An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol, and other triterpenoids from Inonotus obliquus

Article abstract of DOI:10.1016/j.phytochem.2010.07.005

An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol (1), and two lanostane-type triterpenoids, inonotsudiol A (2) and inonotsuoxodiol A (3), were isolated from the sclerotia of Inonotus obliquus. Their structures were determined to be (3S,7S,9R)-

Full text of DOI:10.1016/j.phytochem.2010.07.005

Phytochemistry 71 (2010) 1774–1779
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Phytochemistry
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An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol, and other
triterpenoids from Inonotus obliquus
*
Noriko Handa, Takeshi Yamada, Reiko Tanaka
Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol (1), and two lanostane-type triterpenoids,
inonotsudiol A (2) and inonotsuoxodiol A (3), were isolated from the sclerotia of Inonotus obliquus. Their
structures were determined to be (3S,7S,9R)-3,7-dihydroxy-7(8 ? 9)abeo-lanost-24-en-8-one (1), lano-
sta-8,24-dien-3b,11b-diol (2), and (22R)-3b,22-dihydroxylanosta-8,24-dien-11-one (3) on the basis of
NMR spectroscopy, including 1D and 2D (1H–1H COSY, NOESY, HMQC, HMBC) NMR, and FABMS. Com-
pounds 1–3 showed moderate activity against cultured P388, L1210, HL-60 and KB cells.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 22 January 2010
Received in revised form 21 April 2010
Available online 4 August 2010
Keywords:
Inonotus obliquus
Triterpenoid
Lanostane
Spiroinonotsuoxodiol
(3S,7S,9R)-3,7-Dihydroxy-7(8 ? 9)abeo-
lanost-24-en-8-one
Inonotsudiol A
Inonotsuoxodiol A
Cytotoxicity
Cancer cell
1. Introduction
sta-8,24-dien-21-al. In addition, we reported that inotodiol inhibits
cell proliferation through caspase-3-dependent apoptosis (Nomura
Inonotus obliquus (PERS.: Fr.) Pil. (=Fuscoporia obliqua (PERS.: Fr.)
Aoshima), called kabanoanatake in Japan and chaga or tchaga in
Russia, is a white-rot fungus belonging to the family Hymenochaet-
aceae Donk (Hawksworth et al., 1995) and thrives in Europe, Asia,
and North America (Ellis and Ellis, 1990). It is widely distributed
in Betula platyphylla var. japonica (Japanese name: shirakaba) for-
ests in Hokkaido, Japan (Mizuno et al., 1999). In Eastern Europe,
particularly Russia, the sclerotia of this mushroom have been used
as folk medicine for cancer treatments since the 16th or 17th
century (Shivrina, 1965). Recently, the extract of this fungus was
reported to possess anti-tumor (Nakajima et al., 2009; Song
et al., 2008), anti-oxidant (Kim et al., 2008; Ham et al., 2009; Zheng
et al., 2009; Hu et al., 2009), anti-inflammatory (Kim et al., 2007),
and anti-diabetic (Lee et al., 2006) activities. Previously, we re-
ported the structures of new lanostane-type triterpenoids isolated
from the sclerotia of this mushroom: inonotsuoxides A and B (Nak-
ata et al., 2007), inonotsulides A, B, and C (Taji et al., 2007), inonot-
sutriols A, B, and C (Taji et al., 2008a), lanosta-8,23E-diene-
3b,22R,25-triol, and lanosta-7:9(11),23E-triene-3b,22R,25-triol
(Taji et al., 2008b), as well as the anti-tumor promoting activities
of the most abundant triterpene, inotodiol, and 3b-hydroxylano-
et al., 2008). Careful examination of the sclerotia of I. obliquus has
led to the isolation of an unusual lanostane-type triterpene named
spiroinonotsuoxodiol (1) and two new lanostane-type triterpe-
noids, inonotsudiol A (2) and inonotsuoxodiol A (3). The structures
of new compounds 1–3 were determined on the basis of NMR spec-
troscopy, including 1D and 2D (1H, 1H COSY, NOESY, HMQC, HMBC)
NMR, and FABMS. The cytotoxicity of these compounds was exam-
ined by using the murine P388 leukemia, murine L1210 leukemia,
human HL-60 leukemia, and human KB epidermoid carcinoma cell
lines. In this paper, the structures and biological activities of the
compounds 1–3 are described.
2. Results and discussion
The sclerotia of I. obliquus were extracted with CHCl3 and the
extract was subjected to silica gel column chromatography, med-
ium-pressure liquid chromatography (MPLC), and C18 reversed-
phase high-pressure liquid chromatography (HPLC) to yield three
new triterpenoids (1–3).
The molecular formula of spiroinonotsuoxodiol (1) was deter-
mined to be C30H50O3 (M+; m/z 458.3757) on the basis of HRFABMS.
The IR spectrum showed bands assignable to a hydroxy group
(
mmax 3448 cmꢀ1
)
and
a six-membered ring ketone (mmax
* Corresponding author. Tel./fax: +81 72 690 1084.
