Article pubs.acs.org/jnp
Chemical Constituents from Inonotus obliquus and Their Biological Activities Chao Liu,† Cui Zhao,† Hong-Hui Pan,‡ Jie Kang,† Xiong-Tao Yu,‡ Hong-Qing Wang,† Bao-Ming Li,† Yi-Zhen Xie,§ and Ruo-Yun Chen*,† †
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology, South China (The Ministry-Province Joint Development), Guangzhou 510663, People’s Republic of China § Guangdong Yuewei Edible Fungi Technology Co. Ltd, Guangzhou 510663, People’s Republic of China S Supporting Information *
ABSTRACT: Seven new triterpenes, inonotusol A−G (1−7), one new diterpene, inonotusic acid (8), and 22 known compounds were isolated from Inonotus obliquus. Their structures were elucidated on the basis of spectroscopic analysis, including homonuclear and heteronuclear correlation NMR (1H−1H COSY, ROESY, HSQC, and HMBC) experiments. In in vitro assays, compounds 6 and 8−16 showed hepatoprotective effects against D-galactosamine-induced WB-F344 cell damage, with inhibitory effects from 34.4% to 81.2%. Compounds 7, 17, and 18 exhibited selective cytotoxicities against KB, Bel-7402, or A-549 cell lines. Compounds 16 and 17 showed inhibitory effects against protein tyrosine kinases, with IC50 values of 24.6 and 7.7 μM, respectively.
T
hydroxylanosta-8,24-den-21-al, 21-hydroxylanosterol, 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid, and botulin], six sterols [i.e., ergosta-7,22-dien-3β-ol (13), 24β-ethylcholest-4en-3β-ol (14), ergosta-7-en-3β-ol (15), 5,8-epidioxyergosta6,22-dien-3β-ol, ergostrol, and ergosta-4,6,8(14),22-tetraen-3one], and six phenolic compounds [i.e., vanillic acid (16), protocatechuic aldehyde (17), 4-(3,4-dihydroxyphenyl)but-3en-2-one (18), protocatechuic acid, 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one, and 2,3-dihydroxy-1(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone]. The novelty of new compounds 1−5 mainly stems from the presence of a 21,24-cyclopentanol moiety in the side chain, and 1/2 and 3/4 are stereoisomers, respectively. Details of the structure elucidation and their bioactivities are reported herein.
he medicinal fungus Inonotus obliquus (Pers.: Fr.) Pilat, which belongs to the family Hymenochaetaceae, is widely distributed in Europe, Asia, and North America. In Russia and western Siberia, this mushroom has been used in folk medicine for cancer treatment for more than four centuries.1−3 Recently, the extract of this fungus was reported to possess antitumor,4 antioxidant,5 and anti-inflammatory activities.6 Previous phytochemical studies on I. obliquus have shown that this fungus is rich in triterpenes.3,7−13 During an ongoing search for new bioactive metabolites from fungi, the EtOAc fraction of an ethanolic extract from the sclerotia of I. obliquus was found to be rich in secondary metabolites by TLC and HPLC analysis and exhibited cytotoxicity and inhibitory activity against protein tyrosine kinases (PTKs). Careful purification of the EtOAc fraction by column chromatography and HPLC resulted in seven new lanostane-type triterpenes, inonotusols A−G (1−7), a new abietane-type diterpene, inonotusic acid (8), and 22 known compounds, including 10 triterpenes [i.e., inotodiol (9), lanosterol (10), 3β,22-dihydroxylanosta-8,24-dien-11-one (11), trametenolic acid (12), 3β,22-dihydroxylanosta7,9(11),24-triene, 3β,22-dihydroxylanosta-8,24-dien-7-one, 3β© XXXX American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Compound 1 was obtained as an amorphous powder with [α]25D −114.4. Its molecular formula, C30H48O6, was deduced by HRESIMS (high-resolution electrospray ionization mass Received: July 14, 2013
A
dx.doi.org/10.1021/np400552w | J. Nat. Prod. XXXX, XXX, XXX−XXX
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H3-30 to C-15 by HMBC (heteronuclear multiple bond coherence). The carbonyl group at C-7 was proven by correlations from H-6 to C-7 by HMBC. The 21,24cyclopentanol moiety in the side chain part was proven by correlations between H-17/H-20/H-21/H-24 and H-22b (δH 1.48)/H-23b (δH 1.42) by 1H−1H COSY (two-dimensional homonuclear chemical shift correlated spectroscopy) and longrange correlations from H-20 to C-21; from H-24 to C-21, C22, and C-25; from H3-26 to C-24, C-25, and Me-27; and from H3-27 to C-24, C-25, and Me-26 by HMBC. The relative configuration of 1 was determined by analysis of the ROESY (rotating frame Overhauser effect spectroscopy) spectrum. The correlations between H-3/H3-29, H-11/H3-18, and H-15/H3-18 indicated H-3, H-11, and H-15 were all βoriented. The correlations between H-17/H3-30, H-24/H3-30, H-20/H3-18, and H-21/H-12β/H3-18 confirmed that H-17 and H-24 were α-oriented, and H-20 and H-21 were β-oriented. Moreover, the ECD spectrum of 1 displayed positive and negative Cotton effects at 335 (Δε +4.65) and 254 (Δε −12.87) nm, respectively, which corresponded to the n→π* and π→π* transition of the conjugated 8-en-7-one chromophore. On the basis of the octant rule for the α,β-unsaturated ketone group,14,15 the 5S,10S configurations were assigned to 1. Therefore, inonotusol A was characterized as (−)-(3R,5S,10S,11R,15S,17R,20R,21S,24S)-21,24-cyclopenta3,11,15,21,25-pentahydroxylanosta-8-en-7-one. The molecular formula for compound 2 was also determined as C30H48O6 by HRESIMS. A comparison of the 1H and 13C NMR data (Table 1) of 2 with those for 1 showed they were almost superimposable on those of the lanostane skeleton, and the long-range correlations from H-20 to C-17; from H3-26 to C-24, C-25, and Me-27; and from H3-27 to C-24, C-25, and Me-26 by HMBC were also quite similar to those for 1. The main differences between 2 and 1 were the 1H and 13C NMR shifts of the side chain, such as H-20, H-21, H-24 and C-20, C21, C-24 (shifted by ΔδH +0.68, +0.49, −0.40, and ΔδC −5.8, −4.4, −4.5 ppm, respectively), which indicated the changes of the relative configuration from 2 to 1. The relative configuration of 2 was also determined by analysis of the ROESY spectrum. The correlations between H-3/H3-29, H-11/ H3-18, and H-15/H3-18 indicated H-3, H-11, and H-15 were all β-oriented. The correlations between H-17/H3-18, H-20/H318, H-21/H3-18, and H-24/H3-18 confirmed that H-17, H-20, H-21, and H-24 were all β-oriented. The absolute configuration was assigned as 5S,10S from positive and negative