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Astraeusins A–L, lanostane triterpenoids from the edible mushroom Astraeus odoratus
Tetrahedron xxx (2016) 1e8
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Astraeusins AeL, lanostane triterpenoids from the edible mushroom Astraeus odoratus Masahiko Isaka *, Somporn Palasarn, Prasert Srikitikulchai, Vanicha Vichai, Somjit Komwijit 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
a b s t r a c t
Article history: Received 1 March 2016 Received in revised form 1 April 2016 Accepted 21 April 2016 Available online xxx
Twelve new lanostane triterpenoids, astraeusins AeL (1e12), together with six known compounds, were isolated from the food mushroom Astraeus odoratus. Their structures were elucidated by spectroscopic analysis and chemical correlations. Antibacterial and cytotoxic activities of these lanostanoids were evaluated. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Astraeus odoratus Lanostane Cytotoxicity Antibacterial activity
1. Introduction Astraeus is a small genus of mushrooms belonging to the family Diplocystaceae, which is composed of nine described species including a few edible or medicinal species.1 In Thailand, two species, Astraeus odoratus and Astraeus asiaticus, are popular as food, that are expensive due to the limitation of natural occurrence and the difficulty of industrial production by cultivation. There have been several reports of chemical analyses of this genus. Lanostane triterpenoids are common constituents in mushroom fruiting bodies: astrahygrol, 3-epi-astrahygrol, astrahygrone, and astrakurkrol from Astraeus hygrometricus,2,3 astrapteridone, astrapteridiol, and 3-epiastrapteridiol from Astraeus pteridis.4 Recently, additional lanostanoids, astraororic acids AeD, were isolated from A. odoratus,5 and astrasiaone and astradiate from A. asiaticus6 in Thailand. Some of these lanostanoids were shown to exhibit antimycobacterial (Mycobacterium tuberculosis), antifungal (Candida albicans), and cytotoxic activities, and activity against Leishmania donovani promastigotes, but, in marginal activity level. As part of our research on the utilization of local edible and medicinal mushrooms in Thailand, we have been conducting chemical analysis and evaluation of biological activities.7e9 We report here the results of the chemical investigation of fruiting
* Corresponding author. Tel.: þ66 25646700x3554; fax: þ66 25646707; e-mail address: [email protected] (M. Isaka).
bodies of A. odoratus, purchased at a fresh food market in Surin province, Northeast Thailand. Antibacterial and cytotoxic activities of the isolated compounds were evaluated.
2. Results and discussion Chromatographic fractionations of the extracts by flash silica gel column chromatography and preparative HPLC using reversephase columns led to the isolation of 12 new lanostane triterpenoids, named astraeusins AeL (1e12), together with six known lanostanoids; astraodoric acids A (13), B (14), and D (15),5 artabotryols B (16) and A (17),10 and lanosterol (18) (see Fig. 1). Astraeusin A (1) was isolated as a colorless solid, and its molecular formula was determined to be C30H44O2 by HRESIMS. Its IR spectrum exhibited carbonyl absorption bands at 1702 and 1680 cm1. The 1H and 13C NMR, DEPT, and HMQC data for 1 supported the presence of a ketone (dC 217.6), an aldehyde (dC 195.0; dH 9.40, s), three tetrasubstituted olefinic carbons, three olefinic methines, four quaternary carbons, three methines, eight methylenes, and seven methyl groups (Tables 1 and 2). Elucidation of the lanostane skeleton of 1 was accomplished by analysis of COSY and HMBC data (Fig. 2). Key HMBC data for the tetracyclic ring were the 2 J correlations from the five methyl group singlets (H3-18, H3-19, H3-28, H3-29, and H3-30) to their attached quaternary carbons C13, C-10, C-4, C-4, and C-14, respectively, and their 3J correlations. The C-3 aliphatic ketone (dC 217.6) was assigned on the basis of the
http://dx.doi.org/10.1016/j.tet.2016.04.057 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
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M. Isaka et al. / Tetrahedron xxx (2016) 1e8
Fig. 1. Structures of compounds 1e18.
HMBC correlations from Hb-1, Ha-2, Hb-2, H-5, H3-28 and H3-29 to this carbon. A tetrasubstituted olefin was assigned to C-8/C-9 by the HMBC correlations from H3-30 and H-6 (dH 1.60) to C-8 (dC 135.2), and from H3-19 to C-9 (dC 133.3). The structure of the C-20eC-27 side-chain was elucidated by COSY correlations (C-20eC-24) and HMBC correlations for the a,b-unstaurated aldehyde (C-24eC-27). Thus, the HMBC correlations were observed from formyl proton (dH 9.40, H-26) to C-24, C-25, and C-26, from H3-27 to C-24, C-25, and C-26, and from H-24 to C-26 and C-27. The olefinic geometry of the conjugated diene was determined as 22E,24E by the 1He1H coupling constant for H-22/H-23 (J¼15.0 Hz) and the NOESY correlations of H-24/H-26 and H-23/H3-27. The same relative configuration as lanosterol (18) (C-4, C-10, C-13, C-14, C-17, and C20; ʻnormalʼ configuration) was suggested by the NOESY correlations. The correlations from the equatorial methyl group H3-28 to H-5 suggested their being in a-orientations. NOESY correlations from Hb-6 (axial) to H3-19 and H3-29 and from Hb-2 (axial) to H3-19 and H3-29 were indicative of their being in b-orientations. The NOESY cross-peak for H3-30/H-17 revealed their axial (a) orientations. Intense NOESY correlations of H3-18/H-20, H3-21/Hb-12 (dH
1.70, m), H-22/H-17, and H-22/H3-21 were consistent with the configuration of 17R,20R. Consequently, astraeusin A (1) was identified as a new lanostanoid, (22E,24E)-3-oxolanosta-8,22,24trien-26-al. Astraeusin B (2) was assigned the molecular formula C30H46O2 by HRESIMS. The 1H and 13C NMR spectra displayed similarity to those of 1. The significant difference was the presence of a secondary alcohol (dH 3.23, dd, J¼11.6, 4.4 Hz; dC 79.0) replacing the C-3 ketone in 1. Location of this oxymethine (C-3) was assigned on the basis of the COSY correlations of H-3 with H2-2 and the HMBC correlations from H-3 to C-28 and C-29, and the correlations from Hb-1, Hb-2, H3-28 and H3-29 to C-3. The axial (a) orientation of H-3 was evident from the coupling constants, including the axial-axial coupling (J¼11.6 Hz) with Hb-2, and the intense NOESY correlations of H-3/H-5 and H-3/H3-28. Therefore, astraeusin B (2) was identified as (22E,24E)-3b-hydroxylanosta-8,22,24-trien-26-al. The molecular formula of astraeusin C (3) was determined by HRESIMS to be C34H50O7, which was the same as 2. The only significant difference in the NMR spectroscopic data compared to 2 was the small coupling constants of H-3 (dH 3.42) of 3, resonating as
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M. Isaka et al. / Tetrahedron xxx (2016) 1e8
3
Table 1 13 C NMR spectroscopic data for compounds 1e10 No.
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3-OCOCH3 3-OCOCH3 22-OCOCH3 22-OCOCH3 26-OCH3
36.0 34.6 217.6 47.4 51.2 19.4 26.4 135.2 133.3 37.0 21.1 30.9a 44.9 49.93 30.9a 28.2 49.90 16.2 18.7 41.5 19.8 151.9 123.5 149.6 136.1 195.0 9.4 26.2 21.3 24.3
35.7 27.9 79.0 38.9 50.6 18.3 26.6 134.4 134.7 37.2 21.0 30.9 45.0 49.9 31.1 28.2 50.1 16.2 19.2 41.4 19.8 151.9 123.6 149.5 136.1 194.8 9.4 28.0 15.4 24.3
30.1 25.7 75.9 37.6 44.2 18.1 26.0 133.9 134.8 36.9 20.9 31.0 44.8 49.8b 30.8 28.2 49.8b 16.1 18.9 41.5 19.7 152.1 123.4 149.8 135.9 195.8 9.4 28.0 22,2 24.2
36.1 34.6 217.5 47.4 51.4 19.5 26.4 135.4 133.4 37.0 21.1 31.0 44.6 50.0 31.1 27.6 47.0 15.9 18.7 40.9 11.9 73.4 34.0 122.5 137.3 68.7 13.9 26.3 21.3 24.4
35.6 27.8e 79.0 38.9 50.4 18.3 26.5 134.3 134.5 37.0 21.0 31.1 44.5 49.8 30.8 27.6e 46.9 15.7 19.2 40.9 11.8 73.4 33.9 122.5 137.2 68.7 14.0 28.0 15.4 24.3
30.1 25.8 76.0 37.6 44.3 18.2 26.1 134.0 134.8 37.0 21.0 31.1 44.5 49.9 30.8 27.6 46.9 15.7 19.0 40.9 11.8 73.5 33.9 122.6 137.2 68.8 14.0 28.0 22.2 24.3
35.6 27.91f 79.0 38.9 50.4 18.2 26.5 134.3 134.5 37.0 20.9 31.0 44.4 49.9 30.7 27.8f 46.9 15.6 19.2 39.6 12.9 74.9 31.8 138.8 129.5 171.6 12.4 27.94 15.4 24.3
30.8c 23.3 78.1 31.7 45.3 18.0 26.0 134.0 134.7 36.9 21.0 31.0 44.5 49.9 30.8c 27.61 46.8 15.7 19.0 41.2 11.7 72.8 35.1 141.5 128.6 172.0 12.4 27.57 21.8 24.4 170.9 21.4
36.2 34.6 217.4 47.4 51.4 19.5 26.4 135.4 133.5 37.0 21.2 31.0g 44.4 50.2 31.1g 28.1h 46.8 15.6 18.7 40.5 12.9 68.2 28.3h 123.8 132.3 99.6 18.9 26.3 21.3 24.4
30.2 25.9 76.1 37.7 44.4d 18.3 26.1 134.1 135.0 37.0 21.0 30.9 44.4d 50.1 31.1 28.2i 46.8 15.5 18.9 40.5 12.9 68.3 28.3i 123.8 132.3 99.6 19.0 28.0 22.2 24.3
55.4
55.4
170.7 21.0
a-d
The carbon resonances were overlapped. e-i The carbon assignment may be interchanged.
Table 2 1 H NMR spectroscopic data for astraeusins A (1), B (2), C (3), H (7), and I (8) No.