E-mail address: tanakar@gly.oups.ac.jp (R. Tanaka).
1687 cmꢀ1). The 1H and 13C NMR spectra of 1 (Tables 1 and 2)
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.07.005

N. Handa et al. / Phytochemistry 71 (2010) 1774–1779
1775
Table 1
1H NMR spectroscopic data for compounds 1, 1a, 2, and 3 (500 MHz, CHCl3).a
Table 2
13C NMR spectroscopic data for compounds 1, 1a, 2 and 3 (125 MHz, CHCl3).a
H
1
1a
2
3
Position
1
1a
2
3
1
1b
a
1.97 (m)
1.64 (m)
1.70 (m)
1.79 (m)
3.22 (dd)
1.36 (dd)
2.25 (m)
1.58 (dt)
4.30 (br s)
–
2.00 (m)
1.50 (m)
1.58 (m)
1.75 (m)
1.20 (m)
1.85 (m)
1.93 (m)
1.30 (m)
1.63 (m)
0.66 (s)
1.46 (s)
1.41 (m)
0.91 (d)
1.05 (m)
1.43 (m)
1.86 (m)
2.03 (m)
5.09 (m)
1.69 (s)
1.60 (s)
0.95 (s)
0.95 (s)
1.20 (s)
1.66 (m)
2.00 (m)
1.73 (m)
1.82 (m)
4.46 (dd)
1.45 (m)
2.25 (dt)
1.57 (m)
4.31 (m)
–
1.56 (m)
1.26 (m)
2.01 (m)
1.78 (m)
1.20 (m)
1.86 (m)
1.93 (m)
1.30 (m)
1.63 (m)
0.65 (s)
1.49 (s)
1.41 (m)
0.92 (d)
1.05 (m)
1.41 (m)
1.89 (m)
2.03 (m)
5.09 (m)
1.69 (s)
1.60 (s)
0.84 (s)
1.01 (s)
1.20 (s)
1.39 (m)
2.14 (m)
1.66 (m)
1.72 (m)
3.26 (dd)
0.95 (m)
1.70 (m)
1.56 (m)
2.09 (m)
2.12 (m)
4.65 (dt)
–
1.92 (m)
2.32 (d)
1.21 (m)
1.58 (m)
1.95 (m)
1.37 (m)
1.52 (m)
0.86 (s)
1.11 (s)
1.42 (m)
0.99 (d)
1.03 (m)
1.45 (m)
1.87 (m)
2.04 (m)
5.10 (t)
1.69 (s)
1.61 (s)
1.00 (s)
0.84 (s)
0.87 (s)
1.06 (m)
3.01 (dt)
1.68 (m)
1.63 (m)
3.24 (dd)
0.90 (d)
1.75 (m)
1.46 (m)
2.27 (dd)
2.36 (dd)
–
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
30.6 (t)
28.2 (t)
79.7 (d)
38.2 (s)
50.2 (d)
34.0 (t)
80.6 (d)
215.6 (s)
64.1 (s)
48.9 (s)
29.9 (t)
30.7 (t)
47.5 (s)
61.2 (s)
29.6 (t)
27.0 (t)
50.3 (d)
17.0 (q)
18.4 (q)
35.4 (d)
18.6 (q)
36.0 (t)
24.8 (t)
124.9 (d)
131.2 (s)
25.7 (q)
17.6 (q)
29.6 (q)
16.2 (q)
19.6 (q)
30.3 (t)
24.3 (t)
81.0 (d)
37.3 (s)
50.2 (d)
33.9 (t)
80.4 (d)
215.5 (s)
64.1 (s)
48.6 (s)
29.9 (t)
30.7 (t)
47.6 (s)
61.2 (s)
29.6 (t)
27.0 (t)
50.3 (d)
16.9 (q)
18.5 (q)
35.3 (d)
18.7 (q)
36.0 (t)
24.8 (t)
124.9 (d)
131.2 (s)
25.7 (q)
17.6 (q)
29.5 (q)
17.3 (q)
19.6 (q)
21.2 (q)
171.1 (s)
34.7 (t)
27.7 (t)
79.2 (d)
39.0 (s)
50.9 (d)
18.0 (t)
26.6 (t)
142.8 (s)
133.8 (s)
37.3 (s)
81.6 (d)
36.5 (t)
42.3 (s)
51.0 (s)
30.7 (t)
28.1 (t)
49.9 (d)
16.8 (q)
21.8 (q)
36.1 (d)
18.7 (q)
36.2 (t)
24.9 (t)
125.1 (d)
131.0 (s)
25.7 (q)
17.6 (q)
28.3 (q)
15.4 (q)
24.7 (q)
34.3 (t)
28.0 (t)
78.7 (d)
39.0 (s)
51.8 (d)
17.3 (t)
29.9 (t)
163.9 (s)
139.6 (s)
37.7 (s)
199.0 (s)
51.8 (t)
47.4 (s)
51.2 (s)
31.1 (t)
26.1 (t)
46.9 (d)
16.6 (q)
19.0 (q)
41.3 (d)
12.5 (q)
73.0 (d)
29.3 (t)
120.9 (d)
135.5 (s)
26.0 (q)
18.0 (q)
28.3 (q)
15.6 (q)
25.8 (q)
2a
2b
3
5
6a
6b
7a
7b
11
a
11b
12
12b
15
15b
16
16b
17
18
19
–
a
2.66 (m)
2.48 (d)
1.37 (m)
1.80 (m)
1.87 (m)
1.50 (m)
1.81 (m)
0.86 (s)
1.13 (s)
1.81 (m)
0.92 (d)
3.65 (dt)
–
2.05 (m)
2.08 (m)
5.17 (m)
1.75 (s)
1.66 (s)
1.02 (s)
0.83 (s)
1.10 (s)
a
a
20
21
22A
22B
23A
23B
24
26
27
28
29
3-OCOCH3
3-OCOCH3
30
Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY experiments.