Cotton effects at 335.5 (Δε +10.22) and 254.5 (Δε −27.71) nm, respectively, in the ECD spectrum.14 Therefore, compound 2 was assigned the structure (−)-(3R,5S,10S,11R,15S,17S,20R,21S,24R)-21,24-cyclopenta3,11,15,21,25-pentahydroxylanosta-8-en-7-one, named inonotusol B, which was stereoisomeric at the 17- and 24-positions of 1. Compound 3 was assigned a molecular formula of C30H48O5 by HRESIMS. Its NMR data were similar to those for 1 except for the data of the lanostane skeleton. The two signals at δH 6.45 (d, J = 6.0 Hz, H-7) and δH 5.53 (d, J = 6.0 Hz, H-11) and the four olefinic carbons [δC 122.0 (C-7), 147.4 (C-8), 142.9 (C-9), 117.1 (C- 11)] in the 1H NMR (Table 1) indicated that 3 possessed a 7,9(11)-diene moiety.11 The two oxymethine protons [δH 4.76 (m), 4.25 (dd, J = 5.0, 11.0 Hz)] and the two signals of oxymethylene at δH 4.13 (d, J = 10.5 Hz) and δH 3.67 (d, J = 10.5 Hz) indicated the presence of two hydroxy groups and a hydroxymethyl group. The positions of two hydroxy
spectrometry). The IR spectrum showed absorption bands for the hydroxy (3386 cm−1) and carbonyl (1644 cm−1) groups. The 1H and 13C NMR data (Table 1) revealed resonances for seven methyls (δH 1.65, 1.44, 1.40, 1.25, 1.11, 1.10, 1.07, each 3H, s; δC 19.5, 26.3, 30.7, 20.2, 16.3, 28.2, 19.0), seven methylenes, eight methines including four oxymethines [δH 4.82 (dd, J = 4.5, 9.5 Hz), δC 64.9; δH 4.64 (dd, J = 6.0, 9.0 Hz), δC 74.0; δH 4.09 (t, J = 8.0 Hz), δC 79.5; δH 3.47 (m), δC 77.7], eight quaternary carbons including an oxygenated carbon (δC 72.6), and one α,β-unsaturated ketone group (δC 204.7, 167.7, 141.6). The data of 1 displayed signals characteristic of a lanostane triterpene and were similar to those for 3β,22dihydroxylanosta-8,24-dien-7-one. The evident differences were that 1 had two additional hydroxy groups on the lanostane skeleton and a 21,24-cyclopentanol moiety in the side chain shown by comparing the NMR data (Table 1) with those reported for 21,24-cyclopentalanosta-3β,21,25-triol-8-ene.9 The positions of the three hydroxy groups at C-3, C-11, and C-15 were determined through correlations from H3-28 to C-3; from H-11 to C-8 and C-9; from H-16a (δH 2.21) to C-15; and from B
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Table 1. NMR Spectroscopic Data for Compounds 1−4a 1
2
3
4
position
δC, type
δH (J in Hz)
δC
δH (J in Hz)
δC
1
35.1 CH2
2.57, dt (5.5, 12.5), 2.12b
35.2 CH2
74.5 CH
4.76, m
75.0 CH
4.75, t (7.2)
2
28.9 CH2
1.97 (2H)
28.9 CH2
36.6 CH2
2.17,b 1.64, m
36.7 CH2
2.10, m, 1.61, m
3 4 5 6
77.7 40.0 50.9 37.9
CH C CH CH2
3.47, m
77.7 40.0 50.9 37.9
CH C CH CH2
2.59, m, 2.20b 2.01,b 1.79, m 3.49, t (7.5)
73.5 43.5 43.0 23.7
CH C CH CH2
4.25, dd (5.0, 11.0)
73.6 CH 43.7 C 43.05 CH 23.9 CH2
4.25, dd (4.2, 11.4)
7 8 9 10 11 12
204.7 141.6 167.7 41.8 64.9 44.4
C C C C CH CH2
204.7 141.5 167.5 41.8 65.0 45.9
C C C C CH CH2
122.0 147.4 142.9 52.8 117.1 37.0
CH C C C CH CH2
13 14 15
49.9 C 52.3 C 74.0 CH
2.01, d (3.5) 2.74 (2H)
4.82, dd (4.5, 9.5) 3.27, dd (9.5, 13.5), 2.48, dd (4.5, 13.5)
4.64, dd (6.0, 9.0) b
49.6 C 52.3 C 74.3 CH
16
36.6 CH2
2.21, m, 2.12
17 18 19 20 21 22
49.3 19.0 20.2 49.2 79.5 24.9
CH CH3 CH3 CH CH CH2
2.36, dd (9.0, 18.0) 1.07, s 1.25, s 2.08, m 4.09, t (8.0) 1.73, m, 1.48, dt (3.5, 6.0)
49.4 19.2 20.3 43.4 75.1 22.2
CH CH3 CH3 CH CH CH2
23 24 25 26 27 28
28.7 58.8 72.6 26.3 30.7 16.3
CH2 CH C CH3 CH3 CH3
1.85, m, 1.42, m 2.23, m 1.44, s 1.40, s 1.11, s
29.1 54.3 72.5 30.4 30.6 16.3
CH2 CH C CH3 CH3 CH3
29 30
28.2 CH3 19.5 CH3
1.10, s 1.65, s
28.2 CH3 19.5 CH3
36.8 CH2
2.01b 2.74, m, 2.71b
4.81, m 2.82, m, 2.71b
45.4 C 38.2 C 29.0 CH2
4.63, dd (6.0, 9.0) 2.20,b 2.07, m 1.70, m 0.89, s 1.33, s 2.76, m 4.58, m 2.20,b 1.68, m 1.23 (2H) 1.83, m
δH (J in Hz)
2.07, m 2.31, m, 2.21, m 6.45, d (6.0)
5.53, d (6.0) 2.87, dd (6.5, 17.5), 2.51, d (17.5)
1.87, m, 1.40, br s b
δC
122.4 147.4 142.8 52.8 117.2 38.3
CH C C C CH CH2
δH (J in Hz)
2.05, m 2.32, dd (6.6, 3.6), 2.24, m 6.45, d (6.0)
5.56, d (6.0) 2.87, br d (18.0), 2.36, dd (6.6, 18.0)
45.3 C 38.6 C 29.4 CH2
1.99, m, 1.79, m
40.1 CH2
2.15 (2H)b
40.0 CH2
2.17 (2H)
49.0 18.1 18.6 49.3 79.6 25.0
CH CH3 CH3 CH CH CH2
2.28, m 1.09, s 1.37, s 2.14, m 4.11, t (8.0) 1.74 (2H)
49.5 CH 18.4 CH3 18.7 CH3 43.09 CH 75.5 CH 22.4 CH2
1.73, m 0.91, s 1.38, s 2.68, m 4.58, m 2.15,b 1.69, m
CH2 CH C CH3 CH3 CH2
2.01 (2H) 2.23, m
28.8 54.5 72.5 30.5 30.8 67.8
1.23 (2H) 1.81, m
1.35, s 1.63, s 1.13, s
28.7 59.0 72.5 26.6 30.7 67.7
1.11, s 1.65, s
13.6 CH3 24.1 CH3
1.43, s 1.47, s 4.13, d (10.5), 3.67, d (10.5) 1.13, s 1.19, s
CH2 CH C CH3 CH3 CH2
13.8 CH3 24.3 CH3
1.34, s 1.64, s 4.14, d (10.8), 3.68, d (10.8) 1.16, s 1.25, s
a13 C NMR data (δ) were measured at 125 MHz for 1−3 and at 150 MHz for 4. 1H NMR data (δ) were measured at 500 MHz for 1−3 and at 600 MHz for 4. The assignments were based on 1H−1H COSY, DEPT, HSQC, HMBC, and ROESY experiments. bOverlapped signals
Compound 4 exhibited the same molecular formula (C30H48O5) as 3 by HRESIMS. The 1H and 13C NMR data (Tables 1) of the lanostane skeleton were quite similar to those for 3, and the data for the side chain part were very similar to those for 2, which were also supported by HMBC correlations from H-1 to C-9 and Me-19; from H-3 to Me-29; from H-28a to C-3; from H-28b to C-3 and Me-29; from H-20 to C-16 and Me-18; from H3-26 to C-24 and Me-27; and from H3-27 to C24 and Me-26. The relative configuration of 4 was proven by ROESY. The correlations between H-1/H3-18 and H-3/H3-30 indicated H-1 and H-3 were β-oriented and α-oriented, respectively. The correlations between H-17/H3-18, H-20/ H3-19, H-21/H3-18, and H-24/H3-19 confirmed that H-17, H20, H-21, and H-24 were all β-oriented. Consequently, inonotusol D was assigned the structure (17β,20β,24β)-21,24cyclopenta-1α,3β,21α,25,28-pentahydroxy-5α-lanosta-7,9(11)diene. Compound 5 was shown to have the molecular formula C30H48O5 by HRESIMS, which was determined to be a 15dehydroxy analogue of 2 from its 1H and 13C NMR data (Table
