3.31 (3H, s, 15-OCH3), 3.23 (1H, m, H-3), 2.61 (1H, m, H-9), 2.34 (1H, m, Hβ-1), 2.32 (1H, m, Hα-6), 2.19 (1H, m, Hβ-6), 1.88 (1H, m, Hβ-2), 1.55 (1H, m, Hα-2), 1.35 (1H, dd, J = 12.5, 4.1 Hz, H-5), 1.02 (3H, s, H-13), 0.82 (3H, s, H-14); HRESIMS (m/z): HRESIMS (m/z): 333.1673 [M+Na]+ (calc. for C17H26NaO5, 333.1672).
3.3.3. Gymnodrimane C (3) MeOH Colorless amorphous solid; [α]24 D ‒19 (c 0.10, MeOH); UV λmax −1 (nm) (log ε): 234 (3.44); IR νmax ATR (cm ): 1766, 1713, 1220, 1141, 923; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 301.1423 [M+Na]+ (calc. for C16H22O4Na, 301.1410). 3.3.4. Gymnodrimane D (4) MeOH Colorless powder; [α]23 (nm) D +152 (c 0.10, MeOH); UV λmax (log ε): 232 (3.43); IR νmax ATR (cm−1): 1773, 1686, 1209, 1104, 944; 1 H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 301.1417 [M+Na]+ (calc. for C16H22O4Na, 301.1410).
3.5. Synthesis of the Mosher ester derivatives 9a and 9b Compound 8 (1.0 mg) was treated with (-)-(R)-MTPA-Cl (10 μl) and 4-dimethylaminopyridine (10 mg) in CH2Cl2 (0.2 ml) at room temperature for 16 h. The mixture was diluted with EtOAc and washed with H2O, 0.1 M HCl (×2), and then with 1 M NaHCO3 (×2), and the organic layer was concentrated in vacuo to obtain a pale yellow gum (2.6 mg), which is mainly composed of an (S)-MTPA ester derivative 9a. Similarly, (R)-MTPA ester derivative 9b was prepared from 8 and (+)-(S)-MTPA-Cl.
3.3.5. Gymnodrimane E (5) MeOH Colorless powder; [α]24 (nm) D ‒99 (c 0.10, MeOH); UV λmax −1 (log ε): 227 (3.37); IR νmax ATR (cm ): 1711, 1689, 1652, 1099; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 333.1687 [M+Na]+ (calc. for C17H26O5Na, 333.1672).
3.5.1. (S)-MTPA ester 9a Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.56 (2H, m, phenyl of MTPA), 7.50 (3H, m, phenyl of MTPA), 7.05 (1H, d, J = 6.2 Hz, H-7), 4.89 (1H, s, H-15), 4.86 (1H, dd, J = 11.7, 4.5 Hz, H3), 4.25 (1H, t, J = 9.0 Hz, Hα-11), 3.68 (3H, s, –CO2CH3), 3.55 (3H, s, OCH3 of MTPA), 3.44 (1H, dd, J = 9.0, 5.0 Hz, Hβ-11), 3.17 (3H, s, 15OCH3), 2.67 (2H, m, Hβ-6 and H-9), 2.21 (1H, m, Hα-6), 1.90 (2H, m, Hβ-1 and Hα-2), 1.74 (2H, m, Hβ-2 and H-5), 1.39 (1H, m, Hα-1), 1.04 (3H, s, H-13), 0.93 (3H, s, H-14); HRESIMS (m/z): 549.2084 [M+Na]+ (calc. for C27H33F3O5Na, 549.2071).
3.3.6. Gymnodrimane F (6) MeOH Colorless powder; [α]24 (nm) D +12 (c 0.10, MeOH); UV λmax −1 (log ε): 228 (3.52); IR νmax ATR (cm ): 1685, 1649, 1276, 1106, 1010; 1 H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 333.1681 [M+Na]+ (calc. for C17H26O5Na, 333.1672). 3.3.7. Gymnodrimane G (7) Colorless amorphous solid; [α]23 +81 (c 0.10, MeOH); UV D λmaxMeOH (nm) (log ε): 223 (2.80); IR νmax ATR (cm−1): 1773, 1748, 1188, 1050; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 287.1267 [M+Na]+ (calc. for C15H20O4Na, 287.1254).
3.5.2. (R)-MTPA ester 9b Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.58 (2H, m, phenyl of MTPA), 7.48 (3H, m, phenyl of MTPA), 7.04 (1H, d, J = 6.3 Hz, H-7), 4.94 (1H, s, H-15), 4.85 (1H, dd, J = 10.4, 5.6 Hz, H3), 4.26 (1H, t, J = 9.0 Hz, Hα-11), 3.68 (3H, s, -CO2CH3), 3.59 (3H, s, OCH3 of MTPA), 3.45 (1H, dd, J = 9.0, 5.0 Hz, Hβ-11), 3.18 (3H, s, 15OCH3), 2.67 (1H, m, H-9), 2.64 (1H, m, Hβ-6), 2.17 (1H, m, Hα-6), 1.96 (2H, m, Hβ-1 and Hα-2), 1.93 (1H, m, Hβ-2), 1.73 (1H, dd, J = 12.2, 4.8 Hz, H-5), 1.42 (1H, m, Hα-1), 0.88 (3H, s, H-13), 0.87 (3H, s, H-14); HRESIMS (m/z): 549.2081 [M+Na]+ (calc. for C27H33F3O5Na, 549.2071).