a
Assignments were based on 1H–1H COSY, HMQC, HMBC, and NOESY
a
experiments.
24
22
26
21
18
exhibited signals assignable to five tertiary methyls, one secondary
methyl, two vinyl methyls, nine CH2 groups, six methine groups,
including two oxymethines (dH 3.22 (1H, dd); 4.30 (1H, brs)), one
trisubstituted olefin (dH 5.09 (1H, m), and seven quaternary car-
bons, including an sp2-carbon and a saturated ketone (dC 215.6
(s)). The 1H and 13C NMR spectra showed that the compound had
one more methine and quaternary sp3 carbons but lacked two
methylene groups in comparison with that of the corresponding
lanostane-type triterpenoid. Therefore, 1 seems to be a migrated
lanostane-type triterpenoid. The structure of 1 was determined
from the HMBC and 1H–1H COSY spectra (Fig. 1). The HMBC spec-
trum of 1 showed correlations between: Me-19 (d 1.46) and two
sp3 quaternary carbons, C-9 and C-10, in addition to C-1 and C-5;
H-7 (d 4.30) and C-5, C-6, C-8, C-9, and C-10; H-3 (d 3.22) and C-
1, C-2, C-4, and C-5; Me-18 (d 0.66) and C-12, C-13, C-14, and C-
17; Me-21 (dH 0.91) and C-17, C-20, and C-22; Me-26 (dH 1.69)
and C-24, C-25, and C-27; Me-27 (dH 1.60) and C-24, C-25, and C-
26; Me-28 (dH 0.95) and C-3, C-4, C-5, and C-29; Me-29 (dH 0.95)
and C-3, C-4, C-5, and C-28; and Me-30 (dH 1.20) and C-8, C-13, C-
14, and C-15. In the 1H–1H COSY spectrum, H-7 correlated with
H2-6, and H2-11 correlated with only H2-12; therefore, C-9 is a qua-
ternary sp3 carbon. The FABMS of 1 displayed two characteristic
peaks of spiro-lanostane-type triterpenoid (Tanaka et al., 1992) at
305.2481 [C20H33O2] and 205.1961 [C15H25] (Fig. 1) along with m/z
440 [MꢀH2O]+, 422 [Mꢀ2H2O]+, and 327. When 1 was acetylated
in the usual manner, monoacetate (1a) was obtained in quantitative
yield and the H-3 proton signal was shifted downfield to d 4.46,
indicating that the C-7 hydroxyl group is located in a considerably
sterically hindered position. Based on the molecular formula of
20
25
23
27
17
12
8
19
16
15
11
13
1
4
14
2
10
9
7
H
1H-1H COSY
HMBC
5
3
6
O
OH
30
HO
H
28
29
m/z 205
m/z 305
Fig. 1. Selected 1H–1H COSY and HMBC correlations and EIMS of 1.
having a ketone at C-8 and two hydroxyl groups at C-3 and C-7.
The relative structure of 1 was establishedfrom the NOESY spectrum
(Fig. 2): significant NOE correlations were observed between: Me-19
and H-1b, H-2b, H-6b, and Me-18; H-7 and H-5
Me-30; and H-3 and H-1 , H-5 , and Me-28. Therefore, the configu-
ration of C-9 was R and those of the hydroxyl groups at C-3 and C-7
were S and S. In addition, correlations were found between H-5 and
H-3 , andH-11b; H-17 and Me-30;H-20and H-16, and Me-18;and
H-12b and H-1 , Me-18, and Me-19 which led us to deduce that the
a, H-11a, H-15a, and
a
a
a
a
a
a
C-ring adopted a boat conformation. Therefore, structure 1 was
determined (3S,7S,9R)-3,7-dihydroxy-7(8 ? 9)abeo-lanost-24-en-8-one
(1). 3,7-Dihydroxy-7(8 ? 9)abeo-lanostane-type triterpenoids,
spiroveitchionolide (Tanaka et al., 1992), and spiromarienonols A
and B (Tanaka et al., 2004) were isolated from Abies veitchii and Abies
C30H50O3 and the spectroscopic data mentioned above, it was con-
cluded that 1 possesses a rare 7(8 ? 9)abeo-lanostane skeleton

1776
N. Handa et al. / Phytochemistry 71 (2010) 1774–1779
2
3
1
29
21
4
19
18
10
26
5
22
23
28
9
13
17
12
11
20
24
25
6
27
7
8
16
14
15
30
Fig. 2. Key NOEs correlations in 1 (graphical representation using the program CHEM 3D).