1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 3-OAc 22-OAc
1
2
3
7
8
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
a 1.63, m b 1.98, ddd (13.0, 7.1, 3.5) a 2.40, ddd (15.6, 6.7, 3.5) b 2.57, ddd (15.6, 11.2, 7.1)
a 1.22, m b 1.73, m a 1.64, m b 1.56, m
a 1.58, m b 1.44, m a 1.60, m b 1.92, m
a 1.24, m b 1.70, m a 1.66, m b 1.59, m
a 1.46, m b 1.83, m a 1.63, m b 1.85, m
d 1.61, m 1.65, m; 1.62, m 2.08e2.04 (2H), m 2.08e2.05 (2H), m 1.79, m; 1.70, m 1.62, m; 1.20, m 1.76, m; 1.31, m 1.64, m 0.77, s 1.11, s 2.31, m 1.09, d (6.9) 6.09, dd (15.0, 8.9) 6.47, dd (15.0, 11.2) 6.80, d (11.2) 9.40, s 1.82, s 1.08, s 1.06, s 0.89, s d 2.04, s
3.23, dd (11.6, 4.4) 1.04, dd (12.5, 2.2) 1.69, m; 1.51, m 2.05e2.01 (2H), m 2.05e2.01 (2H), m 1.76, m; 1.65, m 1.60, m; 1.18, m 1.73, m; 1.30, m 1.62, m 0.74 s 0.98, s 2.30, m 1.09, d (6.6) 6.09, dd (15.0, 10.9) 6.46, dd (15.0, 11.1) 6.80, d (11.1) 9.40, s 1.82, d (0.8) 0.99, s 0.80, s 0.88, s 2.07, s d
3.42, br s 1.51, m 1.67, m; 1.56, m 2.05e2.00 (2H), m 2.08e1.99 (2H), m 1.76, m; 1.66, m 1.59, m; 1.17, m 1.73, m; 1.28, m 1.61, m 0.73, s 0.98, s 2.29, m 1.08, d (6.5) 6.09, dd (15.0, 8.9) 6.44, dd (15.0, 11.2) 6.79, d (11.2) 9.38, s 1.82, s 0.95, s 0.86, s 0.88, s d d
3.24, dd (11.7, 4.4) 1.04, dd (12.5, 1.8) 1.68 m; b 1.52, m 2.05e2.00 (2H), m 2.05e2.00 (2H), m 1.71, m; 1.68, m 1.60, m; 1.19, m 1.99, m; 1.33, m 1.58, m 0.68, s 0.97, s 1.54, m 0.98, d (6.8) 5.08, t (7.0) 2.54, m; 2.35, m 6.77, t (7.1) d 1.85, s 0.99, s 0.80, s 0.85, s d d
4.67, s 1.49, m 1.50e1.47 (2H), m 2.04e2.01 (2H), m 2.07e2.02 (2H), m 1.78, m; 1.66, m 1.61, m; 1.24, m 1.98, m; 1.36, m 1.86, m 0.70, s 1.00, s 1.45, m 0.94, d (7.0) 3.86, m 2.48, m; 2.25, m 6.94, t (7.0) d 1.86, s 0.86, s 0.91, s 0.95, s d d
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Fig. 2. COSY and key HMBC correlations for astraeusins A (1) and D (4).
from Hb-1, Hb-2, H-5, H3-28 and H3-29 to C-3. The axial (a) orientation of H-3 was confirmed by the coupling constants, and the NOESY correlation of H-3/Ha-1, H-3/H-5 and H-3/H3-28. Astraeusin F (6) was identified as the 3a-hydroxy isomer (C-3 epimer) of 5. Thus, its NMR spectroscopic data suggested the same tetracyclic ring structure as 3, and the side-chain was the same as 4 and 5. The equatorial (b) orientation of H-3 was confirmed by the small coupling constants (dH 3.43, t, J¼2.8 Hz). The relative and absolute configurations of 5 and 6, including C-22, were established by chemical correlation to the known astraodoric acid B (14), whose structure was previously determined by X-ray crystallographic analysis.5 LiAlH4 reduction of 14 gave 5 (major product), 6, and their 24,25-dihydro derivatives, which indicated that 5 and 6 share the same relative configuration as 14. Based on the established absolute configuration of 14, the absolute configuration of the side-chain of 5 and 6 was confirmed to be 20S,22S. The close resemblance of the NMR spectroscopic data strongly suggested that astraeusin D (4) also shares the same stereochemistry.
a broad singlet with narrow peak width, indicating its equatorial orientation. Therefore, this compound was identified as the C-3 epimer of 2. The molecular formula of astraeusin D (4) was determined by HRESIMS as C30H48O3. The 1H and 13C NMR spectroscopic data (Tables 1 and 3) suggested the same tricyclic ring structure with astraodoric acid B (14), one of the major triterpenoid constituents. The only difference was in the side-chain structure, possessing a hydroxymethyl group (dH 4.02, 2H, s; dC 68.7), replacing the carboxyl group (C-26) in 14. The upfield shifts of H-24 (dH 5.46) and C-24 (dC 122.5), compared with 14, were consistent with the allylic alcohol functional group in 4. The 24E configuration was evident from the intense NOESY correlation of H-24/H2-26. Astraeusin E (5) was assigned the molecular formula C30H50O3 by HRESIMS. The 1H and 13C NMR spectra of 5 suggested that its side-chain was identical to 4, whereas the tetracyclic ring structure was the same as 2. The location of the secondary alcohol (dH 3.23, dd, J¼11.6, 4.4 Hz, H-3; dC 79.0, C-3) was assigned on the basis of the HMBC correlations from H-3 to C-28 and C-29, and the correlations
Table 3 1 H NMR spectroscopic data for astraeusins D (4), E (5), F (6), I (9), and J (10) No.
1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 26-OCH3
4
5
6
9
10
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
a 1.63, m b 1.97, m a 2.40, m b 2.58, m
a 1.23, m b 1.73, m a 1.67, m b 1.59, m
a 1.45, m b 1.59, m a 1.62, m b 1.93, m
a 1.63, m b 1.99, ddd (13.1, 7.1, 3.6) a 2.41, ddd (15.8, 6.7, 3.6) b 2.58, ddd (15.8, 11.2, 7.1)
a 1.59, m b 1.44, m a 1.60, m b 1.94, m
d 1.59, m 1.62, m; 1.60, m 2.09e2.02 (2H), m 2.09e2.02 (2H), m 1.79, m; 1.69, m 1.63, m; 1.23, m 1.97, m; 1.34, m 1.88, m 0.71, s 1.11, s 1.44, m 0.92, d (6.8) 3.72, dd (8.6, 4.8) 2.32, m; 2.06, m 5.46, t (6.8) 4.02 (2H), s 1.68, s 1.08, s 1.06, s 0.91, s d
3.23, dd (11.6, 4.5) 1.05, dd (12.6, 2.0) 1.67, m; 1.52, m 2.06e2.00 (2H), m 2.06e2.00 (2H), m 1.77, m; 1.68, m 1.63, m; 1.22, m 1.97, m; 1.34, m 1.86, m 0.69, s 0.98, s 1.44, m 0.92, d (6.8) 3.73, ddd (8.7, 3.9, 1.2) 2.34, m; 2.07, m 5.47, t (7.1) 4.03 (2H), s 1.69, s 1.00, s 0.81, s 0.90, s d
3.43, t (2.8) 1.51, m 1.60, m; 1.52, m 2.04e2.00 (2H), m 2.07e2.03 (2H), m 1.78, m; 1.68, m 1.61, m; 1.21, m 1.97, m; 1.34, m 1.86, m 0.70, s 0.99, s 1.45, m 0.92, d (6.8) 3.73, m 2.34, m; 2.08, m 5.47, t (7.2) 4.04 (2H), s 1.69, s 0.97, s 0.87, s 0.91, s d
d 1.61, m 1.70, m; 1.62, m 2.10e2.05 (2H), m 2.07e2.02 (2H), m 1.83, m; 1.72, m 1.63, m; 1.23, m 2.06, m; 1.34, m 2.05, m 0.73, s 1.12, s 1.44, m 0.96, d (6.8) 3.94, dd (11.4, 2.5) 2.24, m; 1.60, m 5.66, d (5.3) 4.63, s 1.69, br s 1.09, s 1.07, s 0.92, s 3.46, s
3.43, t (2.6) 1.52, m 1.60, m; 1.52, m 2.05e2.00 (2H), m 2.08e2.00 (2H), m 1.81, m; 1.72, m 1.60, m; 1.21, m 2.06, m; 1.32, m 2.05, m 0.71, s 0.99, s 1.44, m 0.96, d (7.0) 3.94, dd (11.5, 2.5) 2.23, m; 1.62, m 5.66, br d (5.4) 4.63, s 1.69, br s 0.97, s 0.87, s 0.91, s 3.46, s
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M. Isaka et al. / Tetrahedron xxx (2016) 1e8
Astraeusin G (7) had the molecular formula C32H50O5 as determined by HRESIMS. The 1H and 13C NMR spectroscopic data suggested the same side-chain structure as astraodoric acid A (14), with a 22S-acetoxy group and 24E-geometry of the a,b-unsaturated carboxylic acid. NMR data for the tetracyclic core were similar to those of astraeusins B (2) and E (5), possessing a 3b-hydroxy group (dC 79.0, C-3; dH 3.24, dd, J¼11.7, 4.4 Hz, H-3) and a tetrasubstituted olefin (C-8/C-9). The side-chain structure is also common in lanostanoids from mycelial cultures of Ganoderma species.11,12 The NMR spectroscopic data of astraeusin H (8) indicate that its side-chain is the same as astraodoric acid B (14), possessing a 22Shydroxy group and an a,b-unsaturated carboxylic acid. The functional groups of the tetracyclic core were a 3a-acetoxy group (dC 78.1, C-3; dH 4.67, s, H-3) and a tetrasubstituted olefin (C-8/C-9). The molecular formula of astraeusin I (9) was assigned by HRESIMS as C31H48O3. Interpretation of the NMR spectroscopic data indicated that the tetracyclic core of 9 is the same as the 3-oxo8-ene-type compounds 1, 4, 13, and 14. The side-chain contained a methoxy group (dC 55.4; dH 4.63, 3H, s), an acetal methine carbon (dC 99.6; dH 4.63, s), an oxygenated methine carbon (dC 68.2; dH 3.94, dd, J¼11.4, 2.5 Hz), and a trisubstituted olefin. The COSY correlations revealed the connections of C-20eC-24. The HMBC correlations from H3-27 to the olefinic carbons (C-24 and C-25) and the acetal carbon (C-26), and the correlation from the methoxyl protons to the acetal carbon and from H-26 to the methoxy carbon indicated the location of the acetal carbon and the methoxy group. The dihydropyran ring was evident from the HMBC correlation from H-26 to C-22. The NOESY correlation H-22/26-OCH3 and the absence of the cross-peak for H-22/H-26 indicated the cis-relation of H-22 and methoxy group and suggested their pseudoaxial orientations. This methyl acetal derivative is probably an artifact formed from the hemiacetal during the extraction process. On the basis of biosynthetic grounds, a 22S configuration is assigned.