3.4. Synthesis of the methyl ester derivative 8 To a solution of 1 (20 mg) in MeOH (1.5 ml) was added p-TsOH·H2O (1 mg) and the mixture was stirred at room temperature for 15 h. The reaction was terminated by addition of 2 drops of ammonia solution, and the mixture was concentrated by evaporation. The residue was dissolved in EtOAc, washed with H2O, and the organic phase was concentrated under reduced pressure to obtain a colorless gum (21 mg), whose 1H NMR spectroscopic data indicated that it was mainly composed of 2. To a solution of a portion of this reaction product (10 mg) in
3.6. Biological assays Antibacterial activities against Bacillus cereus and Enterococcus faecium were performed using the resazurin microplate assay and the 145
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optical density microplate assay (OD600), respectively (Clinical and Laboratory Standards Institute (CLSI, 2006). Antimycobacterial activity against Mycobacterium tuberculosis H37Ra was evaluated using the green fluorescent protein microplate assay (Changsen et al., 2003). An assay for activity against Plasmodium falciparum (K1, multidrug resistant strain) was performed using the microculture radioisotope technique (Desjardins et al., 1979). Cytotoxic activities against the tumor cell-lines, NCI-H187 (human small-cell lung cancer) and MCF-7 (human breast cancer), were evaluated using the resazurin microplate assay (O’Brien et al., 2000).
Changsen, C., Franzblau, S.G., Palittapongarnpim, P., 2003. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob. Agents Chemother. 47, 3682–3687. Clinical and Laboratory Standards Institute (CLSI), 2006. Method for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically. Approved Standard, M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA, USA. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710–718. Hatfield, G.M., Brady, L.R., 1969. Occurrence of bis-noryangonin in Gymnopilus spectabilis. J. Pharm. Sci. 58, 1298–1299. Isaka, M., Chinthanom, P., Sappan, M., Supothina, S., Vichai, V., Danwisetkanjana, K., Boonpratuang, T., Hyde, K.D., Choeyklin, R., 2017. Antitubercular activity of mycelium-associated Ganoderma lanostanoids. J. Nat. Prod. 80, 1361–1369. Isaka, M., Chinthanom, P., Thummarukcharoen, T., Boonpratuang, T., Choowong, W., 2019. Highly modified lanostane triterpenes from fruiting bodies of the basidiomycete Tomophagus sp. J. Nat. Prod. 82, 1165–1176. Lee, I.K., Cho, S.M., Seok, S.J., Yun, B.S., 2008. Chemical constituents of Gymnopilus spectabilis and their antioxidant activity. Mycobiology 36, 55–59. Nozoe, S., Koike, Y., Tsuji, E., Kusano, G., Seto, H., 1983a. Isolation and structure of gymnoprenols, a novel type of polyisoprenepolyols from Gymnopilus spectabilis. Tetrahedron Lett. 24, 1731–1734. Nozoe, S., Koike, Y., Kusano, G., Seto, H., 1983b. Structure of gymnopilin, a bitter principle of an hallucinogenic mushroom, Gymnopilus spectabilis. Tetrahedron Lett. 24, 1735–1736. O’Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. 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. Tanaka, M., Hashimoto, K., Okuno, T., Shirahama, H., 1993. Neurotoxic oligoisoprenoids of the hallucinogenic mushroom, Gymnopilus spectabilis. Phytochemistry 34, 661–664.
Acknowledgements Financial support from the Thailand Research Fund (Grant No. DBG628008) is gratefully acknowledged. Identification of the fungus used in this study was supported by the National Biobank of Thailand. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.11.016. References Aoyagi, F., Maeno, S., Okuno, T., Matsumoto, H., Ikura, M., Hikichi, K., Matsumoto, T., 1983. Gymnopilins, bitter principles of the big-laughter mushroom Gymnopilus spectabilis. Tetrahedron Lett. 24, 1991–1994.
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Drimane sesquiterpenoids from cultures of the basidiomycete Gymnopilus sp. BCC 19384
T
Masahiko Isakaa,*, Malipan Sappana, Rapheephat Suvannakada, Thitiya Boonpratuangb, Tuksaporn Thummarukcharoenb a b
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand National Biobank of Thailand, 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
ARTICLE INFO
ABSTRACT
Keywords: Gymnopilus Drimane sesquiterpene Hymenogastraceae
Seven previously undescribed drimane sesquiterpenoids, gymnodrimanes A–G (1–7), were isolated from cultures of the basidiomycete Gymnopilus sp. BCC 19384. The structures were elucidated by spectroscopic analysis. The absolute configurations of 1 and 2 were determined by application of the modified Mosher’s method.