mariesii by our group, but those compounds are abieslactone deriv-
atives. Compound 1 seems to be biosynthesized from lanosterol
(5), one of the major components in the sclerotium (Nakata et al.,
2007). Compound 5 is oxidized to give 7-oxo-lanosterol (A), and
this is followed by the Baeyer–Villiger oxidation to furnish inter-
mediate (B). Lactone B is reduced and rearranged to give 1 (Scheme
1).
(dH 1.00) and C-3, C-4, C-5, and C-29; Me-29 (dH 0.84) and C-3,
C-4, C-5, and C-28; Me-30 (dH 0.87) and C-8, C-13, C-14, and C-
15; and H-24 (dH 5.10) and C-22, C-23, C-25, C-26, and C-27. In
the 1H–1H COSY spectrum, H2-12 correlated with an oxymethine
proton at H-11 (dH 4.65). On the basis of the above spectroscopic
data, structure 2 was suggested to be lanosta-8,24-dien-3,11-diol,
and the relative stereostructure was established by NOESY experi-
ment. In the NOESY spectrum (Fig. 3), significant NOE correlations
The molecular formula of inonotsudiol A (2) was determined to
be C30H48O2 (M+; m/z 440.3643) on the basis of HRFABMS. The IR
were observed between: H-3
a
and H-5
a; between H-11 and H-1
a
spectrum indicated the presence of
a
hydroxy group
(
mmax
and H-1b; Me-30 and H-7 , H-12
a
a
, H-15
a
, and H-17 ; and Me-21
a
3436 cmꢀ1). The 1H and 13C NMR spectra of 2 (Tables 1 and 2)
exhibited signals assignable to five tertiary methyls, one secondary
methyl, two vinyl methyls, nine CH2 groups, five methine groups
including two oxymethines [dH 3.26 (1H, dd); 4.65 (1H, dt)], one
trisubstituted olefin [dH 5.10 (1H, t)], four quaternary carbons,
and one tetrasubstituted olefin. In the HMBC spectrum of 2, long-
range correlations were observed between: Me-18 (d 0.86) and
C-12, C-13, C-14, and C-17; H2-12 (d 1.92, 2.32) and C-9, C-11, C-
13, and C-14; Me-19 (dH 1.11) and C-1, C-5, C-9 and C-10; Me-21
(dH 0.99) and C-17, C-20, and C-22; Me-26 (dH 1.69) and C-24, C-
25, and C-27; Me-27 (dH 1.61) and C-24, C-25, and C-26; Me-28
and H-12b and H-23. Therefore, the configurations of the hydroxyl
groups of 2 were established as 3b and 11b. Compound 2 was syn-
thesized from lanosterol (Woodward et al., 1957), and this is its
first report in nature.
Inonotsuoxodiol A (3) was assigned the molecular formula
C
30H48O3 (M+; m/z 456.3602) on the basis of HRFABMS. The UV
and IR spectra showed bands indicating the presence of a hydroxy
group (mmax 3398 cmꢀ1) and an
a,b-unsaturated six-membered
ring ketone (mmax 1655 cmꢀ1; kmax 256 nm). The 1H and 13C NMR
spectra of 3 (Tables 1 and 2) exhibited signals assignable to five
tertiary methyls, one secondary methyl, two vinyl methyls, eight
[O]
[O]
O
O
HO
HO
HO
B
5
A
O
HO
H
9
8
7
O
OH
O
H
HO
CH=O
1
C
7
Scheme 1. Proposed mechanism for bioenergies of 1 from 5.

N. Handa et al. / Phytochemistry 71 (2010) 1774–1779
1777
19
18
21
26
2
29
27
1
10
12
22
9
13
17
20
11
25
23
16
24
3
4
5
8
6
14
15
7
28
30
Fig. 3. Key NOEs correlations in 2 (graphical representation using the program CHEM 3D).