5
Astraeusin J (10) is identified as the 3a-hydroxy congener of 9. The equatorial orientation of H-3 (dH 3.43, t, J¼2.6 Hz) was evident from its coupling constants. All other NMR spectroscopic data were very similar to those of 9. Perenniporiol derivatives, lanostanoids possessing the same side-chain as 9, 10 and corresponding hemiacetals, have been isolated from cultured mycelia of the woodrotting basidiomycete Perenniporia ochroleuca.13,14 The structure of one of these compounds, (26S)-3-O-acetyl-26-O-methylperenniporiol, was unambiguously determined by X-ray crystallographic analysis.13 Astraeusin J (10) is the C-3 epimer of the known (26S)-15-deacetoxy-7,11-dihydro-26-O-methylperenniporiol, and astraeusin I (9) is the 3-oxo analog. The NMR spectroscopic data for the side-chain of 9 and 10 were similar to those of the 26-Omethylperennitol derivatives from P. ochroleuca, which further confirmed the configuration of 20S,21S,26S. The molecular formula of astraeusin K (11) was determined by HRESIMS as C30H48O3. The 1H and 13C NMR spectroscopic data showed resemblance to the known hemiacetal derivative artabotryol B (16),10 displaying a mixture of hemiacetal epimers (ca. 2.5:1 in CDCl3): dC 101.8 (dH 4.25, d, J¼8.2 Hz; major) and dC 95.2 (dH 5.04, d, J¼2.8 Hz; minor) (Table 4). Resonances for the tricyclic core were superimposed between the epimers, and the data were consistent with those of the 3-oxo-8-ene-type lanostanoids (1, 4, 9, 13, and 14). The COSY correlations revealed the C-20eC-27 side-chain carbon linkage for both epimers. The NOESY correlation H-22/H26 for the major epimer indicated the cis relation and axial orientations of these protons. The NOESY correlation H-26/H3-27 and absence of an H-26/H-25 cross-peak and the coupling constant for H-26 and H-25 (J¼8.2 Hz) demonstrated the cis relation of H-26 and CH3-27 and an axial orientation of H-25. On the other hand, a NOESY cross-peak for H-22/H-26 was not observed for the miner epimer. The small coupling constant (J¼2.8 Hz) for H-26 and H-25 indicated an equatorial orientation of H-26. The significant
Table 4 NMR spectroscopic data for astraeusins K (11) and L (12) No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
(26R)-11 [(26S)-11, minor]
(26R)-12 [(26S)-12, minor]
dC, mult.
dH, mult. (J in Hz)
dC, mult.
dH, mult. (J in Hz)
36.0, CH2 34.6, CH2 217.9, qC 47.4, qC 51.2, CH 19.4, CH2 26.3, CH2 135.3, qC 133.2, qC 36.9, qC 21.1, CH2 30.9a, CH2 44.3, qC 49.9, qC 31.0a, CH2 27.6 [27.5], CH2 46.5 [46.6], CH 15.7, CH3 18.7, CH3 40.9 [40.6], CH 13.4 [13.2], CH3 78.5 [69.7], CH 28.6 [29.0], CH2 31.4 [26.1], CH2 37.8 [34.9], CH 101.8 [95.2], CH 16.7 [17.1], CH3 26.1, CH3 21.3, CH3 24.6, CH3
a 1.62, m; b 1.96, m a 2.40, m; b 2.58, ddd (15.4, 11.4, 7.1)
35.6, CH2 27.8, CH2 79.0, CH 38.9, qC 50.4, CH 18.3, CH2 26.5, CH2 134.4, qC 134.5, qC 37.0, qC 21.0, CH2 30.9b, CH2 44.4 [44.3], qC 49.8, qC 31.0b, CH2 27.7 [27.6], CH2 46.5 [46.7], CH 15.6, CH3 19.1, CH3 40.9 [40.6], CH 13.4 [13.1], CH3 78.6 [69.8], CH 28.7 [29.0], CH2 31.4 [26.1], CH2 37.8 [34.9], CH 101.8 [95.3], CH 16.7 [17.1], CH3 28.0, CH3 15.4, CH3 24.5, CH3
a 1.23, m; b 172, m a 1.67, m; b 1.57, m
1.59, m 1.63, m; 1.58, m 2.09e2.06 (2H), m
2.05e2.01 (2H), m 1.79, m; 1.67, m
1.62, 1.96, 1.95, 0.70, 1.10, 1.40, 0.94, 3.45, 1.57, 1.78, 1.31, 4.25, 0.93, 1.08, 1.05, 0.90,
m; 1.21, m m; 1.35, m m [1.88, m] s s m [1.35, m] d (6.6) [0.90, d (6.6)] d (11.3) [3.98, d (11.1)] m; 1.21, m [1.61, m; 1.29, m] m; 1.19, m [1.52, m; 1.27, m] m [1.68, m] d (8.2) [5.04, d (2.8)] d (6.5) [0.90, d (6.6)] s s s
3.23, dd (11.4, 4.4) 1.04, dd (12.6, 1.7) a 1.67, m; b 1.51, m 2.05e2.00 (2H), m
2.05e2.00 (2H), m 1.77, m; 1.66, m
1.62, 1.94, 1.95, 0.67, 0.97, 1.38, 0.95, 3.45, 1.56, 1.76, 1.30, 4.24, 0.94, 0.99, 0.80, 0.90,
a,b
The assignments may be interchanged.
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m; 1.20, m m; 1.35, m m [1.87, m] s s m [1.36, m] d (6.8) [0.89, d (6.1)] d (11.4) [3.97, d (10.8)] m; 1.22, m [1.61, m; 1.29, m] m; 1.19, m [1.54, m; 1.19, m] m [1.68, m] d (8.2) [5.03, d (2.0)] d (6.5) [0.89, d (6.1)] s s s
6
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downfield shift of H-22 of the minor epimer (dH 3.98) when compared with the major epimer (dH 3.45) can be explained by the deshielding of H-22 by the 26-a-OH group (axial). Since the 22S configuration is assignable by correlation to other lanostanoids from the same mushroom extract, the configurations are proposed as 22S,25R,26R for the major epimer, and 22S,25R,26S for the minor epimer. Astraeusin L (12) had the molecular formula C30H50O3 as determined by HRESIMS. The 1H and 13C resonances were similar to those of 11 and 16. The only difference was the presence of a 3b-hydroxy group (dH 3.23, dd, J¼11.7, 4.4 Hz, H-3; dC 79.0, C-3). This compound was therefore identified as the C-3 epimer of artabotryol B (16). The isolated compounds, excluding astraeusin F (6) because of shortage of the sample, were subjected to our bioassay protocols to evaluate cytotoxic activities against the cancer cell-lines (NCI-H187, MCF-7, and KB) and nonmalignant Vero cells, and antibacterial activities against Bacillus cereus and Enterococcus faecium (Table 5). Among the new compounds, compounds 2, 10, and 12 exhibited weak cytotoxic activities. Cytotoxicity of astraodoric acids A (13), B (14), and D (15) and artabotryols A (17) and B (16) were previously reported.5,6 In the present study, some of the isolated compounds showed activities against Gram-positive bacteria. In particular, astraodoric acid A (13) inhibited the proliferation of both B. cereus (MIC 6.25 mg/mL) and E. faecium (MIC 6.25 mg/mL). Astraeusins G (7) and H (8) also showed moderate antibacterial activities. Table 5 Cytotoxic and antibacterial activities of compounds 1e5, 7e18, and standard compounds Compound
Astraeusin A (1) Astraeusin B (2) Astraeusin C (3) Astraeusin D (4) Astraeusin E (5) Astraeusin G (7) Astraeusin H (8) Astraeusin I (9) Astraeusin J (10) Astraeusin K (11) Astraeusin L (12) Astraodoric acid A (13) Astraodoric acid B (14) Astraodoric acid D (15) Artabotryol B (16) Artabotryol A (17) Lanosterol (18) Doxorubicin hydrochloridea Ellipticinea Vancomycin hydrochlorideb Tetracycline hydrochlorideb a b
Cytotoxicity (IC50, mg/mL)
Antibacterial activity (MIC, mg/mL)
NCI-H187 MCF-7 KB
Vero
B. cereus E. faecium
>50 47 >50 >50 >50 >50 >50 >50 19 >50 >50 19 >50 >50 16 >50 >50 0.079
18 49 48 >50 >50 41 35 >50 19 >50 17 48 48 49 9.0 >50 >50 d
>50 >50 >50 >50 >50 25 25 >50 >50 >50 >50 6.25 >50 12.5 >50 >50 >50 d
>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 25 >50 >50 >50 23 >50 >50 7.4
>50 17 >50 >50 >50 >50 >50 >50 18 >50 16 35 >50 >50 13 >50 >50 0.60
d
d d
d
d
d
d
d
d
2.5
1.5
1.1 d 1.0 d
>50 >50 >50 >50 >50 50 12.5 >50 >50 >50 >50 6.25 25 >50 >50 >50 >50 d d d 0.0976
Positive controls for the cytotoxicity assays. Positive controls for the antibacterial assays.
3. Conclusion In conclusion, the present results demonstrate that the edible mushroom A. odoratus is a rich source of lanostane-type triterpenoids. While the structure variation of the tetracyclic core is limited different only at C-3 (3-oxo, 3a-hydroxy, 3b-hydroxy, and 3a-acetoxy), there are diverse structural patterns of the side-chain, including the novel doubly conjugated aldehyde (1e3) and allylic alcohol (4e6) derivatives. Some of the lanostanoids were shown to exhibit activities against Gram-positive bacteria.