groups at C-1 and C-3 were determined through long-range correlations from H-1 to C-9, C-10, and Me-19 and from H-3 to Me-29 by HMBC. The position of the hydroxymethyl group at C-4 was determined through correlations from H-28a (δH 4.13) to C-5 and Me-29 and from H-28b (δH 3.67) to C-3, C4, and Me-29 by HMBC. The relative configuration of 3 was determined by analysis of the ROESY spectrum. The correlations between H-1/H3-18 and H-3/H3-30 indicated H1 and H-3 were β-oriented and α-oriented, respectively. Moreover, the same orientations between H3-29 and H3-19 indicated the hydroxymethyl group was α-oriented. Furthermore, the correlations between H-24/H-17, H-17/H3-30, H20/H3-18, and H-21/H-12β/H3-18 confirmed that H-17 and H-24 were α-oriented, and H-20 and H-21 were β-oriented, which were the same as those of 1. Hence, inonotusol C was assigned the structure (17α,20β,24α)-21,24-cyclopenta1α,3β,21α,25,28-pentahydroxy-5α-lanosta-7,9(11)-diene. To our knowledge, a lanostane triterpene having a 1α,3β-dihydroxy moiety has rarely been isolated from I. obliquus. C
dx.doi.org/10.1021/np400552w | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. NMR Spectroscopic Data for Compounds 5−8a 5
6
7
8
position
δC, type
δH (J in Hz)
δC
δH (J in Hz)
1
35.6 CH2
2.22 (2H)
35.6 CH2
1.73, m, 1.23b
35.5 CH2
1.74, m, 1.24b
37.9 CH2
2
29.3 CH2
27.9 CH2
1.66, m, 1.58, m
27.8 CH2
1.68 (2H)b
18.9 CH2
3
77.9 CH
1.82, m, 1.22, m 3.48, t (7.8)
2.26, br d (13.2), 1.53, dd (3.6, 13.2) 1.76, m, 1.65, m
79.0 CH
3.23, dd (4.5, 11.5)
78.9 CH
3.23, dd (4.4, 11.2)
37.6 CH2
4 5 6
40.2 C 51.5 CH 38.1 CH2
2.00b 2.71 (2H)b
38.9 C 50.4 CH 18.2 CH2
1.05, dd (2.0, 13.0) 1.69,b 1.51, m
38.8 C 50.3 CH 18.2 CH2
1.05, m 1.70,b 1.53, m
47.0 C 38.9 CH 44.9 CH2
7 8 9 10 11 12
200.9 141.7 163.0 41.7 65.4 45.3
C C C C CH CH2
13 14 15 16 17 18 19 20 21 22
48.5 50.1 27.9 44.2 34.6 19.0 20.7 48.8 75.2 22.3
C C CH2 CH2 CH CH3 CH3 CH CH CH2
23
29.1 CH2
24 25 26 27 28 29 30 31
54.5 72.6 30.5 30.8 16.5 28.5 26.0
CH C CH3 CH3 CH3 CH3 CH3
4.83, m 2.80, m, 2.71b
1.72 (2H) 2.57 (2H) 2.04, m 0.86, s 1.36b 2.10, m 4.64, m 1.07, m, 1.00, m 2.00 (2H)b 1.82, m 1.36b 1.64, s 1.13, s 1.11, s 1.56, s
δC
27.1 134.8 134.0 37.1 21.0 26.5
CH2 C C C CH2 CH2
45.0 49.5 30.9 30.9 46.5 15.6 19.1 39.4 167.1 29.8
C C CH2b CH2b CH CH3 CH3 CH C CH2
78.6 CH 122.1 149.0 13.6 12.5 15.4 28.0 24.4 20.5
C C CH3 CH3 CH3 CH3 CH3 CH3
δH (J in Hz)
1.77, m, 1.41, m
2.03 (2H)b 2.03 (2H)b
1.69,b 1.23b 1.69,b 1.23b 1.50, m 0.73, s 0.98b 2.03b 2.45, t (15.0), 1.90, d (3.5) 4.39, dt (3.5, 15.0)
0.98b 1.88, s 1.00, s 0.81, s 0.89, s 1.94, s
δC
27.2 134.5 134.1 37.0 20.9 26.4
CH2 C C C CH2 CH2
44.8 49.4 30.9 30.9 47.2 15.7 19.1 41.1 12.9 69.3
C C CH2b CH2b CH CH3 CH3 CH CH3 CH
37.2 CH2 203.4 144.7 125.8 17.4 15.4 27.9 24.3
C C CH2 CH3 CH3 CH3 CH3
δH (J in Hz)
1.70,b 1.45, m
198.6 131.7 154.2 38.0 124.7 133.1
2.03 (2H)b 2.03 (2H)b
C C C C CH CH
147.2 C 125.4 CH 34.1 CH 24.21 CH3 24.17 CH3 24.0 CH3 17.3 CH3 180.6 C
1.68,b 1.24b 1.68,b 1.24b 1.40, m 0.74, s 0.98, s 1.83, m 0.96, d (6.8) 4.17, m
2.03, dt (3.6, 13.2), 1.87, br d (13.2) 2.94, dd (3.0, 16.8) 3.06, dd (3.0, 13.2), 2.98, m
7.36, d (8.4) 7.46, dd (1.8, 8.4)
8.15, 2.84, 1.17, 1.17, 1.22, 1.50,
d (1.8) m d (7.0) d (7.0) s s
2.78, d (17.2), 2.66, dd (10.4, 17.2)
6.02, 1.89, 0.99, 0.81, 0.88,
s, 5.85, s s s s s
a13 C NMR data (δ) were measured in pyridine-d5 at 150 MHz for 5 and 8 and in CDCl3 at 125 MHz for 6 and at 100 MHz for 7. 1H NMR data (δ) were measured in pyridine-d5 at 600 MHz for 5 and 8 and in CDCl3 at 500 MHz for 6 and at 400 MHz for 7. The assignments were based on 1 H−1H COSY, DEPT, HSQC, HMBC, and ROESY experiments. bOverlapped signals
(dt, J = 3.5, 15.0 Hz), δC 78.6], nine quaternary carbons including four olefinic carbons (δC 149.0, 134.8, 134.0, 122.1), and a carbonyl carbon (δC 167.1). The data for 6 displayed signals characteristic of a lanostane triterpene and were very similar to those reported for 3-hydroxy-8,24-diene-lanosta21,23-lactone.8 The evident difference was that 6 had one additional methyl (δH 1.94, 3H, s; δC 20.5) at C-24, which was proven by the tetrasubstituted olefinic bond [δC 122.1 (C), 149.0 (C)] and the long-range correlations from H3-31 to C-22, C-24, and C-25 by HMBC. The relative configuration of 6 was proven by ROESY. The correlations between H-3/H3-28, H17/H3-30, and H-17/H-21/H-23 indicated H-3, H-17, H-21, and H-23 were all α-oriented. Therefore, inonotusol F was elucidated as (17α,21α,23α)-24-methyl-3β-hydroxy-5α-lanosta8,24-diene-21,23-lactone. Compound 7 was assigned the molecular formula C30H48O3 by HRESIMS. Analysis of the 1H and 13C NMR spectra (Table 2) indicated 7 and 6 had the same lanostane skeleton, but a
2) and the long-range correlations from H3-28 to C-3 and from H-12b to C-11 by HMBC. The relative configuration of 5 was established by ROESY. The correlations between H-3/H3-29 and H-11/H3-18 indicated H-3 and H-11 were β-oriented. The correlations between H-17/H3-19, H-20/H3-18, H-21/H3-18, and H-24/H3-18 confirmed that H-17, H-20, H-21, and H-24 were all β-oriented. Moreover, the Cotton effects in the ECD spectrum were similar to those for 2 (Supporting Information S58). Therefore, compound 5 was assigned the structure (−)-(3R,5S,10S,11R,17S,20R,21S,24R)-21,24-cyclopenta3,11,21,25-tetrahydroxylanosta-8-en-7-one and named inonotusol E. Compound 6 was assigned the molecular formula C31H48O3 by HRESIMS. The 1H and 13C NMR data (Tables 2) revealed resonances for eight methyls (δH 1.94, 1.88, 1.00, 0.98, 0.98, 0.89, 0.81, 0.73, each 3H, s; δC 20.5, 12.5, 15.4, 19.1, 13.6, 24.4, 28.0, 15.6), nine methylenes, five methines including two oxymethines [δH 3.23 (dd, J = 4.5, 11.5 Hz), δC 79.0; δH 4.39 D
dx.doi.org/10.1021/np400552w | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Article
(14),23 ergosta-7-en-3β-ol (15),24 5,8-epidioxyergosta-6,22dien-3β-ol,25 ergostrol,22 ergosta-4,6,8(14),22-tetraen-3-one,22 vanillic acid (16),26 protocatechuic aldehyde (17),27 4-(3,4dihydroxyphenyl)but-3-en-2-one (18),28 protocatechuic acid,29 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one,30 and 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone30 by comparing their spectroscopic data with values reported in the literature. All compounds were bioassayed for their hepatoprotective effects against D-galactosamine-induced toxicity in WB-F344 cells, using the hepatoprotective activity drug bicyclol as the positive control. As shown in Table 3, 10 compounds (10 μM)