CH2 groups, five methine groups, including two oxymethines (dH
higher field than those of the 3,22-di-(R)-MTPA ester (3b) [Dd:
3.24 (1H, dd); 3.65 (1H, dt)) and one trisubstituted olefin (dH
5.17 (1H, m), four sp3 quaternary carbons, one tetrasubstituted ole-
fin, and one unsaturated ketone (dC 199.0 (s)). The structure of 3
was determined from analysis of the HMBC and 1H–1H COSY spec-
tra. The HMBC spectrum of 3 indicated long-range correlations be-
tween: Me-18 (dH 0.86) and each of C-12, C-13, C-14, and C-17;
Me-19 (dH 1.13) and C-1, C-5, C-9, and C-10; Me-21 (dH 0.92) and
C-17, C-20, and C-22; Me-26 (dH 1.75) and C-24, C-25, and C-27;
Me-27 (dH 1.66) and C-24, C-25, and C-26; Me-28 (dH 1.02) and
C-3, C-4, C-5, and C-29; Me-29 (dH 0.83) and C-3, C-4, C-5, and C-
28; Me-30 (dH 1.10) and C-8, C-13, C-14, and C-15; H2-12 (dH
2.48, 2.66) and C-9, C-11, C-13, and C-14; and H-24 (dH 5.17) and
C-22, C-23, C-25, C-26, and C-27. In the 1H–1H COSY spectrum,
the H-20 (dH 1.81) and H2-23 (dH 2.05, 2.08) correlated with H-
22 (dH 3.65), and H2-12 proton correlated with only the geminal
proton. The above spectroscopic data suggested that structure 3
was 3,22-dihydroxylanosta-8,24-diene-11-one. The absolute con-
figuration at C-20 of 3 was established as C-20b because of the sig-
nificant NOEs for H-20 to Me-18, Me-21, and H-16b. One of the
hydroxyl groups was C-3b, as shown by the chemical-shift and
negative], while signals due to protons attached to the C-28 and
C-29-positions of 3a were observed at lower fields than those of
3b [Dd: positive]. Therefore, the absolute configuration at the C-3
position of 3 was determined to be S. On the other hand, signals
due to protons attached to the C-23, C-26, and C-27 positions of
3,22-di-(S)-MTPA ester (3a) were observed at lower fields than
those of 3,22-di-(R)-MTPA ester (3b) [
onance due to the proton attached to the C-21 position in 3a was
observed at a higher field than that of 3b [ d: negative]. Hence,
Dd: positive], while the res-
D
the absolute configuration at the C-22 position of 3 was deter-
mined to be R and the total stereostructure of inonotsuoxodiol A
(3) was determined to be (22R)-3b,22-dihydroxylanosta-8,24-
dien-11-one (3). NOEs were observed from Me-19 to H-2b and
Me-29; from H-5
a
to H-7
a
; from H-6b to Me-19 and Me-29; from
H-7 to Me-30; from H-12
a
a to Me-21 and Me-30; and from H-11b
to Me-18. Therefore, the C rings in 3 adopted a boat conformation.
As a primary screen for anti-tumor activity, the cancer cell
growth inhibitory properties of spiroinonotsuoxodiol (1), inono-
tsudiol A (2), and inonotsuoxodiol A (3) were examined using mur-
ine P388 leukemia, murine L1210 leukemia, and human HL-60
leukemia cell lines. All compounds exhibited moderate cytotoxic
activity against the cancer cell lines (Table 3).
the coupling constants [dH 3.24 (1H, dd, J3,2 = 5.0 Hz and
a
J3,2b = 11.4 Hz); dc 78.7 (d)]. The absolute configuration at C-22 of
3 was deduced to be R because coupling constants were observed
[dH 3.65 (1H, dt, J22,20b = 9.8 Hz and J22,23 = 3.0 Hz); dc 73.0 (d)] and
2.1. Concluding remarks
significant NOEs were noted from H-22 to H-16
a; from H-22 to H-
24; from H-22 to H-17 ; and from H-17 to Me-21. The absolute
a
a
In this study, an unusual lanostane-type triterpenoid, spiro-
inonotsuoxodiol (1), and two new lanostane-type triterpenoids,
inonotsudiol A (2) and inonotsuoxodiol A (3) were isolated from
the sclerotia of I. obliquus. Their structures were determined to
be (3S,7S,9R)-3,7-dihydroxy-7(8 ? 9)abeo-lanost-24-en-8-one (1),
lanosta-8,24-dien-3b,11b-diol (2), and (22R)-3b,22-dihydroxylano-
sta-8,24-dien-11-one (3).
configuration at C-22 was determined by the modified Mosher’s
method (Ohtani et al., 1991). Compound 3 gave 3,22-di-(S)-MTPA
ester (3a) on treatment with [(ꢀ)-MTPACl] in pyridine, and 3,22-
di-(R)-MTPA ester (3b) on treatment with [(+)-MTPACl] in pyri-
dine. As shown in Fig. 4, signals due to protons attached to the
C-19 position of 3,22-di-(S)-MTPA ester (3a) were observed at a
OR
-0.18
OH
+0.09
22
-0.04
+0.02
+0.07
22
O
-0.02
+0.12
O
(-)-or (+)-MTPA
pyridine
3
3
RO
+0.03
HO
+0.09
3a: R=(S)-MTPA
3b: R=(R)-MTPA
3
Fig. 4. 1H chemical-shift differences (
Dd = dS–dR) between the (R)- and (S)-MTPA esters (3a and 3b).