1. Introduction Gymnopilus is a genus of gilled mushroom within the fungal family Hymenogastraceae. Although over 250 species have been described, extensive chemical investigations have been done only for the biglaughter mushroom, Gymnopilus spectabilis (currently named as G. junonius): polyisoprenepolyols such as gymnoprenols and gymnopilins (Aoyagi et al., 1983; Nozoe et al., 1983a,b; Tanaka et al., 1993), and styryl 2-pyranones (Hatfield and Brady, 1969; Lee et al., 2008). There are many other chemically unexplored or rarely investigated species. As part of our continuing search for novel bioactive compounds from basidiomycetes (Isaka et al., 2017, 2019), a strain of Gymnopilus sp. (BCC 19384) was chemically investigated. Extracts from cultures of this strain displayed a unique 1H NMR spectroscopic profile, suggesting the presence of several terpenoid metabolites. Herein, we report the isolation and structure elucidation of seven new drimane sesquiterpenoids, named gymnodrimanes A–G (1–7) (Fig. 1). 2. Results and discussion Gymnodrimane A (1) was isolated as a colorless solid, and its molecular formula was determined to be C15H22O5 by HRESIMS. The 1H and 13C NMR spectroscopic data of 1 suggested that it is a ca. 2:1 mixture (in acetone-d6) of interchangeable isomers. The ratios of the isomers were ca. 2:1 in CDCl3 (poor solubility) and ca. 2:1 in DMSO-d6. Since the 1H and 13C NMR spectra in acetone-d6 displayed good peak dispersions, both isomers were successfully identified by interpretation of the 2D NMR data (Table 1). The NMR analysis and assignments of
⁎
protons and carbons were performed first for the major isomer. The DEPT-135 and HSQC data supported the presence of a carbonyl carbon (δC 168.5), a trisubstituted olefin, an acetal methine carbon (δC 102.0; δH 5.39, s), an oxymethine (δC 78.1; δH 3.28, m), an oxymethylene (δC 71.1; δH 4.19 and 3.64), two quaternary carbons, two methines, three methylenes, and two methyls. A drimane-type carbon skeleton was deduced from the COSY and HMBC data (Fig. 2). Thus, the presence of geminal methyl groups and the location of the secondary alcohol at C-3 were elucidated by the HMBC correlations from both singlet methyl protons, H3-13 (δH 1.04) and H3-14 (δH 0.94), to C-3, C-4, and C-5, and the cross correlations from H3-13 to C-14 and from H3-14 to C-13. The presence of a tetrahydrofuran ring, containing a hemiacetal, was revealed by the HMBC correlations from the hemiacetal proton (H-15) to C-1 and the oxymethylene carbon (C-11), and from the oxymethylene protons (H2-11) to C-8, C-9, C-10, and C-15. Location of the carboxyl group at C-12 was evident from the HMBC correlation from the olefinic proton (H-7) to the carbonyl carbon. The planar structure of the minor isomer was also elucidated similarly by interpretation of the 2D NMR spectra to be the same as the major isomer. Consequently, compound 1 should be a mixture of C-15 epimers. The relative configurations of these isomers were established on the basis of the NOESY data (Fig. 3). NOESY correlations between Hα-1/H-3, Hα-1/H-5, Hα-1/H-9, and H-3/ H-5 of the major isomer suggested axial- and α-orientation of these protons. The major isomer displayed NOESY cross-peaks of the acetal methine proton (H-15) with Hβ-1 and Hβ-2, which indicated an α-orientation of H-15. On the other hand, intense NOESY cross-peaks of H15 with H3-14 and Hβ-6 of the minor isomer demonstrated β-orientation of H-15.
Corresponding author. E-mail address: [email protected] (M. Isaka).
https://doi.org/10.1016/j.phytol.2019.11.016 Received 26 September 2019; Received in revised form 15 November 2019; Accepted 19 November 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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presence of an aldehyde (δC 205.2; δH 9.76) and an acetal (δC 105.1; δH 5.05). Detailed analysis of 2D NMR spectroscopic data revealed a drimane-type carbon skeleton (Fig. 2). Location of the formyl group (C-15) was indicated by the HMBC correlations from the formyl proton (H-15) to C-1 and C-10, and from H2-1, H-5, and H-9 to C-15. The presence of a methyl acetal, locating at C-11 and forming a γ-lactone, was revealed by the COSY correlation of H-9/H-11 and HMBC correlations from the acetal proton (H-11) to C-9, C-10, and the carbonyl carbon (δC 166.3, C12), and from the methoxy protons to C-11. The relative configuration of 3 was determined on the basis of NOESY correlations (Fig. 3). Correlations between Hα-1/H-5, Hα-1/H-9, and Hα-3/H-5 indicated α-orientation of these protons. Key NOESY correlations on the β-face were H-15/H3-14, H-15/Hβ-6, H-15/H-11, and Hβ-1/H-11. The molecular formula of gymnodrimane D (4) was determined to be C16H22O4 by HRESIMS. Its NMR spectra demonstrated the presence of two acetal carbons at δC 111.7 (δH 4.83, s) and δC 102.8 (δH 5.95, d, J = 5.5 Hz) (Table 2). The former acetal carbon (C-15) is bonded to a methoxy group, which was confirmed by the HMBC correlation from the methoxy protons to C-15. The other acetal carbon (C-11) links two five-membered rings, which was revealed by the HMBC correlations from the acetal proton (H-11) to C-8, C-15, and a carbonyl carbon at δC 169.6 (C-12) (Fig. 2). The relative configuration of 4 was elucidated by analysis of NOESY data (Fig. 3). The correlations between Hα-1/H-9 and H-5/H-9 demonstrated axial- and α-orientation of