different side chain. The NMR data for the side chain part, resonances for two methyls [δH 1.89 (3H, s), δC 17.4; δH 0.96 (3H, d, J = 6.8 Hz), δC 12.9], two methylenes including one olefinic methylene [δH 6.02 (1H, s), 5.85 (1H, s); δC 125.8], two methines including one oxymethine [δH 4.17 (1H, m); δC 69.3], two quaternary carbons including one olefinic carbon (δC 144.7), and one carbonyl carbon (δC 203.4), which were similar to those of inonotsutriols D and E,10 indicated 7 was the 24-oxo derivative of inonotsutriol D or E. This was proven by 2D NMR data analysis, particularly by the correlations by HMBC from H-22 to C-24; from H2-23 to C-24; from H-26b (δH 5.85) to C-24, C-25, and Me-27; and from H3-27 to C-24, C-25, and C-26. The relative configuration of 7 was proven by ROESY. The correlations between H-3/H3-28 indicated H-3 was αoriented.10 Therefore, inonotusol G was designated as 3β,22dihydroxy-5α-lanosta-8,25-dien-24-one. We have tried to determine the absolute configuration of C-22 by application of the modified Mosher’s method,3 but failed. The derivative of (S)- or (R)-MTPA esters for the hydroxy group at C-22 could not be obtained from 7 using (−)- or (+)-MTPACl (αmethoxy-α-trifluoromethylphenylacetyl chloride) by stirring at room temperature for 24 h or heating at 60 °C for 30 min. The hydrogen bond effect between the carbonyl group at C-24 and the hydroxy group at C-22 may have hindered the reactions. Compound 8 was obtained as an amorphous powder with [α]25D −19.3. The molecular formula (C20H26O3) with eight degrees of unsaturation was indicated by HRESIMS. The 1H NMR (Table 2) revealed resonances for two methyls (δH 1.50, 1.22, each 3H, s), an isopropyl group [δH 2.84 (1H, m), 1.07 (6H, d, J = 7.0 Hz)], three aromatic proton signals [δH 8.15 (1H, d, J = 1.8 Hz), 7.46 (1H, dd, J = 1.8, 8.4 Hz), 7.36 (1H, d, J = 8.4 Hz)], an aliphatic methine [δH 2.94 (1H, dd, J = 3.0, 16.8 Hz, H-5)], and eight proton signals due to four methylenes. In addition to proton-bearing carbon resonances corresponding to the above units, the 13C NMR spectra exhibited resonances for six quaternary carbons consisting of three aromatic carbon signals (δC 154.2, 147.2, 131.7), a ketone group (δC 198.6), a carboxyl group (δC 180.6), and an aliphatic carbon (δC 47.0). The above-mentioned data were similar to those reported for 12-hydroxy-13-isopropyl-7-oxo-20-oic acid16 and suggest 8 was a 12-dehydroxy analogue. This was supported by HMBC correlations from H-1β to C-20; from H-11 to C-8, C-9, C-10, and C-13; from H-14 to C-7, C-8, and C-12; from H-12 to C-9, C-11, and C-15; from H-15 to C-13, Me-16, and Me-17; and from H3-16, 17 to C-13 and C-15. The relative configuration was established by ROESY. The correlations between H-5 and H3-19 indicated H-5 was αoriented. Moreover, the ECD spectrum of 8 displayed a positive Cotton effect at 328.5 nm (Δε +2.04) and a negative Cotton effect at 295.5 nm (Δε −1.60), indicating a 5S,10S configuration based on the aryl ketone CD rule.17−19 Therefore, compound 8 was assigned the structure (−)-(5S,10S)-13-isopropyl-7-oxo-abieta-8,11,13-trien-20-oic acid, named inonotusic acid. To our knowledge, this is the first report of the occurrence of an abietane-type diterpene in I. obliquus. The isolated known compounds were identified as inotodiol (9),8 lanosterol (10),8 3β,22-dihydroxylanosta-8,24-dien-11one (11),3 trametenolic acid (12),8 3β,22-dihydroxylanosta7,9(11),24-triene,11 3β,22-dihydroxylanosta-8,24-dien-7-one,12 3β-hydroxylanosta-8,24-den-21-al,13 21-hydroxylanosterol,12 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid,20 botulin,21 ergosta-7,22-dien-3β-ol (13),22 24β-ethylcholest-4-en-3β-ol
Table 3. Hepatoprotective Effects of 10 Compounds (10 μM) against D-Galactosamine-Induced Toxicity in WB-F344 Cellsa compound
cell survival rate (% of normal)
inhibition (% of control)e
± ± ± ± ± ± ± ± ± ± ± ± ±
71.9 34.4 56.3 74.8 81.2 75.0 43.8 40.6 37.4 56.5
normal control bicyclol 6 8 9 10 11 12 13 14 15 16
100 68 80 91 79 86 92 94 92 82 81 80 86
3.3 3.1 1.1c 6.9d 3.2d 3.2c 6.3d 2.5c 0.6b 0.5c 5.6d 1.2d 4.6c
Results are expressed as the means ± SD (n = 3; for normal and control, n = 6); bicyclol was used as positive control (10 μM). bp < 0.001. cp < 0.01. dp < 0.05. eInhibition (%) = [(OD(sample) − OD(control))/(OD(normal) − OD(control))] × 100%. a
showed hepatoprotective activities. Compounds 6 and 10−12 exhibited moderate hepatoprotective effects, with inhibitions of >70%, respectively. They were also tested for their cytotoxicity against three human tumor cell lines, Bel-7402 (human hepatoma cell line), A-549 (human lung epithelial cell line), and KB (human nasopharynx cancer cell line), using 5fluorouracil as the positive control. As shown in Table 4, Table 4. Cytotoxicity of Three Compounds against BEL7402, A-549, or KB Human Cell Lines (IC50, μM) 7 17 18 5-fluorouracil
BEL-7402
A-549
KB
>10 3.7 4.7 5.5
>10 3.1 >10 3.1
9.9 >10 >10 8.1
compound 7 exhibited cytotoxicity against the KB cell line, compound 17 exhibited cytotoxicity against Bel-7402 and A549 cell lines, and compound 18 exhibited cytotoxicity against the Bel-7402 cell line, with IC50 values of
Chemical Constituents from Inonotus obliquus and Their Biological Activities Chao Liu,† Cui Zhao,† Hong-Hui Pan,‡ Jie Kang,† Xiong-Tao Yu,‡ Hong-Qing Wang,† Bao-Ming Li,† Yi-Zhen Xie,§ and Ruo-Yun Chen*,† †
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology, South China (The Ministry-Province Joint Development), Guangzhou 510663, People’s Republic of China § Guangdong Yuewei Edible Fungi Technology Co. Ltd, Guangzhou 510663, People’s Republic of China S Supporting Information *
ABSTRACT: Seven new triterpenes, inonotusol A−G (1−7), one new diterpene, inonotusic acid (8), and 22 known compounds were isolated from Inonotus obliquus. Their structures were elucidated on the basis of spectroscopic analysis, including homonuclear and heteronuclear correlation NMR (1H−1H COSY, ROESY, HSQC, and HMBC) experiments. In in vitro assays, compounds 6 and 8−16 showed hepatoprotective effects against D-galactosamine-induced WB-F344 cell damage, with inhibitory effects from 34.4% to 81.2%. Compounds 7, 17, and 18 exhibited selective cytotoxicities against KB, Bel-7402, or A-549 cell lines. Compounds 16 and 17 showed inhibitory effects against protein tyrosine kinases, with IC50 values of 24.6 and 7.7 μM, respectively.