1778
N. Handa et al. / Phytochemistry 71 (2010) 1774–1779
calcd for 458.3764); IR (KBr) mmax cmꢀ1; 3448 (OH), 2962, 1687
(C@O), 1459, 1383, 1019; for 1H and 13C NMR spectroscopic data,
see Tables 1 and 2. FABMS m/z (rel. int.): 458 (28) [M]+, 443 (7),
440 (37) [MꢀH2O]+, 369 (25), 327 (20), 318 (5), 305.2481 (100)
[C20H33O2, calcd for 305.2480]+, 205.1961 (14) [C15H25, calcd for
205.1956]+, 179 (22), 161 (39), 136 (35).
Table 3
Cytotoxity of compounds 1–3 against P388, L1210, HL-60, and KB cell lines.
Compounds
P388
L1210
HL-60
KB
IC50
(
l
M)a
IC50
(l
M)a
IC50
(l
M)a
IC50 (l
M)a
1
2
3
29.5
23.8
15.2
2.5
12.5
23.8
19.7
2.1
30.1
27.2
17.7
3.7
21.2
14.5
89.8
7.7
5-Fluorouracilb
a
3.5. Spiroinonotsuoxodiol monoacetate (1a)
DMSO was used for vehicle.
Positive control.
b
A mixture of compound 1 (8.39 mg) and Ac2O (1 mL) in pyridine
(1 mL) was kept at room temperature overnight, which following
work-up furnished a residue (9.01 mg) that was subjected to HPLC
(ODS, 95% MeOH) to give compound 1a (5.21 mg). M.p. 141–
3. Experimental
143 °C; ½a 2D0
ꢀ171.2 (c 0.079, CHCl3); HRFABMS m/z: 500.3864
ꢂ
3.1. General
[M]+ (C32H52O4, calcd for 500.3858); IR (KBr) mmax cmꢀ1: 3506,
2957, 1724 (OAc), 1459, 1377, 1261, 1153, 1030; for 1H and 13C
NMR spectroscopic data, see Tables 1 and 2. FABMS m/z (rel.
int.): 500 (22) [M]+, 440 (13) [MꢀHOAc]+, 422 (14)
[MꢀHOAcꢀH2O]+, 327 (58), 305 (100), 205 (9), 161 (29), 136 (25).
Melting points were determined on a Yanagimoto micro-melt-
ing point apparatus and are uncorrected. Optical rotations were
measured using a JASCO DIP-1000 digital polarimeter. IR spectra
were recorded using a Perkin–Elmer 1720X FTIR spectrophotome-
ter. 1H and 13C NMR spectra were obtained on a Varian INOVA 500
spectrometer with standard pulse sequences, operating at 500 and
125 MHz, respectively. CDCl3 was used as the solvent and TMS, as
the internal standard. NOESY spectrum was represented based on
Chem 3D program: CS Chem 3D Pro 2000, version 5.0, Cambridge,
MA 02140-237, USA. FABMS were recorded on a Hitachi 4000H
double-focusing mass spectrometer (70 eV). Column chromatogra-
phy (CC) was carried out over silica gel (70–230 mesh, Merck) and
MPLC was carried out with silica gel (230–400 mesh, Merck). HPLC
was run on a JASCO PU-1586 instrument equipped with a differen-
tial refractometer (RI 1531). CC fractions were monitored by TLC
(silica gel 60 F254, Merck). Preparative TLC was carried out on
Merck silica gel F254 plates (20 ꢁ 20 cm, 0.5 mm thick).
3.6. Inonotsudiol A (2)
Colorless crystals; m.p. 123–125 °C; ½a D20
ꢂ
+4 (c 0.089, CHCl3);
HRFABMS m/z: 440.3643 [M]+ (C30H48O2, calcd for 440.3643); IR
(KBr) mmax cmꢀ1: 3436 (OH), 2952, 1458, 1376, 1027; for 1H and
13C NMR spectroscopic data, see Tables 1 and 2. FABMS m/z (rel.
int.): 456 (22) [M]+, 441 (20) [MꢀMe]+, 438 (27) [MꢀH2O]+, 371
(21), 341 (58), 311 (66), 261 (36).
3.7. Inonotsuoxodiol A (3)
Colorless crystals; m.p. 112–114 °C; ½a D21
ꢂ
+51.7 (c 0.092, CHCl3);
3.2. Material
HRFABMS m/z: 456.3602 [M]+ (C30H48O3, calcd for 456.3603); UV
(EtOH) kmax nm: 256.0 (log
e
3.77); IR (KBr) mmax cmꢀ1: 3398
Sclerotia (4 kg) of I. obliquus (PERS.: Fr.) Pil. were purchased
from Salad Melon Co., Ltd. (Nayoro City, Hokkaido, Japan) in April,
2005, and the extraction and preliminary separation procedures
were as previously mentioned (Nakata et al., 2007). A voucher
specimen (IO-02) is deposited at the Herbarium of the Laboratory
of Medicinal Chemistry, Osaka University of Pharmaceutical
Sciences.