4. Experimental section 4.1. 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. 4.2. Fungal material The fresh mushroom fruiting bodies of A. odoratus were purchased at the Chong Chom fresh food market, Surin Province, Thailand, on 25 June 2014, and identified by one of the authors (P.S.). A voucher specimen has been deposited at the Bangkok BIOTEC Herbarium, Pathumthani as BBH 40443. 4.3. Extraction and isolation Fruiting bodies of A. odoratus (wet weight 3.5 kg) were chopped into small pieces and macerated in MeOH (8 L) in a 20 L solvent container bottle at room temperature for 30 days. The mixture was filtered and the residue was washed with MeOH (2 L). The MeOH solution (total ca. 10 L) was separated into ca. 1 L portions. Each potion was diluted with hexane (800 mL) and H2O (50 mL), shaken in a separating funnel, and the layers were separated. The hexane (upper) layers were concentrated and combined to obtain a yellow gum (extract A; 3.50 g). Each MeOH (bottom) layer was partially concentrated by evaporation and the combined residues were extracted three times with EtOAc (35 L) and concentrated under reduced pressure to obtain a dark brown gum (extract B; 30.3 g). A portion of extract B (17.3 g) was subjected to silica gel column chromatography (CC) eluting with a step gradient of (28% aqueous ammonia solution/MeOH¼1:9)dCH2Cl2 from 0:100 to 15:85 (0:100, 1:99, 3:97, 6:94, then 15:85) to obtain five pooled fractions: Fr-B1 (4.4 g), Fr-B2 (3.0 g), Fr-B3 (3.2 g), Fr-B4 (3.6 g), and Fr-B5 (3.5 g). Fr-B1 was further fractionated by silica gel CC ((ammonia solution/MeOH¼1:9)dCH2Cl2) and preparative HPLC using a reversed phase column (Waters Prep Nova-PakÔ HR C18, 25100 mm, 6 mm; mobile phase MeCN/H2O¼95:5 for 10 min, then 100:0; flow rate 10 mL/min) to furnish 1 (33 mg), 2 (12 mg), 3 (90 mg), 9 (32 mg), 10 (51 mg), 17 (690 mg), and ergosterol (20 mg). Fr-B2 was also fractionated by combination of silica gel CC and preparative HPLC to yield pure compounds: 1 (18 mg), 2 (28 mg), 3 (30 mg), 6 (1.8 mg), 11 (70 mg), 12 (31 mg), 16 (620 mg), and 17 (260 mg). Fr-B3 was subjected to purification by silica gel CC and preparative HPLC to furnish 1 (17 mg), 5 (14 mg), 11 (310 mg), 14 (30 mg), 16 (615 mg), and 17 (370 mg). Purification of Fr-B4 by silica gel CC and preparative HPLC yielded 4 (66 mg), 5 (30 mg), 7 (6.9 mg), 8 (16 mg), 13 (367 mg), 14 (201 mg), and 15 (180 mg). Three known compounds were purified from Fr-B5: 13 (23 mg), 14 (808 mg), and 15 (68 mg). Extract A was fractionated by silica gel CC ((ammonia solution/ MeOH¼1:9)dCH2Cl2), and the fractions were purified by silica gel CC and preparative HPLC to yield 2 (9 mg), 11 (9 mg), 12 (60 mg), 16 (428 mg), 17 (562 mg), 18 (6 mg), and ergosterol (1.22 g). 4.3.1. Astraeusin A (1). Colorless solid; ½aD 24 þ67 (c 0.215, CHCl3); UV (MeOH) lmax (log 3) 221 sh (3.45), 266 (3.69), 290 sh (3.62) nm; IR (ATR) nmax 2959, 1702, 1680, 1631, 1378, 1217, 1116, 1012, 974 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz,
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CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 459.3243 [MþNa]þ (calcd for C30H44O2Na, 459.3234). 4.3.2. Astraeusin B (2). Colorless solid; ½aD 25 þ55 (c 0.23, CHCl3); UV (MeOH) lmax (log 3) 216 (3.35), 266 (3.71), 295 (3.66) nm; IR (ATR) nmax 3471, 2935, 1678, 1631, 1372, 1218, 1027 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 461.3382 [MþNa]þ (calcd for C30H46O2Na, 461.3390). 4.3.3. Astraeusin C (3). Colorless gum; ½aD 24 þ54 (c 0.21, CHCl3); UV (MeOH) lmax (log 3) 215 (3.32), 265 (3.71), 295 (3.66) nm; IR (ATR) nmax 3471, 2941, 1678, 1631, 1372, 1217, 975 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 461.3394 [MþNa]þ (calcd for C30H46O2Na, 461.3390). 4.3.4. Astraeusin D (4). Colorless gum; ½aD 25 þ56 (c 0.21, CHCl3); UV (MeOH) lmax (log 3) 215 (3.32), 276 (3.07) nm; IR (ATR) nmax 3399, 2945, 1701, 1457, 1376, 1013 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 479.3493 [MþNa]þ (calcd for C30H48O3Na, 479.3496). 4.3.5. Astraeusin E (5). Colorless solid; ½aD 25 þ49 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 214 (3.28) nm; IR (ATR) nmax 3338, 2942, 1697, 1454, 1372, 1028, 1012, 983 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 481.3646 [MþNa]þ (calcd for C30H50O3Na, 481.3652). 4.3.6. Astraeusin F (6). Colorless solid; ½aD 23 þ27 (c 0.075, CHCl3); UV (MeOH) lmax (log 3) 217 (3.48) nm; IR (ATR) nmax 3342, 1728, 1457, 1352 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 481.3653 [MþNa]þ (calcd for C30H50O3Na, 481.3652). 4.3.7. Astraeusin G (7). Colorless gum; ½aD 23 þ45 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 220 (3.60) nm; IR (ATR) nmax 3396, 2925, 1733, 1710, 1373, 1239, 1220, 1025 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 537.3570 [MþNa]þ (calcd for C32H50O5Na, 537.3550). 4.3.8. Astraeusin H (8). Colorless solid; ½aD 23 þ3 (c 0.15, CHCl3); UV (MeOH) lmax (log 3) 227 (3.68) nm; IR (ATR) nmax 2945, 1712, 1687, 1375, 1248 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 537.3552 [MþNa]þ (calcd for C32H50O5Na, 537.3550). 4.3.9. Astraeusin I (9). Colorless solid; ½aD 23 þ81 (c 0.215, CHCl3); UV (MeOH) lmax (log 3) 215 (3.10) nm; IR (ATR) nmax 2946, 1706, 1453, 1381, 1094, 1055, 1033, 960 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 491.3490 [MþNa]þ (calcd for C31H48O3Na, 491.3496). 4.3.10. Astraeusin J (10). Colorless solid; ½aD 23 þ59 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 215 (3.30) nm; IR (ATR) nmax 2946, 1706, 1454, 1381, 1093, 1055, 1033, 960 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 493.3655 [MþNa]þ (calcd for C31H50O3Na, 493.3652). 4.3.11. Astraeusin K (11). Colorless solid; ½aD 24 þ76 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 215 (3.16) nm; IR (ATR) nmax 3420, 2945,
7
1700, 1485, 1374, 1219, 1044 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 4; HRMS (ESI-TOF) m/z 479.3491 [MþNa]þ (calcd for C30H48O3Na, 479.3496). 4.3.12. Astraeusin L (12). Colorless solid; ½aD 24 þ41 (c 0.205, CHCl3); UV (MeOH) lmax (log 3) 215 (3.04) nm; IR (ATR) nmax 3410, 2939, 1458, 1372, 1151, 1061, 1043, 1024 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 4; HRMS (ESI-TOF) m/z 481.3656 [MþNa]þ (calcd for C30H50O3Na, 481.3652). 4.4. LiAlH4 reduction of astraodoric acid B (14) To a solution of 14 (50 mg, 0.106 mmol) in THF (1.5 mL) was added a small portion of LiAlH4 (ca.30 mg), and the mixture was stirred at room temperature for 1.5 h. The reaction was quenched by slow addition of H2O (0.5 mL), and extracted with EtOAc (21 mL). The combined organic fractions were dried (MgSO4) and filtered. The filtrate was concentrated under reduced pressure to leave a pale yellow gum (37 mg), which was subjected to preparative HPLC (MeCN/H2O¼90:10) to furnish 5 (15.7 mg, 32%), 24,25-dihydro-5 (6.0 mg, 12%), 6 (1.9 mg, 4%), and 24,25-dihydro-6 (0.9 mg, 2%). 4.5. Biological assays Cytotoxic activities against the tumor cell-lines: NCI-H187 (human small-cell lung cancer), MCF-7 (human breast cancer), and KB (oral human epidermoid carcinoma), were evaluated using the resazurin microplate assay.15 Cytotoxicity to Vero cells (African green monkey kidney fibroblasts) was performed using the green fluorescent protein (GFP)-based microplate assay.16 Antibacterial activities against B. cereus and E. faecium were performed using the resazurin microplate assay and the optical density microplate assay (OD600), respectively.17 Acknowledgements Financial support from the National Science and Technology Development Agency (Grant No. P-13-00856) is gratefully acknowledged. Supplementary data Supplementary data (NMR spectra of compounds 1e12.) associated with this article can be found in the online version, at http:// dx.doi.org/10.1016/j.tet.2016.04.057. References and notes 1. Phosri, C.; Martin, M. P.; Sihanonth, P.; Whalley, A. J. S.; Watling, R. Mycol. Res. 2007, 111, 276e286. 2. Takaishi, Y.; Murakami, Y.; Ohashi, T.; Nakano, K.; Murakami, K.; Tomimatsu, T. Phytochemistry 1987, 26, 2341e2344. 3. Lai, T. K.; Biswas, G.; Chatterjee, S.; Dutta, A.; Pal, C.; Banerji, J.; Bhuvanesh, N.; Reibenspies, J. H.; Acharya, K. Chem. Biodiv 2012, 9, 1517e1524. 4. Stanikunaite, R.; Radwan, M. M.; Trappe, J. M.; Fronczek, F.; Ross, S. A. J. Nat. Prod. 2008, 71, 2077e2079. 5. Arpha, K.; Phosri, C.; Suwannasai, N.; Mongkolthanaruk, W.; Sodngam, S. J. Agric. Food Chem. 2012, 60, 9834e9841. 6. Pimjuk, P.; Phosri, C.; Wauke, T.; McCloskey, S. Phytochem. Lett. 2015, 14, 79e83. 7. Isaka, M.; Sappan, M.; Rachtawee, P.; Boonpratuang, T. Phytochem. Lett. 2011, 4, 106e108. 8. Isaka, M.; Palasarn, S.; Sappan, M.; Srichomthong, K.; Karunarathuna, S. C.; Hyde, K. D. Nat. Prod. Comm. 2015, 8, 1391e1393. 9. Isaka, M.; Chinthanom, P.; Kongthong, S.; Srichomthong, K.; Choeyklin, R. Phytochemistry 2013, 87, 133e139. 10. Gupta, C.; Prasad, S.; Sahai, M.; Asai, T.; Hara, N.; Fujimoto, Y. Helv. Chim. Acta 2010, 93, 1925e1932.
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11. Baby, S.; Johnson, A. J.; Govindan, B. Phytochemistry 2015, 114, 66e101. 12. Isaka, M.; Chinthanom, P.; Sappan, M.; Danwisetkanjana, K.; Boonpratuang, T.; Choeyklin, R. J. Nat. Prod. 2016, 79, 161e169. 13. Hirotani, M.; Ino, C.; Furuya, T.; Shiro, M. Phytochemistry 1984, 23, 1129e1134. 14. Ino, C.; Hirotani, M.; Furuya, T. Phytochemistry 1984, 23, 2885e2888. 15. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421e5426.