T
hydroxylanosta-8,24-den-21-al, 21-hydroxylanosterol, 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid, and botulin], six sterols [i.e., ergosta-7,22-dien-3β-ol (13), 24β-ethylcholest-4en-3β-ol (14), ergosta-7-en-3β-ol (15), 5,8-epidioxyergosta6,22-dien-3β-ol, ergostrol, and ergosta-4,6,8(14),22-tetraen-3one], and six phenolic compounds [i.e., vanillic acid (16), protocatechuic aldehyde (17), 4-(3,4-dihydroxyphenyl)but-3en-2-one (18), protocatechuic acid, 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one, and 2,3-dihydroxy-1(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone]. The novelty of new compounds 1−5 mainly stems from the presence of a 21,24-cyclopentanol moiety in the side chain, and 1/2 and 3/4 are stereoisomers, respectively. Details of the structure elucidation and their bioactivities are reported herein.
he medicinal fungus Inonotus obliquus (Pers.: Fr.) Pilat, which belongs to the family Hymenochaetaceae, is widely distributed in Europe, Asia, and North America. In Russia and western Siberia, this mushroom has been used in folk medicine for cancer treatment for more than four centuries.1−3 Recently, the extract of this fungus was reported to possess antitumor,4 antioxidant,5 and anti-inflammatory activities.6 Previous phytochemical studies on I. obliquus have shown that this fungus is rich in triterpenes.3,7−13 During an ongoing search for new bioactive metabolites from fungi, the EtOAc fraction of an ethanolic extract from the sclerotia of I. obliquus was found to be rich in secondary metabolites by TLC and HPLC analysis and exhibited cytotoxicity and inhibitory activity against protein tyrosine kinases (PTKs). Careful purification of the EtOAc fraction by column chromatography and HPLC resulted in seven new lanostane-type triterpenes, inonotusols A−G (1−7), a new abietane-type diterpene, inonotusic acid (8), and 22 known compounds, including 10 triterpenes [i.e., inotodiol (9), lanosterol (10), 3β,22-dihydroxylanosta-8,24-dien-11-one (11), trametenolic acid (12), 3β,22-dihydroxylanosta7,9(11),24-triene, 3β,22-dihydroxylanosta-8,24-dien-7-one, 3β© XXXX American Chemical Society and American Society of Pharmacognosy
■
RESULTS AND DISCUSSION Compound 1 was obtained as an amorphous powder with [α]25D −114.4. Its molecular formula, C30H48O6, was deduced by HRESIMS (high-resolution electrospray ionization mass Received: July 14, 2013
A
dx.doi.org/10.1021/np400552w | J. Nat. Prod. XXXX, XXX, XXX−XXX
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H3-30 to C-15 by HMBC (heteronuclear multiple bond coherence). The carbonyl group at C-7 was proven by correlations from H-6 to C-7 by HMBC. The 21,24cyclopentanol moiety in the side chain part was proven by correlations between H-17/H-20/H-21/H-24 and H-22b (δH 1.48)/H-23b (δH 1.42) by 1H−1H COSY (two-dimensional homonuclear chemical shift correlated spectroscopy) and longrange correlations from H-20 to C-21; from H-24 to C-21, C22, and C-25; from H3-26 to C-24, C-25, and Me-27; and from H3-27 to C-24, C-25, and Me-26 by HMBC. The relative configuration of 1 was determined by analysis of the ROESY (rotating frame Overhauser effect spectroscopy) spectrum. The correlations between H-3/H3-29, H-11/H3-18, and H-15/H3-18 indicated H-3, H-11, and H-15 were all βoriented. The correlations between H-17/H3-30, H-24/H3-30, H-20/H3-18, and H-21/H-12β/H3-18 confirmed that H-17 and H-24 were α-oriented, and H-20 and H-21 were β-oriented. Moreover, the ECD spectrum of 1 displayed positive and negative Cotton effects at 335 (Δε +4.65) and 254 (Δε −12.87) nm, respectively, which corresponded to the n→π* and π→π* transition of the conjugated 8-en-7-one chromophore. On the basis of the octant rule for the α,β-unsaturated ketone group,14,15 the 5S,10S configurations were assigned to 1. Therefore, inonotusol A was characterized as (−)-(3R,5S,10S,11R,15S,17R,20R,21S,24S)-21,24-cyclopenta3,11,15,21,25-pentahydroxylanosta-8-en-7-one. The molecular formula for compound 2 was also determined as C30H48O6 by HRESIMS. A comparison of the 1H and 13C NMR data (Table 1) of 2 with those for 1 showed they were almost superimposable on those of the lanostane skeleton, and the long-range correlations from H-20 to C-17; from H3-26 to C-24, C-25, and Me-27; and from H3-27 to C-24, C-25, and Me-26 by HMBC were also quite similar to those for 1. The main differences between 2 and 1 were the 1H and 13C NMR shifts of the side chain, such as H-20, H-21, H-24 and C-20, C21, C-24 (shifted by ΔδH +0.68, +0.49, −0.40, and ΔδC −5.8, −4.4, −4.5 ppm, respectively), which indicated the changes of the relative configuration from 2 to 1. The relative configuration of 2 was also determined by analysis of the ROESY spectrum. The correlations between H-3/H3-29, H-11/ H3-18, and H-15/H3-18 indicated H-3, H-11, and H-15 were all β-oriented. The correlations between H-17/H3-18, H-20/H318, H-21/H3-18, and H-24/H3-18 confirmed that H-17, H-20, H-21, and H-24 were all β-oriented. The absolute configuration was assigned as 5S,10S from positive and negative Cotton effects at 335.5 (Δε +10.22) and 254.5 (Δε −27.71) nm, respectively, in the ECD spectrum.14 Therefore, compound 2 was assigned the structure (−)-(3R,5S,10S,11R,15S,17S,20R,21S,24R)-21,24-cyclopenta3,11,15,21,25-pentahydroxylanosta-8-en-7-one, named inonotusol B, which was stereoisomeric at the 17- and 24-positions of 1. Compound 3 was assigned a molecular formula of C30H48O5 by HRESIMS. Its NMR data were similar to those for 1 except for the data of the lanostane skeleton. The two signals at δH 6.45 (d, J = 6.0 Hz, H-7) and δH 5.53 (d, J = 6.0 Hz, H-11) and the four olefinic carbons [δC 122.0 (C-7), 147.4 (C-8), 142.9 (C-9), 117.1 (C- 11)] in the 1H NMR (Table 1) indicated that 3 possessed a 7,9(11)-diene moiety.11 The two oxymethine protons [δH 4.76 (m), 4.25 (dd, J = 5.0, 11.0 Hz)] and the two signals of oxymethylene at δH 4.13 (d, J = 10.5 Hz) and δH 3.67 (d, J = 10.5 Hz) indicated the presence of two hydroxy groups and a hydroxymethyl group. The positions of two hydroxy