these protons. Intense cross-peak between H-9 and H-11 indicated a cis ring junction of the five-membered rings. The NOESY correlations between H-15/Hβ1, H-15/Hβ-2, and H3-14/15-OCH3, and the absence of the correlation between H3-14 and H-15 revealed β-orientation of 15-OCH3 (15S configuration). A cross-peak of Hβ-1 and H-11 further supported the proposed relative configuration. The molecular formula of gymnodrimane E (5) was determined to be C17H26O5 by HRESIMS. Interpretation of the 2D NMR spectroscopic data revealed the presence of two methyl acetal groups at δC 107.8 (C15) and δC 106.5 (C-11). A tetrahydrofuran ring, involving these acetal carbons, C-9, and C-10, was elucidated by the HMBC correlations from H-15 to C-1, C-5, and C-11, from H-11 to C-8, C-10, and C-15, and from H-9 to C-1, C-7, C-8, C-10, and C-11. The relative configuration of C-5/ C-9/C-10/C-11/C-15 was suggested by the NOESY correlations between H-15/H3-14, H-15/Hβ-6, H-15/H-11, and Hα-1/H-9. The small vicinal coupling constant for H-9 and H-11 (H-15 resonated as a singlet) in the 1 H NMR spectrum, and the very low cross-peak intensity of H-9/H-11 in the NOESY spectrum were consistent with the trans relation of these protons. Presence of an allylic coupling of H-7/H-9 (J =3.0 Hz) was confirmed by their COSY correlation. The molecular formula of gymnodrimane F (6), determined by HRESIMS, was the same as 5. Its 1H and 13C NMR spectroscopic data were similar to those of 5. Analysis of the 2D NMR data (COSY and HMBC) revealed the same planar structure as 5. NOESY correlations between Hα-1/H-5, Hα-1/H-9, and H-5/H-9 demonstrated the same relative configuration of C-5/C-9/C-10 as 5. A β-orientation of 15-OCH3 was deduced from the NOESY correlations between 15-OCH3/H3-14, H15/Hβ-1, and H-15/Hβ-2. Similarly to 5, a trans relation of H-9 and H11 (α-orientation of 11-OCH3) was suggested by the small coupling constant value for H-9 and H-11 (singlet), and their very low NOESY cross-peak intensity. NOESY correlations between H-11/15-OCH3 and H-15/11-OCH3 further supported the trans relation of the methoxy groups. Consequently, gymnodrimane F (6) was identified as the C-15 epimer of gymnodrimane E (5). The molecular formula of gymnodrimane G (7) was determined by HRESIMS as C15H20O4. The NMR spectroscopic data of 7 demonstrated the presence of two carbonyl carbons. Location of one of them at C-12 (δC 175.2), forming a γ-lactone, was deduced from the HMBC correlations from H-8 and H2-11 to C-12 (Fig. 2). Location of the other carbonyl group C-15 (δC 174.5), bridging C-7 and C-10 to form a δ-lactone, was revealed by the HMBC correlations from Hα-1, H-5, H-7, and H-9 to C-15. The relative configuration was determined on the basis of NOESY
Fig. 1. Structures of compounds 1–8, 9a, and 9b.
Gymnodrimane B (2) was assigned the molecular formula C16H24O5 by HRESIMS. The 1H and 13C NMR spectra displayed similarity to those of the major isomer (15β-OH) of 1. The difference was the presence of a methoxy group (δC 54.1; δH 3.07), replacing 15-OH of 1 (15β-OH). Location of the methoxy group was confirmed by the HMBC correlation from the methoxy protons to the acetal carbon (δC 109.6, C-15). The relative configuration of 2 was elucidated by analysis of NOESY data to be the same as 1 (15β-OH isomer). Thus, NOESY correlations between Hα-1/H-3, Hα-1/H-5, Hα-1/H-9, and H-3/H-5 demonstrated α-orientation of these protons. Intense NOESY correlation of H-15/Hβ-2, weaker correlations of H-15/H3-14 and H-15/Hβ-1, and the absence of the H15/Hβ-6 cross-peak indicated α-orientation of H-15. The structural relation between 1 and 2 was confirmed by a chemical correlation, and the absolute configuration of the secondary alcohol carbon (C-3) was determined by application of the modified Mosher’s method (Ohtani et al., 1991). Treatment of 1 with pTsOH·H2O in dry MeOH gave a mixture of 2 (major) and 15-epi-2 (minor), which was subsequently converted to the methyl esters 8 (major) and 15-epi-8 (minor) and separated. The methyl ester 8 was converted to Mosher ester derivatives 9a and 9b (Fig. 4). The Δδ-values indicated 3S-configuration of 8. Consequently, the absolute configuration of 1 was determined to be 3S,5S,9R,10R, and the absolute configuration of 2 was assigned to be 3S,5S,9R,10R,15S. The Mosher esters 9a and 9b were produced each as a single diastereomer, which indicated that the substrate 1 was not a nonequivalent mixture of enantiomers (enantiomerically pure). Therefore, it is not unreasonable to propose that other cometabolites, gymnodrimanes C–G (3–7), whose structures discussed below, should share the same absolute configuration of the drimane core (C-5/C-9/C-10) with 1 and 2 as shown in Fig. 1. The molecular formula of gymnodrimane C (3) was determined by HRESIMS to be C16H22O4. The NMR spectroscopic data indicated the 142
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Table 1 1 H and 13C NMR spectroscopic data for compounds 1 (acetone-d6), 2 (acetone-d6), and 3 (CDCl3). 1 : 15β-OH
1 : 15α-OH
2
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
1
38.2
α 1.21, m; β 1.83, dt (13.4, 3.4)
33.2
α 1.30, m; β 2.44, dt (13.2, 3.4)
38.7
α 1.23, m; β 1.82, m
32.1
2 3
29.6 78.1
α 1.64, m; β 1.64, m 3.28, m
30.3 78.0
α 1.57, m; β 2.04, m 3.24, m
30.0 78.5
α 1.65, m; β 1.67, m 3.29, dd (9.4, 6.7)
18.3 41.5
4 5 6 7 8 9 10 11