16. Hunt, L.; Jordan, M.; De Jesus, M.; Wurm, F. M. Biotechnol. Bioeng. 1999, 65, 201e205. 17. Clinical and Laboratory Standards Institute (CLSI) Method for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically Approved Standard, M7eA7; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2006.
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Astraeusins AeL, lanostane triterpenoids from the edible mushroom Astraeus odoratus Masahiko Isaka *, Somporn Palasarn, Prasert Srikitikulchai, Vanicha Vichai, Somjit Komwijit 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
a b s t r a c t
Article history: Received 1 March 2016 Received in revised form 1 April 2016 Accepted 21 April 2016 Available online xxx
Twelve new lanostane triterpenoids, astraeusins AeL (1e12), together with six known compounds, were isolated from the food mushroom Astraeus odoratus. Their structures were elucidated by spectroscopic analysis and chemical correlations. Antibacterial and cytotoxic activities of these lanostanoids were evaluated. Ó 2016 Elsevier Ltd. All rights reserved.
Keywords: Astraeus odoratus Lanostane Cytotoxicity Antibacterial activity
1. Introduction Astraeus is a small genus of mushrooms belonging to the family Diplocystaceae, which is composed of nine described species including a few edible or medicinal species.1 In Thailand, two species, Astraeus odoratus and Astraeus asiaticus, are popular as food, that are expensive due to the limitation of natural occurrence and the difficulty of industrial production by cultivation. There have been several reports of chemical analyses of this genus. Lanostane triterpenoids are common constituents in mushroom fruiting bodies: astrahygrol, 3-epi-astrahygrol, astrahygrone, and astrakurkrol from Astraeus hygrometricus,2,3 astrapteridone, astrapteridiol, and 3-epiastrapteridiol from Astraeus pteridis.4 Recently, additional lanostanoids, astraororic acids AeD, were isolated from A. odoratus,5 and astrasiaone and astradiate from A. asiaticus6 in Thailand. Some of these lanostanoids were shown to exhibit antimycobacterial (Mycobacterium tuberculosis), antifungal (Candida albicans), and cytotoxic activities, and activity against Leishmania donovani promastigotes, but, in marginal activity level. As part of our research on the utilization of local edible and medicinal mushrooms in Thailand, we have been conducting chemical analysis and evaluation of biological activities.7e9 We report here the results of the chemical investigation of fruiting
* Corresponding author. Tel.: þ66 25646700x3554; fax: þ66 25646707; e-mail address: [email protected] (M. Isaka).
bodies of A. odoratus, purchased at a fresh food market in Surin province, Northeast Thailand. Antibacterial and cytotoxic activities of the isolated compounds were evaluated.
2. Results and discussion Chromatographic fractionations of the extracts by flash silica gel column chromatography and preparative HPLC using reversephase columns led to the isolation of 12 new lanostane triterpenoids, named astraeusins AeL (1e12), together with six known lanostanoids; astraodoric acids A (13), B (14), and D (15),5 artabotryols B (16) and A (17),10 and lanosterol (18) (see Fig. 1). Astraeusin A (1) was isolated as a colorless solid, and its molecular formula was determined to be C30H44O2 by HRESIMS. Its IR spectrum exhibited carbonyl absorption bands at 1702 and 1680 cm1. The 1H and 13C NMR, DEPT, and HMQC data for 1 supported the presence of a ketone (dC 217.6), an aldehyde (dC 195.0; dH 9.40, s), three tetrasubstituted olefinic carbons, three olefinic methines, four quaternary carbons, three methines, eight methylenes, and seven methyl groups (Tables 1 and 2). Elucidation of the lanostane skeleton of 1 was accomplished by analysis of COSY and HMBC data (Fig. 2). Key HMBC data for the tetracyclic ring were the 2 J correlations from the five methyl group singlets (H3-18, H3-19, H3-28, H3-29, and H3-30) to their attached quaternary carbons C13, C-10, C-4, C-4, and C-14, respectively, and their 3J correlations. The C-3 aliphatic ketone (dC 217.6) was assigned on the basis of the
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Fig. 1. Structures of compounds 1e18.
HMBC correlations from Hb-1, Ha-2, Hb-2, H-5, H3-28 and H3-29 to this carbon. A tetrasubstituted olefin was assigned to C-8/C-9 by the HMBC correlations from H3-30 and H-6 (dH 1.60) to C-8 (dC 135.2), and from H3-19 to C-9 (dC 133.3). The structure of the C-20eC-27 side-chain was elucidated by COSY correlations (C-20eC-24) and HMBC correlations for the a,b-unstaurated aldehyde (C-24eC-27). Thus, the HMBC correlations were observed from formyl proton (dH 9.40, H-26) to C-24, C-25, and C-26, from H3-27 to C-24, C-25, and C-26, and from H-24 to C-26 and C-27. The olefinic geometry of the conjugated diene was determined as 22E,24E by the 1He1H coupling constant for H-22/H-23 (J¼15.0 Hz) and the NOESY correlations of H-24/H-26 and H-23/H3-27. The same relative configuration as lanosterol (18) (C-4, C-10, C-13, C-14, C-17, and C20; ʻnormalʼ configuration) was suggested by the NOESY correlations. The correlations from the equatorial methyl group H3-28 to H-5 suggested their being in a-orientations. NOESY correlations from Hb-6 (axial) to H3-19 and H3-29 and from Hb-2 (axial) to H3-19 and H3-29 were indicative of their being in b-orientations. The NOESY cross-peak for H3-30/H-17 revealed their axial (a) orientations. Intense NOESY correlations of H3-18/H-20, H3-21/Hb-12 (dH
1.70, m), H-22/H-17, and H-22/H3-21 were consistent with the configuration of 17R,20R. Consequently, astraeusin A (1) was identified as a new lanostanoid, (22E,24E)-3-oxolanosta-8,22,24trien-26-al. Astraeusin B (2) was assigned the molecular formula C30H46O2 by HRESIMS. The 1H and 13C NMR spectra displayed similarity to those of 1. The significant difference was the presence of a secondary alcohol (dH 3.23, dd, J¼11.6, 4.4 Hz; dC 79.0) replacing the C-3 ketone in 1. Location of this oxymethine (C-3) was assigned on the basis of the COSY correlations of H-3 with H2-2 and the HMBC correlations from H-3 to C-28 and C-29, and the correlations from Hb-1, Hb-2, H3-28 and H3-29 to C-3. The axial (a) orientation of H-3 was evident from the coupling constants, including the axial-axial coupling (J¼11.6 Hz) with Hb-2, and the intense NOESY correlations of H-3/H-5 and H-3/H3-28. Therefore, astraeusin B (2) was identified as (22E,24E)-3b-hydroxylanosta-8,22,24-trien-26-al. The molecular formula of astraeusin C (3) was determined by HRESIMS to be C34H50O7, which was the same as 2. The only significant difference in the NMR spectroscopic data compared to 2 was the small coupling constants of H-3 (dH 3.42) of 3, resonating as
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M. Isaka et al. / Tetrahedron xxx (2016) 1e8
3
Table 1 13 C NMR spectroscopic data for compounds 1e10 No.
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3-OCOCH3 3-OCOCH3 22-OCOCH3 22-OCOCH3 26-OCH3
36.0 34.6 217.6 47.4 51.2 19.4 26.4 135.2 133.3 37.0 21.1 30.9a 44.9 49.93 30.9a 28.2 49.90 16.2 18.7 41.5 19.8 151.9 123.5 149.6 136.1 195.0 9.4 26.2 21.3 24.3
35.7 27.9 79.0 38.9 50.6 18.3 26.6 134.4 134.7 37.2 21.0 30.9 45.0 49.9 31.1 28.2 50.1 16.2 19.2 41.4 19.8 151.9 123.6 149.5 136.1 194.8 9.4 28.0 15.4 24.3
30.1 25.7 75.9 37.6 44.2 18.1 26.0 133.9 134.8 36.9 20.9 31.0 44.8 49.8b 30.8 28.2 49.8b 16.1 18.9 41.5 19.7 152.1 123.4 149.8 135.9 195.8 9.4 28.0 22,2 24.2
36.1 34.6 217.5 47.4 51.4 19.5 26.4 135.4 133.4 37.0 21.1 31.0 44.6 50.0 31.1 27.6 47.0 15.9 18.7 40.9 11.9 73.4 34.0 122.5 137.3 68.7 13.9 26.3 21.3 24.4
35.6 27.8e 79.0 38.9 50.4 18.3 26.5 134.3 134.5 37.0 21.0 31.1 44.5 49.8 30.8 27.6e 46.9 15.7 19.2 40.9 11.8 73.4 33.9 122.5 137.2 68.7 14.0 28.0 15.4 24.3
30.1 25.8 76.0 37.6 44.3 18.2 26.1 134.0 134.8 37.0 21.0 31.1 44.5 49.9 30.8 27.6 46.9 15.7 19.0 40.9 11.8 73.5 33.9 122.6 137.2 68.8 14.0 28.0 22.2 24.3
35.6 27.91f 79.0 38.9 50.4 18.2 26.5 134.3 134.5 37.0 20.9 31.0 44.4 49.9 30.7 27.8f 46.9 15.6 19.2 39.6 12.9 74.9 31.8 138.8 129.5 171.6 12.4 27.94 15.4 24.3
30.8c 23.3 78.1 31.7 45.3 18.0 26.0 134.0 134.7 36.9 21.0 31.0 44.5 49.9 30.8c 27.61 46.8 15.7 19.0 41.2 11.7 72.8 35.1 141.5 128.6 172.0 12.4 27.57 21.8 24.4 170.9 21.4
36.2 34.6 217.4 47.4 51.4 19.5 26.4 135.4 133.5 37.0 21.2 31.0g 44.4 50.2 31.1g 28.1h 46.8 15.6 18.7 40.5 12.9 68.2 28.3h 123.8 132.3 99.6 18.9 26.3 21.3 24.4
30.2 25.9 76.1 37.7 44.4d 18.3 26.1 134.1 135.0 37.0 21.0 30.9 44.4d 50.1 31.1 28.2i 46.8 15.5 18.9 40.5 12.9 68.3 28.3i 123.8 132.3 99.6 19.0 28.0 22.2 24.3
55.4
55.4
170.7 21.0
a-d
The carbon resonances were overlapped. e-i The carbon assignment may be interchanged.
Table 2 1 H NMR spectroscopic data for astraeusins A (1), B (2), C (3), H (7), and I (8) No.