spectrometry). The IR spectrum showed absorption bands for the hydroxy (3386 cm−1) and carbonyl (1644 cm−1) groups. The 1H and 13C NMR data (Table 1) revealed resonances for seven methyls (δH 1.65, 1.44, 1.40, 1.25, 1.11, 1.10, 1.07, each 3H, s; δC 19.5, 26.3, 30.7, 20.2, 16.3, 28.2, 19.0), seven methylenes, eight methines including four oxymethines [δH 4.82 (dd, J = 4.5, 9.5 Hz), δC 64.9; δH 4.64 (dd, J = 6.0, 9.0 Hz), δC 74.0; δH 4.09 (t, J = 8.0 Hz), δC 79.5; δH 3.47 (m), δC 77.7], eight quaternary carbons including an oxygenated carbon (δC 72.6), and one α,β-unsaturated ketone group (δC 204.7, 167.7, 141.6). The data of 1 displayed signals characteristic of a lanostane triterpene and were similar to those for 3β,22dihydroxylanosta-8,24-dien-7-one. The evident differences were that 1 had two additional hydroxy groups on the lanostane skeleton and a 21,24-cyclopentanol moiety in the side chain shown by comparing the NMR data (Table 1) with those reported for 21,24-cyclopentalanosta-3β,21,25-triol-8-ene.9 The positions of the three hydroxy groups at C-3, C-11, and C-15 were determined through correlations from H3-28 to C-3; from H-11 to C-8 and C-9; from H-16a (δH 2.21) to C-15; and from B
dx.doi.org/10.1021/np400552w | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Spectroscopic Data for Compounds 1−4a 1
2
3
4
position
δC, type
δH (J in Hz)
δC
δH (J in Hz)
δC
1
35.1 CH2
2.57, dt (5.5, 12.5), 2.12b
35.2 CH2
74.5 CH
4.76, m
75.0 CH
4.75, t (7.2)
2
28.9 CH2
1.97 (2H)
28.9 CH2
36.6 CH2
2.17,b 1.64, m
36.7 CH2
2.10, m, 1.61, m
3 4 5 6
77.7 40.0 50.9 37.9
CH C CH CH2
3.47, m
77.7 40.0 50.9 37.9
CH C CH CH2
2.59, m, 2.20b 2.01,b 1.79, m 3.49, t (7.5)
73.5 43.5 43.0 23.7
CH C CH CH2
4.25, dd (5.0, 11.0)
73.6 CH 43.7 C 43.05 CH 23.9 CH2
4.25, dd (4.2, 11.4)
7 8 9 10 11 12
204.7 141.6 167.7 41.8 64.9 44.4
C C C C CH CH2
204.7 141.5 167.5 41.8 65.0 45.9
C C C C CH CH2
122.0 147.4 142.9 52.8 117.1 37.0
CH C C C CH CH2
13 14 15
49.9 C 52.3 C 74.0 CH
2.01, d (3.5) 2.74 (2H)
4.82, dd (4.5, 9.5) 3.27, dd (9.5, 13.5), 2.48, dd (4.5, 13.5)
4.64, dd (6.0, 9.0) b
49.6 C 52.3 C 74.3 CH
16
36.6 CH2
2.21, m, 2.12
17 18 19 20 21 22
49.3 19.0 20.2 49.2 79.5 24.9
CH CH3 CH3 CH CH CH2
2.36, dd (9.0, 18.0) 1.07, s 1.25, s 2.08, m 4.09, t (8.0) 1.73, m, 1.48, dt (3.5, 6.0)
49.4 19.2 20.3 43.4 75.1 22.2
CH CH3 CH3 CH CH CH2
23 24 25 26 27 28
28.7 58.8 72.6 26.3 30.7 16.3
CH2 CH C CH3 CH3 CH3
1.85, m, 1.42, m 2.23, m 1.44, s 1.40, s 1.11, s
29.1 54.3 72.5 30.4 30.6 16.3
CH2 CH C CH3 CH3 CH3
29 30
28.2 CH3 19.5 CH3
1.10, s 1.65, s
28.2 CH3 19.5 CH3
36.8 CH2
2.01b 2.74, m, 2.71b
4.81, m 2.82, m, 2.71b
45.4 C 38.2 C 29.0 CH2
4.63, dd (6.0, 9.0) 2.20,b 2.07, m 1.70, m 0.89, s 1.33, s 2.76, m 4.58, m 2.20,b 1.68, m 1.23 (2H) 1.83, m
δH (J in Hz)
2.07, m 2.31, m, 2.21, m 6.45, d (6.0)
5.53, d (6.0) 2.87, dd (6.5, 17.5), 2.51, d (17.5)
1.87, m, 1.40, br s b
δC
122.4 147.4 142.8 52.8 117.2 38.3
CH C C C CH CH2
δH (J in Hz)
2.05, m 2.32, dd (6.6, 3.6), 2.24, m 6.45, d (6.0)
5.56, d (6.0) 2.87, br d (18.0), 2.36, dd (6.6, 18.0)
45.3 C 38.6 C 29.4 CH2
1.99, m, 1.79, m
40.1 CH2
2.15 (2H)b
40.0 CH2
2.17 (2H)
49.0 18.1 18.6 49.3 79.6 25.0
CH CH3 CH3 CH CH CH2
2.28, m 1.09, s 1.37, s 2.14, m 4.11, t (8.0) 1.74 (2H)
49.5 CH 18.4 CH3 18.7 CH3 43.09 CH 75.5 CH 22.4 CH2
1.73, m 0.91, s 1.38, s 2.68, m 4.58, m 2.15,b 1.69, m
CH2 CH C CH3 CH3 CH2
2.01 (2H) 2.23, m
28.8 54.5 72.5 30.5 30.8 67.8
1.23 (2H) 1.81, m
1.35, s 1.63, s 1.13, s
28.7 59.0 72.5 26.6 30.7 67.7
1.11, s 1.65, s
13.6 CH3 24.1 CH3
1.43, s 1.47, s 4.13, d (10.5), 3.67, d (10.5) 1.13, s 1.19, s
CH2 CH C CH3 CH3 CH2
13.8 CH3 24.3 CH3
1.34, s 1.64, s 4.14, d (10.8), 3.68, d (10.8) 1.16, s 1.25, s
a13 C NMR data (δ) were measured at 125 MHz for 1−3 and at 150 MHz for 4. 1H NMR data (δ) were measured at 500 MHz for 1−3 and at 600 MHz for 4. The assignments were based on 1H−1H COSY, DEPT, HSQC, HMBC, and ROESY experiments. bOverlapped signals
Compound 4 exhibited the same molecular formula (C30H48O5) as 3 by HRESIMS. The 1H and 13C NMR data (Tables 1) of the lanostane skeleton were quite similar to those for 3, and the data for the side chain part were very similar to those for 2, which were also supported by HMBC correlations from H-1 to C-9 and Me-19; from H-3 to Me-29; from H-28a to C-3; from H-28b to C-3 and Me-29; from H-20 to C-16 and Me-18; from H3-26 to C-24 and Me-27; and from H3-27 to C24 and Me-26. The relative configuration of 4 was proven by ROESY. The correlations between H-1/H3-18 and H-3/H3-30 indicated H-1 and H-3 were β-oriented and α-oriented, respectively. The correlations between H-17/H3-18, H-20/ H3-19, H-21/H3-18, and H-24/H3-19 confirmed that H-17, H20, H-21, and H-24 were all β-oriented. Consequently, inonotusol D was assigned the structure (17β,20β,24β)-21,24cyclopenta-1α,3β,21α,25,28-pentahydroxy-5α-lanosta-7,9(11)diene. Compound 5 was shown to have the molecular formula C30H48O5 by HRESIMS, which was determined to be a 15dehydroxy analogue of 2 from its 1H and 13C NMR data (Table