39.8 46.8 25.5 142.4 132.7 47.2 48.1 71.1
α 1.04, dt (4.8, 12.4); β 2.38, m α 1.51, m; β 1.48, m α 1.30, dt (6.3, 13.7); β 1.50, m
12 13 14 15 11-OCH3 15-OCH3
168.5 27.3 15.9 102.0
1.54, dd (12.4, 4.9) α 2.16, m; β 2.82, m 7.10, d (6.2) 2.59, m α 4.19, t (8.9); β 3.64, dd (8.9, 5.0) 1.04, s 0.94, s 5.39, s
39.6 47.5 22.6 142.0 132.2 53.7 48.0 67.1 168.2 27.2 14.2 98.4
1.37, dd (12.0, 4.0) α 2.33, m; β 2.18, m 7.16, d (6.4) 2.61, m α 4.01, t (8,9); β 3.64, dd (8.9, 5.9) 1.03, s 0.92, s 5.34, s
40.1 47.2 25.7 141.8 133.0 47.5 48.6 71.7 168.7 27.4 15.7 109.6 54.1
3
1.52, dd (12.2, 4.8) α 2.15, m; β 2.65, m 7.07, d (6.3) 2.60, m α 4.20, t (9.0); β 3.47, dd (9.0, 5.0) 1.03, s 0.84, s 4.83, s 3.07, s
33.4 49.2 24.8 135.6 126.8 53.6 47.6 105.1 166.3 31.2 20.3 205.2 58.0
1.82, dd (11.7, 6.3) α 2.72, m; β 2.54, m 6.92, m 2.74, m 5.05, d (6.1) 0.98, 0.79, 9.76, 3.55,
s s d (1.2) s
3. Experimental 3.1. General procedures Optical rotations were determined using a JASCO P-1030 digital polarimeter. UV spectra were recorded on an Analytik-jena SPEKOL 1200 spectrophotometer. FTIR spectra were acquired using a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured using a Bruker micrOTOF mass spectrometer. Preparative HPLC was performed on a Waters 600 System equipped with a Waters 2296 photodiode array detector (Waters, Milford, USA). Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and Silica gel 60H (particle size, 90 % < 45 μM; Merck KGaA, Darmstadt, Germany) were used for column chromatography. 3.2. Fungal material The mushroom specimens, growing on soil, were collected in Khao Phanoen Thung trail, Prachin Buri Province, Thailand, on December 1, 2005. The living culture is preserved in the BIOTEC Culture Collection as BCC 19384. On the basis of the ITS rDNA sequence data (GenBank accession number: MN220537) and phylogenetic algorithm used Maximum Likelihood (ML) to infer the evolutionary relationship of all taxa related under this genus, including this strain (BCC 19384) was identified as the genus Gymnopilus (Hymenogastraceae), but it was not assignable to the species level.
Fig. 2. COSY and HMBC correlations for 1, 3, 4, and 7.
3.3. Fermentation, extraction, and isolation
correlations (Fig. 3). Correlations between H-9/Hα-1, H-9/H-5, and H9/H-8 indicated α-orientation of these protons. The present study demonstrates that Gymnopilus is also a source of drimane-type sesquiterpenoids. Since the methyl acetal derivatives 2–6 were isolated through non-MeOH extraction/isolation procedures (see Experimental), it can be concluded that they were not isolation artifacts. The isolated compounds were tested for antibacterial (Bacillus cereus and Enterococcus faecium), antimycobacterial (Mycobacterium tuberculosis H37Ra), and antiplasmodial (Plasmodium falciparum K1) activities, and cytotoxicity to cancer cell-lines (MCF-7 and NCI-H187). However, all tested compounds were inactive in these biological assays at a concentration of 50 μg/ml (only for the antiplasmodial assay, the tested sample concentration was 10 μg/ml).
The fungus BCC 19384 was maintained on potato dextrose agar at 25 °C. The agar was cut into small plugs and inoculated into four 250-ml Erlenmeyer flasks containing 25 ml of malt extract broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g/l, maltose 1.8 g/l, and dextrose 6.0 g/ l). After incubation at 25 °C for 7 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1000-ml Erlenmeyer flask containing 250 ml of the same liquid medium (MEB), and incubated at 25 °C for 7 days on a rotary shaker (200 rpm). The secondary cultures were pooled, and each 25 ml portion was transferred into one of 40 1000-ml Erlenmeyer flasks containing 250 ml of MEB. The final fermentation was carried out at 25 °C for 95 days under static conditions. The cultures were filtered to separate broth and mycelia. The filtrate 143
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Fig. 3. Key NOESY correlations for 1, 3, 4, and 7.
Fraction 2 (3.32 g) contained terpenoid metabolites. This fraction was further separated by silica gel CC (3.5 × 15 cm, EtOAc/CH2Cl2, step gradient elution from 0:100 to 60:40) to obtain eleven fractions: fractions 2-1–2-11. Fraction 2-2 (479 mg) was purified by preparative HPLC using a reversed-phase column (Phenomenex Luna C18 100A, 21.2 × 250 mm, 10 μm; mobile phase MeCN/H2O, 70.30; flow rate 8 ml/min) to yield 3 (285 mg). Fraction 2–4 (15 mg) was further separated by silica gel CC (1.5 × 15 cm, acetone/CH2Cl2, step gradient elution from 0:100 to 10:90) to yield 7 (3.0 mg). Fraction 2–7 (370 mg) was purified by preparative HPLC (Phenomenex Luna; MeCN/H2O, 70.30) to yield 5 (11 mg). Considering a possibility that the methyl acetal derivatives (3 and 5) were formed during the chromatographic separation processes using MeOH, another half portion of the broth extract (5.0 g) was subjected to non-MeOH isolation procedures. The extract was fractionated by silica gel CC (3.5 × 15 cm, EtOAc/CH2Cl2, step gradient elution from 0:100 to 60:40) to obtain ten fractions: fractions 1–10. Fraction 2 (228 mg) was further separated by preparative HPLC (Phenomenex Luna; MeCN/ H2O, 70.30) to yield 3 (12 mg) and 4 (159 mg). Fraction 5 (538 mg) was purified by preparative HPLC (Merck LiChroCART 250-10, 21.2 × 250 mm, 10 μm; MeCN/H2O, 52:48; flow rate 4 ml/min) to
Fig. 4. Δδ-Values (δS–δR) of the Mosher esters 9a and 9b.