1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 3-OAc 22-OAc
1
2
3
7
8
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
a 1.63, m b 1.98, ddd (13.0, 7.1, 3.5) a 2.40, ddd (15.6, 6.7, 3.5) b 2.57, ddd (15.6, 11.2, 7.1)
a 1.22, m b 1.73, m a 1.64, m b 1.56, m
a 1.58, m b 1.44, m a 1.60, m b 1.92, m
a 1.24, m b 1.70, m a 1.66, m b 1.59, m
a 1.46, m b 1.83, m a 1.63, m b 1.85, m
d 1.61, m 1.65, m; 1.62, m 2.08e2.04 (2H), m 2.08e2.05 (2H), m 1.79, m; 1.70, m 1.62, m; 1.20, m 1.76, m; 1.31, m 1.64, m 0.77, s 1.11, s 2.31, m 1.09, d (6.9) 6.09, dd (15.0, 8.9) 6.47, dd (15.0, 11.2) 6.80, d (11.2) 9.40, s 1.82, s 1.08, s 1.06, s 0.89, s d 2.04, s
3.23, dd (11.6, 4.4) 1.04, dd (12.5, 2.2) 1.69, m; 1.51, m 2.05e2.01 (2H), m 2.05e2.01 (2H), m 1.76, m; 1.65, m 1.60, m; 1.18, m 1.73, m; 1.30, m 1.62, m 0.74 s 0.98, s 2.30, m 1.09, d (6.6) 6.09, dd (15.0, 10.9) 6.46, dd (15.0, 11.1) 6.80, d (11.1) 9.40, s 1.82, d (0.8) 0.99, s 0.80, s 0.88, s 2.07, s d
3.42, br s 1.51, m 1.67, m; 1.56, m 2.05e2.00 (2H), m 2.08e1.99 (2H), m 1.76, m; 1.66, m 1.59, m; 1.17, m 1.73, m; 1.28, m 1.61, m 0.73, s 0.98, s 2.29, m 1.08, d (6.5) 6.09, dd (15.0, 8.9) 6.44, dd (15.0, 11.2) 6.79, d (11.2) 9.38, s 1.82, s 0.95, s 0.86, s 0.88, s d d
3.24, dd (11.7, 4.4) 1.04, dd (12.5, 1.8) 1.68 m; b 1.52, m 2.05e2.00 (2H), m 2.05e2.00 (2H), m 1.71, m; 1.68, m 1.60, m; 1.19, m 1.99, m; 1.33, m 1.58, m 0.68, s 0.97, s 1.54, m 0.98, d (6.8) 5.08, t (7.0) 2.54, m; 2.35, m 6.77, t (7.1) d 1.85, s 0.99, s 0.80, s 0.85, s d d
4.67, s 1.49, m 1.50e1.47 (2H), m 2.04e2.01 (2H), m 2.07e2.02 (2H), m 1.78, m; 1.66, m 1.61, m; 1.24, m 1.98, m; 1.36, m 1.86, m 0.70, s 1.00, s 1.45, m 0.94, d (7.0) 3.86, m 2.48, m; 2.25, m 6.94, t (7.0) d 1.86, s 0.86, s 0.91, s 0.95, s d d
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4
M. Isaka et al. / Tetrahedron xxx (2016) 1e8
Fig. 2. COSY and key HMBC correlations for astraeusins A (1) and D (4).
from Hb-1, Hb-2, H-5, H3-28 and H3-29 to C-3. The axial (a) orientation of H-3 was confirmed by the coupling constants, and the NOESY correlation of H-3/Ha-1, H-3/H-5 and H-3/H3-28. Astraeusin F (6) was identified as the 3a-hydroxy isomer (C-3 epimer) of 5. Thus, its NMR spectroscopic data suggested the same tetracyclic ring structure as 3, and the side-chain was the same as 4 and 5. The equatorial (b) orientation of H-3 was confirmed by the small coupling constants (dH 3.43, t, J¼2.8 Hz). The relative and absolute configurations of 5 and 6, including C-22, were established by chemical correlation to the known astraodoric acid B (14), whose structure was previously determined by X-ray crystallographic analysis.5 LiAlH4 reduction of 14 gave 5 (major product), 6, and their 24,25-dihydro derivatives, which indicated that 5 and 6 share the same relative configuration as 14. Based on the established absolute configuration of 14, the absolute configuration of the side-chain of 5 and 6 was confirmed to be 20S,22S. The close resemblance of the NMR spectroscopic data strongly suggested that astraeusin D (4) also shares the same stereochemistry.
a broad singlet with narrow peak width, indicating its equatorial orientation. Therefore, this compound was identified as the C-3 epimer of 2. The molecular formula of astraeusin D (4) was determined by HRESIMS as C30H48O3. The 1H and 13C NMR spectroscopic data (Tables 1 and 3) suggested the same tricyclic ring structure with astraodoric acid B (14), one of the major triterpenoid constituents. The only difference was in the side-chain structure, possessing a hydroxymethyl group (dH 4.02, 2H, s; dC 68.7), replacing the carboxyl group (C-26) in 14. The upfield shifts of H-24 (dH 5.46) and C-24 (dC 122.5), compared with 14, were consistent with the allylic alcohol functional group in 4. The 24E configuration was evident from the intense NOESY correlation of H-24/H2-26. Astraeusin E (5) was assigned the molecular formula C30H50O3 by HRESIMS. The 1H and 13C NMR spectra of 5 suggested that its side-chain was identical to 4, whereas the tetracyclic ring structure was the same as 2. The location of the secondary alcohol (dH 3.23, dd, J¼11.6, 4.4 Hz, H-3; dC 79.0, C-3) was assigned on the basis of the HMBC correlations from H-3 to C-28 and C-29, and the correlations
Table 3 1 H NMR spectroscopic data for astraeusins D (4), E (5), F (6), I (9), and J (10) No.
1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 26-OCH3
4
5
6
9
10
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
dH, mult. (J in Hz)
a 1.63, m b 1.97, m a 2.40, m b 2.58, m
a 1.23, m b 1.73, m a 1.67, m b 1.59, m
a 1.45, m b 1.59, m a 1.62, m b 1.93, m
a 1.63, m b 1.99, ddd (13.1, 7.1, 3.6) a 2.41, ddd (15.8, 6.7, 3.6) b 2.58, ddd (15.8, 11.2, 7.1)
a 1.59, m b 1.44, m a 1.60, m b 1.94, m
d 1.59, m 1.62, m; 1.60, m 2.09e2.02 (2H), m 2.09e2.02 (2H), m 1.79, m; 1.69, m 1.63, m; 1.23, m 1.97, m; 1.34, m 1.88, m 0.71, s 1.11, s 1.44, m 0.92, d (6.8) 3.72, dd (8.6, 4.8) 2.32, m; 2.06, m 5.46, t (6.8) 4.02 (2H), s 1.68, s 1.08, s 1.06, s 0.91, s d
3.23, dd (11.6, 4.5) 1.05, dd (12.6, 2.0) 1.67, m; 1.52, m 2.06e2.00 (2H), m 2.06e2.00 (2H), m 1.77, m; 1.68, m 1.63, m; 1.22, m 1.97, m; 1.34, m 1.86, m 0.69, s 0.98, s 1.44, m 0.92, d (6.8) 3.73, ddd (8.7, 3.9, 1.2) 2.34, m; 2.07, m 5.47, t (7.1) 4.03 (2H), s 1.69, s 1.00, s 0.81, s 0.90, s d
3.43, t (2.8) 1.51, m 1.60, m; 1.52, m 2.04e2.00 (2H), m 2.07e2.03 (2H), m 1.78, m; 1.68, m 1.61, m; 1.21, m 1.97, m; 1.34, m 1.86, m 0.70, s 0.99, s 1.45, m 0.92, d (6.8) 3.73, m 2.34, m; 2.08, m 5.47, t (7.2) 4.04 (2H), s 1.69, s 0.97, s 0.87, s 0.91, s d
d 1.61, m 1.70, m; 1.62, m 2.10e2.05 (2H), m 2.07e2.02 (2H), m 1.83, m; 1.72, m 1.63, m; 1.23, m 2.06, m; 1.34, m 2.05, m 0.73, s 1.12, s 1.44, m 0.96, d (6.8) 3.94, dd (11.4, 2.5) 2.24, m; 1.60, m 5.66, d (5.3) 4.63, s 1.69, br s 1.09, s 1.07, s 0.92, s 3.46, s
3.43, t (2.6) 1.52, m 1.60, m; 1.52, m 2.05e2.00 (2H), m 2.08e2.00 (2H), m 1.81, m; 1.72, m 1.60, m; 1.21, m 2.06, m; 1.32, m 2.05, m 0.71, s 0.99, s 1.44, m 0.96, d (7.0) 3.94, dd (11.5, 2.5) 2.23, m; 1.62, m 5.66, br d (5.4) 4.63, s 1.69, br s 0.97, s 0.87, s 0.91, s 3.46, s
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M. Isaka et al. / Tetrahedron xxx (2016) 1e8
Astraeusin G (7) had the molecular formula C32H50O5 as determined by HRESIMS. The 1H and 13C NMR spectroscopic data suggested the same side-chain structure as astraodoric acid A (14), with a 22S-acetoxy group and 24E-geometry of the a,b-unsaturated carboxylic acid. NMR data for the tetracyclic core were similar to those of astraeusins B (2) and E (5), possessing a 3b-hydroxy group (dC 79.0, C-3; dH 3.24, dd, J¼11.7, 4.4 Hz, H-3) and a tetrasubstituted olefin (C-8/C-9). The side-chain structure is also common in lanostanoids from mycelial cultures of Ganoderma species.11,12 The NMR spectroscopic data of astraeusin H (8) indicate that its side-chain is the same as astraodoric acid B (14), possessing a 22Shydroxy group and an a,b-unsaturated carboxylic acid. The functional groups of the tetracyclic core were a 3a-acetoxy group (dC 78.1, C-3; dH 4.67, s, H-3) and a tetrasubstituted olefin (C-8/C-9). The molecular formula of astraeusin I (9) was assigned by HRESIMS as C31H48O3. Interpretation of the NMR spectroscopic data indicated that the tetracyclic core of 9 is the same as the 3-oxo8-ene-type compounds 1, 4, 13, and 14. The side-chain contained a methoxy group (dC 55.4; dH 4.63, 3H, s), an acetal methine carbon (dC 99.6; dH 4.63, s), an oxygenated methine carbon (dC 68.2; dH 3.94, dd, J¼11.4, 2.5 Hz), and a trisubstituted olefin. The COSY correlations revealed the connections of C-20eC-24. The HMBC correlations from H3-27 to the olefinic carbons (C-24 and C-25) and the acetal carbon (C-26), and the correlation from the methoxyl protons to the acetal carbon and from H-26 to the methoxy carbon indicated the location of the acetal carbon and the methoxy group. The dihydropyran ring was evident from the HMBC correlation from H-26 to C-22. The NOESY correlation H-22/26-OCH3 and the absence of the cross-peak for H-22/H-26 indicated the cis-relation of H-22 and methoxy group and suggested their pseudoaxial orientations. This methyl acetal derivative is probably an artifact formed from the hemiacetal during the extraction process. On the basis of biosynthetic grounds, a 22S configuration is assigned.