groups at C-1 and C-3 were determined through long-range correlations from H-1 to C-9, C-10, and Me-19 and from H-3 to Me-29 by HMBC. The position of the hydroxymethyl group at C-4 was determined through correlations from H-28a (δH 4.13) to C-5 and Me-29 and from H-28b (δH 3.67) to C-3, C4, and Me-29 by HMBC. The relative configuration of 3 was determined by analysis of the ROESY spectrum. The correlations between H-1/H3-18 and H-3/H3-30 indicated H1 and H-3 were β-oriented and α-oriented, respectively. Moreover, the same orientations between H3-29 and H3-19 indicated the hydroxymethyl group was α-oriented. Furthermore, the correlations between H-24/H-17, H-17/H3-30, H20/H3-18, and H-21/H-12β/H3-18 confirmed that H-17 and H-24 were α-oriented, and H-20 and H-21 were β-oriented, which were the same as those of 1. Hence, inonotusol C was assigned the structure (17α,20β,24α)-21,24-cyclopenta1α,3β,21α,25,28-pentahydroxy-5α-lanosta-7,9(11)-diene. To our knowledge, a lanostane triterpene having a 1α,3β-dihydroxy moiety has rarely been isolated from I. obliquus. C
dx.doi.org/10.1021/np400552w | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. NMR Spectroscopic Data for Compounds 5−8a 5
6
7
8
position
δC, type
δH (J in Hz)
δC
δH (J in Hz)
1
35.6 CH2
2.22 (2H)
35.6 CH2
1.73, m, 1.23b
35.5 CH2
1.74, m, 1.24b
37.9 CH2
2
29.3 CH2
27.9 CH2
1.66, m, 1.58, m
27.8 CH2
1.68 (2H)b
18.9 CH2
3
77.9 CH
1.82, m, 1.22, m 3.48, t (7.8)
2.26, br d (13.2), 1.53, dd (3.6, 13.2) 1.76, m, 1.65, m
79.0 CH
3.23, dd (4.5, 11.5)
78.9 CH
3.23, dd (4.4, 11.2)
37.6 CH2
4 5 6
40.2 C 51.5 CH 38.1 CH2
2.00b 2.71 (2H)b
38.9 C 50.4 CH 18.2 CH2
1.05, dd (2.0, 13.0) 1.69,b 1.51, m
38.8 C 50.3 CH 18.2 CH2
1.05, m 1.70,b 1.53, m
47.0 C 38.9 CH 44.9 CH2
7 8 9 10 11 12
200.9 141.7 163.0 41.7 65.4 45.3
C C C C CH CH2
13 14 15 16 17 18 19 20 21 22
48.5 50.1 27.9 44.2 34.6 19.0 20.7 48.8 75.2 22.3
C C CH2 CH2 CH CH3 CH3 CH CH CH2
23
29.1 CH2
24 25 26 27 28 29 30 31
54.5 72.6 30.5 30.8 16.5 28.5 26.0
CH C CH3 CH3 CH3 CH3 CH3
4.83, m 2.80, m, 2.71b
1.72 (2H) 2.57 (2H) 2.04, m 0.86, s 1.36b 2.10, m 4.64, m 1.07, m, 1.00, m 2.00 (2H)b 1.82, m 1.36b 1.64, s 1.13, s 1.11, s 1.56, s
δC
27.1 134.8 134.0 37.1 21.0 26.5
CH2 C C C CH2 CH2
45.0 49.5 30.9 30.9 46.5 15.6 19.1 39.4 167.1 29.8
C C CH2b CH2b CH CH3 CH3 CH C CH2
78.6 CH 122.1 149.0 13.6 12.5 15.4 28.0 24.4 20.5
C C CH3 CH3 CH3 CH3 CH3 CH3
δH (J in Hz)
1.77, m, 1.41, m
2.03 (2H)b 2.03 (2H)b
1.69,b 1.23b 1.69,b 1.23b 1.50, m 0.73, s 0.98b 2.03b 2.45, t (15.0), 1.90, d (3.5) 4.39, dt (3.5, 15.0)
0.98b 1.88, s 1.00, s 0.81, s 0.89, s 1.94, s
δC
27.2 134.5 134.1 37.0 20.9 26.4
CH2 C C C CH2 CH2
44.8 49.4 30.9 30.9 47.2 15.7 19.1 41.1 12.9 69.3
C C CH2b CH2b CH CH3 CH3 CH CH3 CH
37.2 CH2 203.4 144.7 125.8 17.4 15.4 27.9 24.3
C C CH2 CH3 CH3 CH3 CH3
δH (J in Hz)
1.70,b 1.45, m
198.6 131.7 154.2 38.0 124.7 133.1
2.03 (2H)b 2.03 (2H)b
C C C C CH CH
147.2 C 125.4 CH 34.1 CH 24.21 CH3 24.17 CH3 24.0 CH3 17.3 CH3 180.6 C
1.68,b 1.24b 1.68,b 1.24b 1.40, m 0.74, s 0.98, s 1.83, m 0.96, d (6.8) 4.17, m
2.03, dt (3.6, 13.2), 1.87, br d (13.2) 2.94, dd (3.0, 16.8) 3.06, dd (3.0, 13.2), 2.98, m
7.36, d (8.4) 7.46, dd (1.8, 8.4)
8.15, 2.84, 1.17, 1.17, 1.22, 1.50,
d (1.8) m d (7.0) d (7.0) s s
2.78, d (17.2), 2.66, dd (10.4, 17.2)
6.02, 1.89, 0.99, 0.81, 0.88,
s, 5.85, s s s s s
a13 C NMR data (δ) were measured in pyridine-d5 at 150 MHz for 5 and 8 and in CDCl3 at 125 MHz for 6 and at 100 MHz for 7. 1H NMR data (δ) were measured in pyridine-d5 at 600 MHz for 5 and 8 and in CDCl3 at 500 MHz for 6 and at 400 MHz for 7. The assignments were based on 1 H−1H COSY, DEPT, HSQC, HMBC, and ROESY experiments. bOverlapped signals
(dt, J = 3.5, 15.0 Hz), δC 78.6], nine quaternary carbons including four olefinic carbons (δC 149.0, 134.8, 134.0, 122.1), and a carbonyl carbon (δC 167.1). The data for 6 displayed signals characteristic of a lanostane triterpene and were very similar to those reported for 3-hydroxy-8,24-diene-lanosta21,23-lactone.8 The evident difference was that 6 had one additional methyl (δH 1.94, 3H, s; δC 20.5) at C-24, which was proven by the tetrasubstituted olefinic bond [δC 122.1 (C), 149.0 (C)] and the long-range correlations from H3-31 to C-22, C-24, and C-25 by HMBC. The relative configuration of 6 was proven by ROESY. The correlations between H-3/H3-28, H17/H3-30, and H-17/H-21/H-23 indicated H-3, H-17, H-21, and H-23 were all α-oriented. Therefore, inonotusol F was elucidated as (17α,21α,23α)-24-methyl-3β-hydroxy-5α-lanosta8,24-diene-21,23-lactone. Compound 7 was assigned the molecular formula C30H48O3 by HRESIMS. Analysis of the 1H and 13C NMR spectra (Table 2) indicated 7 and 6 had the same lanostane skeleton, but a
2) and the long-range correlations from H3-28 to C-3 and from H-12b to C-11 by HMBC. The relative configuration of 5 was established by ROESY. The correlations between H-3/H3-29 and H-11/H3-18 indicated H-3 and H-11 were β-oriented. The correlations between H-17/H3-19, H-20/H3-18, H-21/H3-18, and H-24/H3-18 confirmed that H-17, H-20, H-21, and H-24 were all β-oriented. Moreover, the Cotton effects in the ECD spectrum were similar to those for 2 (Supporting Information S58). Therefore, compound 5 was assigned the structure (−)-(3R,5S,10S,11R,17S,20R,21S,24R)-21,24-cyclopenta3,11,21,25-tetrahydroxylanosta-8-en-7-one and named inonotusol E. Compound 6 was assigned the molecular formula C31H48O3 by HRESIMS. The 1H and 13C NMR data (Tables 2) revealed resonances for eight methyls (δH 1.94, 1.88, 1.00, 0.98, 0.98, 0.89, 0.81, 0.73, each 3H, s; δC 20.5, 12.5, 15.4, 19.1, 13.6, 24.4, 28.0, 15.6), nine methylenes, five methines including two oxymethines [δH 3.23 (dd, J = 4.5, 11.5 Hz), δC 79.0; δH 4.39 D
dx.doi.org/10.1021/np400552w | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