(broth) was extracted with EtOAc (2 × 8 l), and the combined organic layer was concentrated under reduced pressure to obtain a brown gum (10.2 g). A half portion of this extract (5.0 g) was subjected to fractionation by column chromatography (CC) on Sephadex LH-20 (4.0 × 60 cm, MeOH) to obtain four pooled fractions: fractions 1–4. Table 2 1 H and 13C NMR spectroscopic data for compounds 4–7 (CDCl3). 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 11-OCH3 15-OCH3
5
6
7
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
36.5 19.2 42.1 33.5 45.9 27.1 142.9 125.0 51.9 46.0 102.8 169.6 31.1 20.8 111.7
α 1.18, m; β 1.80, m 1.62–1.58 (2 H), m α 1.34, m;β 1.54, m
α 1.18, m; β 2.71, m α 1.38, m; β 1.93, m α 1.21, m; β 1.40, m
29.8 18.3 40.7 34.1 46.0 27.3 74.9 43.6 44.5 43.3 68.1 175.2 30.7 19.3 174.5
α 0.96, dt (4.4, 13.4); β 2.28, m α 1.55, m; β 1.84, m α 1.17, dt (3.7, 12.9); β 1.54, m
3.19, s
38.9 20.1 42.1 33.3 46.4 25.4 146.4 127.7 54.0 46.0 109.2 171.6 31.8 21.4 110.1 56.4 54.7
α 1.12, m; β 2.20, m α 1.52, m; β 1.62, m α 1.28, dt (4.1, 13.3); β 1.47, m
55.9
36.7 20.6 41.9 33.6 47.9 22.7 146.4 129.0 57.9 46.8 106.5 171.8 32.4 19.5 107.8 55.1 56.8
1.87, dd (10.4, 7.7) α 2.41, m; β 2.35, m 6.94, dt (4.1, 3.0) 2.81, m 5.95, d (5.5) 0.96, s 0.80, s 4.83, s
1.32, dd (12.5, 3.4) α 2.36, m; β 2.08, m 7.38, d (6.6) 2.91, br s 4.61, s 0.92, 0.83, 4.86, 3.38, 3.46,
s s s s s
1.51, m α 2.17, m; β 2.61, m 7.30, br d (4.1) 2.58, br s 4.87, s 0.91, 0.83, 4.95, 3.44, 3.22,
144
s s s s s
1.59, dd (10.4, 6.8) α 1.90, m; β 2.01, ddd (14.6, 6.8, 4.0) 5.08, dt (4.0, 1.9) 2.87, dd (11.0, 1.1) 2.72, ddd (11.0, 9.7, 4.9) α 4.40, dd (10.4, 9.7); β 4.02, dd (10.4, 4.9) 0.88, s 0.81, s
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yield 6 (32 mg). Fraction 6 (380 mg) was subjected to preparative HPLC (Phenomenex Luna; MeCN/H2O, 70:30) to yield 5 (25 mg). Fraction 7 (826 mg) was purified by preparative HPLC (Merck LiChroCART; MeCN/H2O, 55:45) to furnish 2 (11 mg). Fraction 8 (826 mg) was also purified by preparative HPLC (Phenomenex Luna; MeCN/H2O, 70:30) to yield 1 (596 mg).
CH2Cl2–MeOH (1:1, 1.0 ml) was added a solution of (trimethylsilyl) diazomethane in hexane (0.6 M, 0.40 ml) and the mixture was stirred at room temperature for 1 h. The reaction was quenched by addition of AcOH (150 μl), then 2 drops of ammonia solution was added, and the mixture was concentrated under reduced pressure to leave a pale yellow gum, which was purified by preparative HPLC (MeCN/H2O) to furnish the methyl ester derivatives 8 (5.3 mg) and 15-epi-8 (1.2 mg).
3.3.1. Gymnodrimane A (1) MeOH Colorless powder; [α]24 (nm) D ‒82 (c 0.10, MeOH); UV λmax (log ε): 230 (3.41); IR νmax ATR (cm−1): 3391, 1686, 1650, 1260, 1037; 1 H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6) data, Table 1; HRESIMS (m/z): 305.1364 [M+Na]+ (calc. for C15H22O5Na, 305.1359).