5
Astraeusin J (10) is identified as the 3a-hydroxy congener of 9. The equatorial orientation of H-3 (dH 3.43, t, J¼2.6 Hz) was evident from its coupling constants. All other NMR spectroscopic data were very similar to those of 9. Perenniporiol derivatives, lanostanoids possessing the same side-chain as 9, 10 and corresponding hemiacetals, have been isolated from cultured mycelia of the woodrotting basidiomycete Perenniporia ochroleuca.13,14 The structure of one of these compounds, (26S)-3-O-acetyl-26-O-methylperenniporiol, was unambiguously determined by X-ray crystallographic analysis.13 Astraeusin J (10) is the C-3 epimer of the known (26S)-15-deacetoxy-7,11-dihydro-26-O-methylperenniporiol, and astraeusin I (9) is the 3-oxo analog. The NMR spectroscopic data for the side-chain of 9 and 10 were similar to those of the 26-Omethylperennitol derivatives from P. ochroleuca, which further confirmed the configuration of 20S,21S,26S. The molecular formula of astraeusin K (11) was determined by HRESIMS as C30H48O3. The 1H and 13C NMR spectroscopic data showed resemblance to the known hemiacetal derivative artabotryol B (16),10 displaying a mixture of hemiacetal epimers (ca. 2.5:1 in CDCl3): dC 101.8 (dH 4.25, d, J¼8.2 Hz; major) and dC 95.2 (dH 5.04, d, J¼2.8 Hz; minor) (Table 4). Resonances for the tricyclic core were superimposed between the epimers, and the data were consistent with those of the 3-oxo-8-ene-type lanostanoids (1, 4, 9, 13, and 14). The COSY correlations revealed the C-20eC-27 side-chain carbon linkage for both epimers. The NOESY correlation H-22/H26 for the major epimer indicated the cis relation and axial orientations of these protons. The NOESY correlation H-26/H3-27 and absence of an H-26/H-25 cross-peak and the coupling constant for H-26 and H-25 (J¼8.2 Hz) demonstrated the cis relation of H-26 and CH3-27 and an axial orientation of H-25. On the other hand, a NOESY cross-peak for H-22/H-26 was not observed for the miner epimer. The small coupling constant (J¼2.8 Hz) for H-26 and H-25 indicated an equatorial orientation of H-26. The significant
Table 4 NMR spectroscopic data for astraeusins K (11) and L (12) No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
(26R)-11 [(26S)-11, minor]
(26R)-12 [(26S)-12, minor]
dC, mult.
dH, mult. (J in Hz)
dC, mult.
dH, mult. (J in Hz)
36.0, CH2 34.6, CH2 217.9, qC 47.4, qC 51.2, CH 19.4, CH2 26.3, CH2 135.3, qC 133.2, qC 36.9, qC 21.1, CH2 30.9a, CH2 44.3, qC 49.9, qC 31.0a, CH2 27.6 [27.5], CH2 46.5 [46.6], CH 15.7, CH3 18.7, CH3 40.9 [40.6], CH 13.4 [13.2], CH3 78.5 [69.7], CH 28.6 [29.0], CH2 31.4 [26.1], CH2 37.8 [34.9], CH 101.8 [95.2], CH 16.7 [17.1], CH3 26.1, CH3 21.3, CH3 24.6, CH3
a 1.62, m; b 1.96, m a 2.40, m; b 2.58, ddd (15.4, 11.4, 7.1)
35.6, CH2 27.8, CH2 79.0, CH 38.9, qC 50.4, CH 18.3, CH2 26.5, CH2 134.4, qC 134.5, qC 37.0, qC 21.0, CH2 30.9b, CH2 44.4 [44.3], qC 49.8, qC 31.0b, CH2 27.7 [27.6], CH2 46.5 [46.7], CH 15.6, CH3 19.1, CH3 40.9 [40.6], CH 13.4 [13.1], CH3 78.6 [69.8], CH 28.7 [29.0], CH2 31.4 [26.1], CH2 37.8 [34.9], CH 101.8 [95.3], CH 16.7 [17.1], CH3 28.0, CH3 15.4, CH3 24.5, CH3
a 1.23, m; b 172, m a 1.67, m; b 1.57, m
1.59, m 1.63, m; 1.58, m 2.09e2.06 (2H), m
2.05e2.01 (2H), m 1.79, m; 1.67, m
1.62, 1.96, 1.95, 0.70, 1.10, 1.40, 0.94, 3.45, 1.57, 1.78, 1.31, 4.25, 0.93, 1.08, 1.05, 0.90,
m; 1.21, m m; 1.35, m m [1.88, m] s s m [1.35, m] d (6.6) [0.90, d (6.6)] d (11.3) [3.98, d (11.1)] m; 1.21, m [1.61, m; 1.29, m] m; 1.19, m [1.52, m; 1.27, m] m [1.68, m] d (8.2) [5.04, d (2.8)] d (6.5) [0.90, d (6.6)] s s s
3.23, dd (11.4, 4.4) 1.04, dd (12.6, 1.7) a 1.67, m; b 1.51, m 2.05e2.00 (2H), m
2.05e2.00 (2H), m 1.77, m; 1.66, m
1.62, 1.94, 1.95, 0.67, 0.97, 1.38, 0.95, 3.45, 1.56, 1.76, 1.30, 4.24, 0.94, 0.99, 0.80, 0.90,
a,b
The assignments may be interchanged.
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m; 1.20, m m; 1.35, m m [1.87, m] s s m [1.36, m] d (6.8) [0.89, d (6.1)] d (11.4) [3.97, d (10.8)] m; 1.22, m [1.61, m; 1.29, m] m; 1.19, m [1.54, m; 1.19, m] m [1.68, m] d (8.2) [5.03, d (2.0)] d (6.5) [0.89, d (6.1)] s s s
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downfield shift of H-22 of the minor epimer (dH 3.98) when compared with the major epimer (dH 3.45) can be explained by the deshielding of H-22 by the 26-a-OH group (axial). Since the 22S configuration is assignable by correlation to other lanostanoids from the same mushroom extract, the configurations are proposed as 22S,25R,26R for the major epimer, and 22S,25R,26S for the minor epimer. Astraeusin L (12) had the molecular formula C30H50O3 as determined by HRESIMS. The 1H and 13C resonances were similar to those of 11 and 16. The only difference was the presence of a 3b-hydroxy group (dH 3.23, dd, J¼11.7, 4.4 Hz, H-3; dC 79.0, C-3). This compound was therefore identified as the C-3 epimer of artabotryol B (16). The isolated compounds, excluding astraeusin F (6) because of shortage of the sample, were subjected to our bioassay protocols to evaluate cytotoxic activities against the cancer cell-lines (NCI-H187, MCF-7, and KB) and nonmalignant Vero cells, and antibacterial activities against Bacillus cereus and Enterococcus faecium (Table 5). Among the new compounds, compounds 2, 10, and 12 exhibited weak cytotoxic activities. Cytotoxicity of astraodoric acids A (13), B (14), and D (15) and artabotryols A (17) and B (16) were previously reported.5,6 In the present study, some of the isolated compounds showed activities against Gram-positive bacteria. In particular, astraodoric acid A (13) inhibited the proliferation of both B. cereus (MIC 6.25 mg/mL) and E. faecium (MIC 6.25 mg/mL). Astraeusins G (7) and H (8) also showed moderate antibacterial activities. Table 5 Cytotoxic and antibacterial activities of compounds 1e5, 7e18, and standard compounds Compound
Astraeusin A (1) Astraeusin B (2) Astraeusin C (3) Astraeusin D (4) Astraeusin E (5) Astraeusin G (7) Astraeusin H (8) Astraeusin I (9) Astraeusin J (10) Astraeusin K (11) Astraeusin L (12) Astraodoric acid A (13) Astraodoric acid B (14) Astraodoric acid D (15) Artabotryol B (16) Artabotryol A (17) Lanosterol (18) Doxorubicin hydrochloridea Ellipticinea Vancomycin hydrochlorideb Tetracycline hydrochlorideb a b
Cytotoxicity (IC50, mg/mL)
Antibacterial activity (MIC, mg/mL)
NCI-H187 MCF-7 KB
Vero
B. cereus E. faecium
>50 47 >50 >50 >50 >50 >50 >50 19 >50 >50 19 >50 >50 16 >50 >50 0.079
18 49 48 >50 >50 41 35 >50 19 >50 17 48 48 49 9.0 >50 >50 d
>50 >50 >50 >50 >50 25 25 >50 >50 >50 >50 6.25 >50 12.5 >50 >50 >50 d
>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 25 >50 >50 >50 23 >50 >50 7.4
>50 17 >50 >50 >50 >50 >50 >50 18 >50 16 35 >50 >50 13 >50 >50 0.60
d
d d
d
d
d
d
d
d
2.5
1.5
1.1 d 1.0 d
>50 >50 >50 >50 >50 50 12.5 >50 >50 >50 >50 6.25 25 >50 >50 >50 >50 d d d 0.0976
Positive controls for the cytotoxicity assays. Positive controls for the antibacterial assays.
3. Conclusion In conclusion, the present results demonstrate that the edible mushroom A. odoratus is a rich source of lanostane-type triterpenoids. While the structure variation of the tetracyclic core is limited different only at C-3 (3-oxo, 3a-hydroxy, 3b-hydroxy, and 3a-acetoxy), there are diverse structural patterns of the side-chain, including the novel doubly conjugated aldehyde (1e3) and allylic alcohol (4e6) derivatives. Some of the lanostanoids were shown to exhibit activities against Gram-positive bacteria.