(14),23 ergosta-7-en-3β-ol (15),24 5,8-epidioxyergosta-6,22dien-3β-ol,25 ergostrol,22 ergosta-4,6,8(14),22-tetraen-3-one,22 vanillic acid (16),26 protocatechuic aldehyde (17),27 4-(3,4dihydroxyphenyl)but-3-en-2-one (18),28 protocatechuic acid,29 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one,30 and 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone30 by comparing their spectroscopic data with values reported in the literature. All compounds were bioassayed for their hepatoprotective effects against D-galactosamine-induced toxicity in WB-F344 cells, using the hepatoprotective activity drug bicyclol as the positive control. As shown in Table 3, 10 compounds (10 μM)
different side chain. The NMR data for the side chain part, resonances for two methyls [δH 1.89 (3H, s), δC 17.4; δH 0.96 (3H, d, J = 6.8 Hz), δC 12.9], two methylenes including one olefinic methylene [δH 6.02 (1H, s), 5.85 (1H, s); δC 125.8], two methines including one oxymethine [δH 4.17 (1H, m); δC 69.3], two quaternary carbons including one olefinic carbon (δC 144.7), and one carbonyl carbon (δC 203.4), which were similar to those of inonotsutriols D and E,10 indicated 7 was the 24-oxo derivative of inonotsutriol D or E. This was proven by 2D NMR data analysis, particularly by the correlations by HMBC from H-22 to C-24; from H2-23 to C-24; from H-26b (δH 5.85) to C-24, C-25, and Me-27; and from H3-27 to C-24, C-25, and C-26. The relative configuration of 7 was proven by ROESY. The correlations between H-3/H3-28 indicated H-3 was αoriented.10 Therefore, inonotusol G was designated as 3β,22dihydroxy-5α-lanosta-8,25-dien-24-one. We have tried to determine the absolute configuration of C-22 by application of the modified Mosher’s method,3 but failed. The derivative of (S)- or (R)-MTPA esters for the hydroxy group at C-22 could not be obtained from 7 using (−)- or (+)-MTPACl (αmethoxy-α-trifluoromethylphenylacetyl chloride) by stirring at room temperature for 24 h or heating at 60 °C for 30 min. The hydrogen bond effect between the carbonyl group at C-24 and the hydroxy group at C-22 may have hindered the reactions. Compound 8 was obtained as an amorphous powder with [α]25D −19.3. The molecular formula (C20H26O3) with eight degrees of unsaturation was indicated by HRESIMS. The 1H NMR (Table 2) revealed resonances for two methyls (δH 1.50, 1.22, each 3H, s), an isopropyl group [δH 2.84 (1H, m), 1.07 (6H, d, J = 7.0 Hz)], three aromatic proton signals [δH 8.15 (1H, d, J = 1.8 Hz), 7.46 (1H, dd, J = 1.8, 8.4 Hz), 7.36 (1H, d, J = 8.4 Hz)], an aliphatic methine [δH 2.94 (1H, dd, J = 3.0, 16.8 Hz, H-5)], and eight proton signals due to four methylenes. In addition to proton-bearing carbon resonances corresponding to the above units, the 13C NMR spectra exhibited resonances for six quaternary carbons consisting of three aromatic carbon signals (δC 154.2, 147.2, 131.7), a ketone group (δC 198.6), a carboxyl group (δC 180.6), and an aliphatic carbon (δC 47.0). The above-mentioned data were similar to those reported for 12-hydroxy-13-isopropyl-7-oxo-20-oic acid16 and suggest 8 was a 12-dehydroxy analogue. This was supported by HMBC correlations from H-1β to C-20; from H-11 to C-8, C-9, C-10, and C-13; from H-14 to C-7, C-8, and C-12; from H-12 to C-9, C-11, and C-15; from H-15 to C-13, Me-16, and Me-17; and from H3-16, 17 to C-13 and C-15. The relative configuration was established by ROESY. The correlations between H-5 and H3-19 indicated H-5 was αoriented. Moreover, the ECD spectrum of 8 displayed a positive Cotton effect at 328.5 nm (Δε +2.04) and a negative Cotton effect at 295.5 nm (Δε −1.60), indicating a 5S,10S configuration based on the aryl ketone CD rule.17−19 Therefore, compound 8 was assigned the structure (−)-(5S,10S)-13-isopropyl-7-oxo-abieta-8,11,13-trien-20-oic acid, named inonotusic acid. To our knowledge, this is the first report of the occurrence of an abietane-type diterpene in I. obliquus. The isolated known compounds were identified as inotodiol (9),8 lanosterol (10),8 3β,22-dihydroxylanosta-8,24-dien-11one (11),3 trametenolic acid (12),8 3β,22-dihydroxylanosta7,9(11),24-triene,11 3β,22-dihydroxylanosta-8,24-dien-7-one,12 3β-hydroxylanosta-8,24-den-21-al,13 21-hydroxylanosterol,12 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid,20 botulin,21 ergosta-7,22-dien-3β-ol (13),22 24β-ethylcholest-4-en-3β-ol
Table 3. Hepatoprotective Effects of 10 Compounds (10 μM) against D-Galactosamine-Induced Toxicity in WB-F344 Cellsa compound
cell survival rate (% of normal)
inhibition (% of control)e
± ± ± ± ± ± ± ± ± ± ± ± ±
71.9 34.4 56.3 74.8 81.2 75.0 43.8 40.6 37.4 56.5
normal control bicyclol 6 8 9 10 11 12 13 14 15 16
100 68 80 91 79 86 92 94 92 82 81 80 86
3.3 3.1 1.1c 6.9d 3.2d 3.2c 6.3d 2.5c 0.6b 0.5c 5.6d 1.2d 4.6c
Results are expressed as the means ± SD (n = 3; for normal and control, n = 6); bicyclol was used as positive control (10 μM). bp < 0.001. cp < 0.01. dp < 0.05. eInhibition (%) = [(OD(sample) − OD(control))/(OD(normal) − OD(control))] × 100%. a
showed hepatoprotective activities. Compounds 6 and 10−12 exhibited moderate hepatoprotective effects, with inhibitions of >70%, respectively. They were also tested for their cytotoxicity against three human tumor cell lines, Bel-7402 (human hepatoma cell line), A-549 (human lung epithelial cell line), and KB (human nasopharynx cancer cell line), using 5fluorouracil as the positive control. As shown in Table 4, Table 4. Cytotoxicity of Three Compounds against BEL7402, A-549, or KB Human Cell Lines (IC50, μM) 7 17 18 5-fluorouracil
BEL-7402
A-549
KB
>10 3.7 4.7 5.5
>10 3.1 >10 3.1
9.9 >10 >10 8.1
compound 7 exhibited cytotoxicity against the KB cell line, compound 17 exhibited cytotoxicity against Bel-7402 and A549 cell lines, and compound 18 exhibited cytotoxicity against the Bel-7402 cell line, with IC50 values of