3.4.1. Gymnodrimane B methyl ester 8 Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.05 (1H, d, J = 6.3 Hz, H-7), 4.84 (1H, s, H-15), 4.22 (1H, t, J = 9.0 Hz, Hα-11), 3.66 (3H, s, –CO2CH3), 3.62 (1H, d, J = 5.3 Hz, 3-OH), 3.42 (1H, dd, J = 9.0, 5.0 Hz, Hβ-11), 3.28 (1H, m, H-3), 3.06 (3H, s, 15-OCH3), 2.64 (1H, m, Hβ-6), 2.59 (1H, m, H-9), 2.16 (1H, m, Hα-6), 1.82 (1H, m, Hβ1), 1.69-1.62 (2H, m, Hα-2 and Hβ-2), 1.52 (1H, dd, J = 12.4, 4.8 Hz, H5), 1.24 (1H, m, Hα-1), 1.03 (3H, s, H-13), 0.83 (3H, s, H-14); HRESIMS (m/z): 333.1670 [M+Na]+ (calc. for C17H26NaO5, 333.1672).
3.3.2. Gymnodrimane B (2) MeOH Colorless powder; [α]24 (nm) D ‒25 (c 0.10, MeOH); UV λmax −1 (log ε): 231 (3.43); IR νmax ATR (cm ): 3410, 1703, 1651, 1104, 1043; 1 H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6) data, Table 1; HRESIMS (m/z): 319.1523 [M+Na]+ (calc. for C16H24O5Na, 319.1516).
3.4.2. 15-Epi-8 Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.14 (1H, d, J = 6.5 Hz, H-7), 4.77 (1H, s, H-15), 3.88 (1H, dd, J = 8.8, 4.6 Hz, Hα11), 3.68 (3H, s, –CO2CH3), 3.46 (1H, d, J = 5.3 Hz, 3-OH), 3.40 (d, J = 8.8 Hz, Hβ-11), 3.31 (3H, s, 15-OCH3), 3.23 (1H, m, H-3), 2.61 (1H, m, H-9), 2.34 (1H, m, Hβ-1), 2.32 (1H, m, Hα-6), 2.19 (1H, m, Hβ-6), 1.88 (1H, m, Hβ-2), 1.55 (1H, m, Hα-2), 1.35 (1H, dd, J = 12.5, 4.1 Hz, H-5), 1.02 (3H, s, H-13), 0.82 (3H, s, H-14); HRESIMS (m/z): HRESIMS (m/z): 333.1673 [M+Na]+ (calc. for C17H26NaO5, 333.1672).
3.3.3. Gymnodrimane C (3) MeOH Colorless amorphous solid; [α]24 D ‒19 (c 0.10, MeOH); UV λmax −1 (nm) (log ε): 234 (3.44); IR νmax ATR (cm ): 1766, 1713, 1220, 1141, 923; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 301.1423 [M+Na]+ (calc. for C16H22O4Na, 301.1410). 3.3.4. Gymnodrimane D (4) MeOH Colorless powder; [α]23 (nm) D +152 (c 0.10, MeOH); UV λmax (log ε): 232 (3.43); IR νmax ATR (cm−1): 1773, 1686, 1209, 1104, 944; 1 H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 301.1417 [M+Na]+ (calc. for C16H22O4Na, 301.1410).
3.5. Synthesis of the Mosher ester derivatives 9a and 9b Compound 8 (1.0 mg) was treated with (-)-(R)-MTPA-Cl (10 μl) and 4-dimethylaminopyridine (10 mg) in CH2Cl2 (0.2 ml) at room temperature for 16 h. The mixture was diluted with EtOAc and washed with H2O, 0.1 M HCl (×2), and then with 1 M NaHCO3 (×2), and the organic layer was concentrated in vacuo to obtain a pale yellow gum (2.6 mg), which is mainly composed of an (S)-MTPA ester derivative 9a. Similarly, (R)-MTPA ester derivative 9b was prepared from 8 and (+)-(S)-MTPA-Cl.
3.3.5. Gymnodrimane E (5) MeOH Colorless powder; [α]24 (nm) D ‒99 (c 0.10, MeOH); UV λmax −1 (log ε): 227 (3.37); IR νmax ATR (cm ): 1711, 1689, 1652, 1099; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 333.1687 [M+Na]+ (calc. for C17H26O5Na, 333.1672).
3.5.1. (S)-MTPA ester 9a Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.56 (2H, m, phenyl of MTPA), 7.50 (3H, m, phenyl of MTPA), 7.05 (1H, d, J = 6.2 Hz, H-7), 4.89 (1H, s, H-15), 4.86 (1H, dd, J = 11.7, 4.5 Hz, H3), 4.25 (1H, t, J = 9.0 Hz, Hα-11), 3.68 (3H, s, –CO2CH3), 3.55 (3H, s, OCH3 of MTPA), 3.44 (1H, dd, J = 9.0, 5.0 Hz, Hβ-11), 3.17 (3H, s, 15OCH3), 2.67 (2H, m, Hβ-6 and H-9), 2.21 (1H, m, Hα-6), 1.90 (2H, m, Hβ-1 and Hα-2), 1.74 (2H, m, Hβ-2 and H-5), 1.39 (1H, m, Hα-1), 1.04 (3H, s, H-13), 0.93 (3H, s, H-14); HRESIMS (m/z): 549.2084 [M+Na]+ (calc. for C27H33F3O5Na, 549.2071).
3.3.6. Gymnodrimane F (6) MeOH Colorless powder; [α]24 (nm) D +12 (c 0.10, MeOH); UV λmax −1 (log ε): 228 (3.52); IR νmax ATR (cm ): 1685, 1649, 1276, 1106, 1010; 1 H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 333.1681 [M+Na]+ (calc. for C17H26O5Na, 333.1672). 3.3.7. Gymnodrimane G (7) Colorless amorphous solid; [α]23 +81 (c 0.10, MeOH); UV D λmaxMeOH (nm) (log ε): 223 (2.80); IR νmax ATR (cm−1): 1773, 1748, 1188, 1050; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS (m/z): 287.1267 [M+Na]+ (calc. for C15H20O4Na, 287.1254).