(OH), 2966, 1655 (C@C–C@O), 1459, 1376, 1288, 1031; for 1H
and 13C NMR spectroscopic data, see Tables 1 and 2. FABMS m/z
(rel. int.): 456 (48) [M]+, 386 (100), 369 (10), 339 (11), 290 (17),
261 (15), 235 (35), 135 (9).
3.8. Preparation of (S)- and (R)-MTPA esters (3a and 3b) from 3
3.3. Isolation procedure
A solution of 3 (2.57 mg) in pyridine (1 mL) was treated with
(ꢀ)-MTPACl (2.0 mg), and the mixture was stirred at room temp.
for 24 h. The solvent was resonance off under reduced pressure
and the residue was purified by HPLC (reversed-phase silica gel)
[MEOH–H2O (95:5, v/v)] to give (S)-MTPA ester derivative (3a,
2.27 mg) as an amorphous powder. Using a similar procedure,
the (R)-MTPA ester derivative (3b, 2.07 mg) was obtained from 3
(3.67 mg) using (+)-MTPACl (2.0 mg).
(S)-MTPA ester derivative (3a); white powder; FABMS m/z (rel.
int.): 889 ([M+H]+, 37.7%). HRFABMS m/z: 889.4480 [M+H]+ (calcd
for C50H62F6O7: 888.4396). 1H NMR d ppm (CDCl3): 0.84 (6H, s, H3-
18 and 29), 0.86 (3H, s, H3-28), 0.94 (3H, d, J = 6.3 Hz, H3-21), 1.12
(3H, s, H3-30), 1.16 (3H, s, H3-19), 1.50 (3H, s, H3-27), 1.58 (3H, s,
H3-26), 4.76 (1H, dd, J = 11.8, 4.9 Hz, H-3), 4.91 (1H, m, H-24),
5.12 (1H, dt, J = 9.6, 3.3 Hz, H-22).
(R)-MTPA ester derivative (3b); white powder; FABMS m/z (rel.
int.): 889 ([M+H]+, 25.8%). HRFABMS m/z: 889.4472 [M+H]+ (calcd
for C50H62F6O7: 888.4396). 1H NMR d ppm (CDCl3): 0.73 (3H, d, J =
6.6 Hz, H3-21), 0.82 (3H, s, H3-18), 0.86 (3H, s, H3-29), 0.95 (3H, s,
H3-28), 1.11 (3H, s, H3-30), 1.14 (3H, s, H3-19), 1.59 (3H, s, H3-27),
1.70 (3H, s, H3-26), 4.73 (1H, dd, J = 11.8, 4.7 Hz, H-3), 5.10 (2H, m,
H-22 and 24).
Preliminary silica gel CC was performed to separate the CHCl3
extract (153.9 g) of the sclerotia of I. obliquus into five fractions
(residues A–E) Residue C was further purified using silica gel CC
as reported previously (Taji et al., 2007, 2008a, 2008b). Residue
C1 (Fr. nos. 21–47 (4.98 g) of residue C) was applied to a silica
gel (70–230 mesh, 500 g) column using hexane:EtOAc = 5:1–3:1
to give residues C1-1 (Fr. nos. 68–74, 128.37 mg), C1-2 (Fr. nos.
75–77, 97.57 mg), C1-3 (Fr. nos. 78–81), C1-4 (Fr. nos. 82–83),
C1-5 (Fr. nos. 84–88), and C1-6 (Fr. nos. 89–92). Residue C1-1
was separated by HPLC (ODS, MeOH–H2O (95:5, v/v)) to give com-
pounds 1 (22.8 mg), 4 (15.4 mg), and 2 (11.8 mg). Residue C1-6
was separated by HPLC (ODS, MeOH–H2O (95:5, v/v)) to give com-
pound 3 (17.4 mg). Compound 4 was identified as lanosta-8,23-
diene-3b,25-diol on the basis of published data (Leong and
Harrison, 1999).
3.4. Spiroinonotsuoxodiol (1)
Colorless crystals; m.p. 133–135 °C (from MeOH–CHCl3); ½a D16
ꢂ
ꢀ66.3 (c 0.101, CHCl3); HRFABMS m/z: 458.3757 [M]+ (C30H50O3,

N. Handa et al. / Phytochemistry 71 (2010) 1774–1779
1779
Kim, H.-G., Yoon, D.-H., Kim, C.-H., Shrertha, B., Chang, W.-C., Lim, S.-Y., Lee, W.-H.,
Han, S.-G., Lee, J.-O., Lim, M.-H., Kim, G.-Y., Choi, S., Song, W.-O., Sung, J.-M.,
Hwang, K.-C., Kim, T.-W., 2007. Ethanol extract of Inonotus obliquus inhibits
lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells. J.
Med. Food 10, 80–89.