4. Experimental section 4.1. 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. 4.2. Fungal material The fresh mushroom fruiting bodies of A. odoratus were purchased at the Chong Chom fresh food market, Surin Province, Thailand, on 25 June 2014, and identified by one of the authors (P.S.). A voucher specimen has been deposited at the Bangkok BIOTEC Herbarium, Pathumthani as BBH 40443. 4.3. Extraction and isolation Fruiting bodies of A. odoratus (wet weight 3.5 kg) were chopped into small pieces and macerated in MeOH (8 L) in a 20 L solvent container bottle at room temperature for 30 days. The mixture was filtered and the residue was washed with MeOH (2 L). The MeOH solution (total ca. 10 L) was separated into ca. 1 L portions. Each potion was diluted with hexane (800 mL) and H2O (50 mL), shaken in a separating funnel, and the layers were separated. The hexane (upper) layers were concentrated and combined to obtain a yellow gum (extract A; 3.50 g). Each MeOH (bottom) layer was partially concentrated by evaporation and the combined residues were extracted three times with EtOAc (35 L) and concentrated under reduced pressure to obtain a dark brown gum (extract B; 30.3 g). A portion of extract B (17.3 g) was subjected to silica gel column chromatography (CC) eluting with a step gradient of (28% aqueous ammonia solution/MeOH¼1:9)dCH2Cl2 from 0:100 to 15:85 (0:100, 1:99, 3:97, 6:94, then 15:85) to obtain five pooled fractions: Fr-B1 (4.4 g), Fr-B2 (3.0 g), Fr-B3 (3.2 g), Fr-B4 (3.6 g), and Fr-B5 (3.5 g). Fr-B1 was further fractionated by silica gel CC ((ammonia solution/MeOH¼1:9)dCH2Cl2) and preparative HPLC using a reversed phase column (Waters Prep Nova-PakÔ HR C18, 25100 mm, 6 mm; mobile phase MeCN/H2O¼95:5 for 10 min, then 100:0; flow rate 10 mL/min) to furnish 1 (33 mg), 2 (12 mg), 3 (90 mg), 9 (32 mg), 10 (51 mg), 17 (690 mg), and ergosterol (20 mg). Fr-B2 was also fractionated by combination of silica gel CC and preparative HPLC to yield pure compounds: 1 (18 mg), 2 (28 mg), 3 (30 mg), 6 (1.8 mg), 11 (70 mg), 12 (31 mg), 16 (620 mg), and 17 (260 mg). Fr-B3 was subjected to purification by silica gel CC and preparative HPLC to furnish 1 (17 mg), 5 (14 mg), 11 (310 mg), 14 (30 mg), 16 (615 mg), and 17 (370 mg). Purification of Fr-B4 by silica gel CC and preparative HPLC yielded 4 (66 mg), 5 (30 mg), 7 (6.9 mg), 8 (16 mg), 13 (367 mg), 14 (201 mg), and 15 (180 mg). Three known compounds were purified from Fr-B5: 13 (23 mg), 14 (808 mg), and 15 (68 mg). Extract A was fractionated by silica gel CC ((ammonia solution/ MeOH¼1:9)dCH2Cl2), and the fractions were purified by silica gel CC and preparative HPLC to yield 2 (9 mg), 11 (9 mg), 12 (60 mg), 16 (428 mg), 17 (562 mg), 18 (6 mg), and ergosterol (1.22 g). 4.3.1. Astraeusin A (1). Colorless solid; ½aD 24 þ67 (c 0.215, CHCl3); UV (MeOH) lmax (log 3) 221 sh (3.45), 266 (3.69), 290 sh (3.62) nm; IR (ATR) nmax 2959, 1702, 1680, 1631, 1378, 1217, 1116, 1012, 974 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz,
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CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 459.3243 [MþNa]þ (calcd for C30H44O2Na, 459.3234). 4.3.2. Astraeusin B (2). Colorless solid; ½aD 25 þ55 (c 0.23, CHCl3); UV (MeOH) lmax (log 3) 216 (3.35), 266 (3.71), 295 (3.66) nm; IR (ATR) nmax 3471, 2935, 1678, 1631, 1372, 1218, 1027 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 461.3382 [MþNa]þ (calcd for C30H46O2Na, 461.3390). 4.3.3. Astraeusin C (3). Colorless gum; ½aD 24 þ54 (c 0.21, CHCl3); UV (MeOH) lmax (log 3) 215 (3.32), 265 (3.71), 295 (3.66) nm; IR (ATR) nmax 3471, 2941, 1678, 1631, 1372, 1217, 975 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 461.3394 [MþNa]þ (calcd for C30H46O2Na, 461.3390). 4.3.4. Astraeusin D (4). Colorless gum; ½aD 25 þ56 (c 0.21, CHCl3); UV (MeOH) lmax (log 3) 215 (3.32), 276 (3.07) nm; IR (ATR) nmax 3399, 2945, 1701, 1457, 1376, 1013 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 479.3493 [MþNa]þ (calcd for C30H48O3Na, 479.3496). 4.3.5. Astraeusin E (5). Colorless solid; ½aD 25 þ49 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 214 (3.28) nm; IR (ATR) nmax 3338, 2942, 1697, 1454, 1372, 1028, 1012, 983 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 481.3646 [MþNa]þ (calcd for C30H50O3Na, 481.3652). 4.3.6. Astraeusin F (6). Colorless solid; ½aD 23 þ27 (c 0.075, CHCl3); UV (MeOH) lmax (log 3) 217 (3.48) nm; IR (ATR) nmax 3342, 1728, 1457, 1352 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 481.3653 [MþNa]þ (calcd for C30H50O3Na, 481.3652). 4.3.7. Astraeusin G (7). Colorless gum; ½aD 23 þ45 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 220 (3.60) nm; IR (ATR) nmax 3396, 2925, 1733, 1710, 1373, 1239, 1220, 1025 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 537.3570 [MþNa]þ (calcd for C32H50O5Na, 537.3550). 4.3.8. Astraeusin H (8). Colorless solid; ½aD 23 þ3 (c 0.15, CHCl3); UV (MeOH) lmax (log 3) 227 (3.68) nm; IR (ATR) nmax 2945, 1712, 1687, 1375, 1248 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 2; HRMS (ESI-TOF) m/z 537.3552 [MþNa]þ (calcd for C32H50O5Na, 537.3550). 4.3.9. Astraeusin I (9). Colorless solid; ½aD 23 þ81 (c 0.215, CHCl3); UV (MeOH) lmax (log 3) 215 (3.10) nm; IR (ATR) nmax 2946, 1706, 1453, 1381, 1094, 1055, 1033, 960 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 491.3490 [MþNa]þ (calcd for C31H48O3Na, 491.3496). 4.3.10. Astraeusin J (10). Colorless solid; ½aD 23 þ59 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 215 (3.30) nm; IR (ATR) nmax 2946, 1706, 1454, 1381, 1093, 1055, 1033, 960 cm1; for 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Tables 1 and 3; HRMS (ESI-TOF) m/z 493.3655 [MþNa]þ (calcd for C31H50O3Na, 493.3652). 4.3.11. Astraeusin K (11). Colorless solid; ½aD 24 þ76 (c 0.20, CHCl3); UV (MeOH) lmax (log 3) 215 (3.16) nm; IR (ATR) nmax 3420, 2945,
7
1700, 1485, 1374, 1219, 1044 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 4; HRMS (ESI-TOF) m/z 479.3491 [MþNa]þ (calcd for C30H48O3Na, 479.3496). 4.3.12. Astraeusin L (12). Colorless solid; ½aD 24 þ41 (c 0.205, CHCl3); UV (MeOH) lmax (log 3) 215 (3.04) nm; IR (ATR) nmax 3410, 2939, 1458, 1372, 1151, 1061, 1043, 1024 cm1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 4; HRMS (ESI-TOF) m/z 481.3656 [MþNa]þ (calcd for C30H50O3Na, 481.3652). 4.4. LiAlH4 reduction of astraodoric acid B (14) To a solution of 14 (50 mg, 0.106 mmol) in THF (1.5 mL) was added a small portion of LiAlH4 (ca.30 mg), and the mixture was stirred at room temperature for 1.5 h. The reaction was quenched by slow addition of H2O (0.5 mL), and extracted with EtOAc (21 mL). The combined organic fractions were dried (MgSO4) and filtered. The filtrate was concentrated under reduced pressure to leave a pale yellow gum (37 mg), which was subjected to preparative HPLC (MeCN/H2O¼90:10) to furnish 5 (15.7 mg, 32%), 24,25-dihydro-5 (6.0 mg, 12%), 6 (1.9 mg, 4%), and 24,25-dihydro-6 (0.9 mg, 2%). 4.5. Biological assays Cytotoxic activities against the tumor cell-lines: NCI-H187 (human small-cell lung cancer), MCF-7 (human breast cancer), and KB (oral human epidermoid carcinoma), were evaluated using the resazurin microplate assay.15 Cytotoxicity to Vero cells (African green monkey kidney fibroblasts) was performed using the green fluorescent protein (GFP)-based microplate assay.16 Antibacterial activities against B. cereus and E. faecium were performed using the resazurin microplate assay and the optical density microplate assay (OD600), respectively.17 Acknowledgements Financial support from the National Science and Technology Development Agency (Grant No. P-13-00856) is gratefully acknowledged. Supplementary data Supplementary data (NMR spectra of compounds 1e12.) associated with this article can be found in the online version, at http:// dx.doi.org/10.1016/j.tet.2016.04.057. References and notes 1. Phosri, C.; Martin, M. P.; Sihanonth, P.; Whalley, A. J. S.; Watling, R. Mycol. Res. 2007, 111, 276e286. 2. Takaishi, Y.; Murakami, Y.; Ohashi, T.; Nakano, K.; Murakami, K.; Tomimatsu, T. Phytochemistry 1987, 26, 2341e2344. 3. Lai, T. K.; Biswas, G.; Chatterjee, S.; Dutta, A.; Pal, C.; Banerji, J.; Bhuvanesh, N.; Reibenspies, J. H.; Acharya, K. Chem. Biodiv 2012, 9, 1517e1524. 4. Stanikunaite, R.; Radwan, M. M.; Trappe, J. M.; Fronczek, F.; Ross, S. A. J. Nat. Prod. 2008, 71, 2077e2079. 5. Arpha, K.; Phosri, C.; Suwannasai, N.; Mongkolthanaruk, W.; Sodngam, S. J. Agric. Food Chem. 2012, 60, 9834e9841. 6. Pimjuk, P.; Phosri, C.; Wauke, T.; McCloskey, S. Phytochem. Lett. 2015, 14, 79e83. 7. Isaka, M.; Sappan, M.; Rachtawee, P.; Boonpratuang, T. Phytochem. Lett. 2011, 4, 106e108. 8. Isaka, M.; Palasarn, S.; Sappan, M.; Srichomthong, K.; Karunarathuna, S. C.; Hyde, K. D. Nat. Prod. Comm. 2015, 8, 1391e1393. 9. Isaka, M.; Chinthanom, P.; Kongthong, S.; Srichomthong, K.; Choeyklin, R. Phytochemistry 2013, 87, 133e139. 10. Gupta, C.; Prasad, S.; Sahai, M.; Asai, T.; Hara, N.; Fujimoto, Y. Helv. Chim. Acta 2010, 93, 1925e1932.
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16. Hunt, L.; Jordan, M.; De Jesus, M.; Wurm, F. M. Biotechnol. Bioeng. 1999, 65, 201e205. 17. Clinical and Laboratory Standards Institute (CLSI) Method for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically Approved Standard, M7eA7; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2006.
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