3.5.2. (R)-MTPA ester 9b Pale yellow gum; 1H NMR (400 MHz, acetone-d6) δ 7.58 (2H, m, phenyl of MTPA), 7.48 (3H, m, phenyl of MTPA), 7.04 (1H, d, J = 6.3 Hz, H-7), 4.94 (1H, s, H-15), 4.85 (1H, dd, J = 10.4, 5.6 Hz, H3), 4.26 (1H, t, J = 9.0 Hz, Hα-11), 3.68 (3H, s, -CO2CH3), 3.59 (3H, s, OCH3 of MTPA), 3.45 (1H, dd, J = 9.0, 5.0 Hz, Hβ-11), 3.18 (3H, s, 15OCH3), 2.67 (1H, m, H-9), 2.64 (1H, m, Hβ-6), 2.17 (1H, m, Hα-6), 1.96 (2H, m, Hβ-1 and Hα-2), 1.93 (1H, m, Hβ-2), 1.73 (1H, dd, J = 12.2, 4.8 Hz, H-5), 1.42 (1H, m, Hα-1), 0.88 (3H, s, H-13), 0.87 (3H, s, H-14); HRESIMS (m/z): 549.2081 [M+Na]+ (calc. for C27H33F3O5Na, 549.2071).
3.4. Synthesis of the methyl ester derivative 8 To a solution of 1 (20 mg) in MeOH (1.5 ml) was added p-TsOH·H2O (1 mg) and the mixture was stirred at room temperature for 15 h. The reaction was terminated by addition of 2 drops of ammonia solution, and the mixture was concentrated by evaporation. The residue was dissolved in EtOAc, washed with H2O, and the organic phase was concentrated under reduced pressure to obtain a colorless gum (21 mg), whose 1H NMR spectroscopic data indicated that it was mainly composed of 2. To a solution of a portion of this reaction product (10 mg) in
3.6. Biological assays Antibacterial activities against Bacillus cereus and Enterococcus faecium were performed using the resazurin microplate assay and the 145
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optical density microplate assay (OD600), respectively (Clinical and Laboratory Standards Institute (CLSI, 2006). Antimycobacterial activity against Mycobacterium tuberculosis H37Ra was evaluated using the green fluorescent protein microplate assay (Changsen et al., 2003). An assay for activity against Plasmodium falciparum (K1, multidrug resistant strain) was performed using the microculture radioisotope technique (Desjardins et al., 1979). Cytotoxic activities against the tumor cell-lines, NCI-H187 (human small-cell lung cancer) and MCF-7 (human breast cancer), were evaluated using the resazurin microplate assay (O’Brien et al., 2000).
Changsen, C., Franzblau, S.G., Palittapongarnpim, P., 2003. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob. Agents Chemother. 47, 3682–3687. Clinical and Laboratory Standards Institute (CLSI), 2006. Method for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically. Approved Standard, M7-A7. Clinical and Laboratory Standards Institute, Wayne, PA, USA. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710–718. Hatfield, G.M., Brady, L.R., 1969. Occurrence of bis-noryangonin in Gymnopilus spectabilis. J. Pharm. Sci. 58, 1298–1299. Isaka, M., Chinthanom, P., Sappan, M., Supothina, S., Vichai, V., Danwisetkanjana, K., Boonpratuang, T., Hyde, K.D., Choeyklin, R., 2017. Antitubercular activity of mycelium-associated Ganoderma lanostanoids. J. Nat. Prod. 80, 1361–1369. Isaka, M., Chinthanom, P., Thummarukcharoen, T., Boonpratuang, T., Choowong, W., 2019. Highly modified lanostane triterpenes from fruiting bodies of the basidiomycete Tomophagus sp. J. Nat. Prod. 82, 1165–1176. Lee, I.K., Cho, S.M., Seok, S.J., Yun, B.S., 2008. Chemical constituents of Gymnopilus spectabilis and their antioxidant activity. Mycobiology 36, 55–59. Nozoe, S., Koike, Y., Tsuji, E., Kusano, G., Seto, H., 1983a. Isolation and structure of gymnoprenols, a novel type of polyisoprenepolyols from Gymnopilus spectabilis. Tetrahedron Lett. 24, 1731–1734. Nozoe, S., Koike, Y., Kusano, G., Seto, H., 1983b. Structure of gymnopilin, a bitter principle of an hallucinogenic mushroom, Gymnopilus spectabilis. Tetrahedron Lett. 24, 1735–1736. O’Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. 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. Tanaka, M., Hashimoto, K., Okuno, T., Shirahama, H., 1993. Neurotoxic oligoisoprenoids of the hallucinogenic mushroom, Gymnopilus spectabilis. Phytochemistry 34, 661–664.
Acknowledgements Financial support from the Thailand Research Fund (Grant No. DBG628008) is gratefully acknowledged. Identification of the fungus used in this study was supported by the National Biobank of Thailand. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.11.016. References Aoyagi, F., Maeno, S., Okuno, T., Matsumoto, H., Ikura, M., Hikichi, K., Matsumoto, T., 1983. Gymnopilins, bitter principles of the big-laughter mushroom Gymnopilus spectabilis. Tetrahedron Lett. 24, 1991–1994.
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