Kim, M.-Y., Seguin, P., Ahn, J.-K., Kim, J.-J., Chun, S.-C., Kim, E.-H., Seo, S.-H., Kang, E.-
Y., Kim, S.-L., Park, Y.-J., 2008. Phenolic compound concentration and
antioxidant activities of edible and medicinal mushrooms from Korea. J. Agric.
Food Chem. 56, 7265–7270.
3.9. Assay for cytotoxicity to P388, L1210, HL-60 and KB cell lines
Cytotoxic activities of spiroinonotsuoxodiol (1), inonotsudiol A
(2), and inonotsuoxodiol A (3) were examined with the 3-(4,5-di-
methyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
method. P388, L1210, HL-60 and KB cells were cultured in Eagle’s
Minimum Essential Medium (10% fetal calf serum) at 37 °C in 5%
CO2. The test material was dissolved in dimethyl sulfoxide (DMSO)
Lee, K.-K., Kim, J.-L., Kwon, H.-K., Joung, E.-M., Lee, D.-S., Jeong, S.-O., 2006. Inonotus
obliquus WI0628, extracts having antidiabetic activity. Repub. Korean Kongkae
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to make a concentration of 10
lM, and the solution was diluted
with the medium to yield concentrations of 200, 20, and 2
lM,
Leong, Y.-W., Harrison, L.J., 1999. (20R,23E)-Eupha-8, 23-diene-3b, 25-diol from
Tripetalum cymosuum. Phytochemistry 50, 849–857.
respectively. Each solution was combined with each cell suspen-
sion (1 ꢁ 105 cells mLꢀ1) in the medium, respectively. After incu-
bating at 37 °C for 72 h in 5% CO2, the grown cells were labeled
with 4 mg mLꢀ1 MTT in phosphate-buffered saline (PBS), and the
absorbance of formazan dissolved in 20% sodium dodecyl sulfate
(SDS) in 0.1 N HCl was measured at 540 nm using a microplate
reader (Model 450) (Bio-Rad Laboratories, Inc., Tokyo, Japan). Each
absorbance value was expressed as percentage relative to that of
the control cell suspension that was prepared without the test
substance using the same procedure as that described above. All
assays were performed three times, semilogarithmic plots were
constructed from the averaged data, and the effective dose of the
substance required to inhibit cell growth by 50% (IC50) was
determined.
Mizuno, T., Zhuang, C., Abe, K., Okamoto, H., Kiho, T., Ukai, S., Leclerc, S., Meijer, L.,
1999. Antitumor and hypoglycemic activities of polysaccharides from sclerotia
and mycelia of Inonotus obliquus (PERS.: Fr.) Pil. (Aphyllophoromycetideae). Int.
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Nakajima, Y., Nishida, H., Matsugo, S., Konishi, T., 2009. Cancer cell cytotoxicity of
extracts and small phenolic compounds from Chaga [Inonotus obliquus
(persoon) Pilat]. J. Med. Food 12, 501–507.
Nakata, T., Yamada, T., Taji, S., Ohishi, H., Wada, S., Tokuda, H., Sakuma, K., Tanaka,
R., 2007. Structure determination of inonotsuoxides A and B and in vivo anti-
tumor promoting activity of inotodiol from the sclerotia of Inonotus obliquus.
Bioorg. Med. Chem. 15, 257–264.
Nomura, M., Takahashi, T., Uesugi, A., Tanaka, R., Kobayashi, S., 2008. Inotodiol, a
lanostane triterpenoid, from Inonotus obliquus inhibits cell proliferation through
caspase-3-dependent apoptosis. Anticancer Res. 28, 2691–2696.
Ohtani, I., Kusumi, T., Kashman, Y., Kakizawa, H., 1991. High-field FT NMR
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terpenoids. J. Am. Chem. Soc. 113, 4092–4096.
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Nauka Press, Moscow-Leningrad, 198pp (in Russian).
Compounds 1–3 had purities of over 99%.
Song, Y., Hui, J., Kou, W., Xin, R., Jia, F., Wang, N., Hu, F., Zhang, H., Liu, H., 2008.
Identification of Inonotus obliquus and analysis of antioxidation and antitumor
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Acknowledgement
Taji, S., Yamada, T., In, Y., Wada, S., Usami, Y., Sakuma, K., Tanaka, R., 2007. Three
new lanostane triterpenoids from Inonotus obliquus. Helv. Chim. Acta 90, 2047–
2057.
Taji, S., Yamada, T., Tanaka, R., 2008a. Three new lanostane triterpenoids,
inonotsutriols A, B, and C from Inonotus obliquus. Helv. Chim. Acta 91, 1513–
1524.
This study was supported by a Grant-in-aid for High Technology
from the Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan.
Taji, S., Yamada, T., Wada, S., Tokuda, H., Sakuma, K., Tanaka, R., 2008b. Lanostane-
type triterpenoids from the sclerotia of Inonotus obliquus possessing anti-tumor
promoting activity. Eur. J. Med. Chem. 43, 2373–2379.
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