Cadinane-type sesquiterpenoids, strobilols E-K, from the liquid culture of Strobilurus ohshimae

Article abstract of DOI:10.1002/hlca.200890173

New cadinane sequiterpenoids, strobilols E-K (5 - 11, resp.) have been isolated from a liquid culture of Strobilurus ohshimae. Their structures have been established on the basis of spectral analyses.

Full text of DOI:10.1002/hlca.200890173

Helvetica Chimica Acta – Vol. 91 (2008)
1595
Cadinane-Type Sesquiterpenoids, Strobilols E – K, from the Liquid Culture of
Strobilurus ohshimae
by Fuminori Hiramatsua), Tetsuya Murayamaa), Takuya Kosekia), Katsuhide Okadab), and
Yoshihito Shiono*a)
a) Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka,
Yamagata 997-8555, Japan (fax: þ 81-235-28-2873; e-mail: yshiono@tds1.tr.yamagata-u.ac.jp)
b) Faculty of Education, Art and Science, Yamagata University, Yamagata, Yamagata 990-8560, Japan
New cadinane sequiterpenoids, strobilols E – K (5 – 11, resp.) have been isolated from a liquid
culture of Strobilurus ohshimae. Their structures have been established on the basis of spectral analyses.
Introduction. – In recent years, mushrooms have become attractive objects as
functional food and as source for biologically active compounds due to their diverse
nutritional, medicinal, and pharmacological properties. In addition, the biological
importance of the edible mushrooms arises from their chemical components, including
especially various biologically active polysaccharides and polyphenolic compounds [1][2].
Strobilurus ohshimae, a wild edible mushroom, which belongs to Tricholomataceae
family, is widely distributed over the cedar forest area of Japan. The Japanese name for
S. ohshimae is sugiedatake. This wild mushroom has traditionally been eaten by specific
groups of local people and seasonally in Japan. However, little is known about the
chemical composition and nutritional and pharmacological values of S. ohshimae.
During our search for bioactive natural products from edible wild mushrooms, we have
investigated the chemical constituents of the fresh fruiting bodies of S. ohshimae
collected in Japan, and recently isolated cadinane-sesquiterpenes, strobilols A (1), B
(2), C (3), and D (4) [3]. Strobilol A showed moderate activity against Altemia salina
(LD50 value of 100 mm). Subsequently, we investigated the possibility of growing S.
ohshimae in liquid fermentation, comparing the chemical compositions of mycelium
with those of fresh fruiting bodies. Two new drimane sesquiterpenes, strobilactones A
and B were isolated from the liquid cultured of S. ohshimae, which are not produced in
the fruiting bodies of this fungus [4]. In our ongoing investigation of chemical
compositions of the liquid culture of S. ohshimae, we have isolated seven new cadinane-
sesquiterpenes, strobilols E – K (5 – 11, resp.), together with the known compounds 1 –
4. In this report, we describe the isolation and structure elucidation of 5 – 11.
Results and Discussion. – The culture broth (5.0 l) of Strobilurus ohshimae was
filtered, and the cultured filtrate was extracted with AcOEt. The AcOEt extract was
chromatographed on silica gel and separated into 13 fractions. Further purification by
column chromatography yielded strobilol J (10) from Fr. 5, and strobilols H (8) and I
(9) from Fr. 6, as well as strobilols E (5), F (6), G (7), and K (11) from Fr. 8 and 9.
ꢀ 2008 Verlag Helvetica Chimica Acta AG, Zürich

1596
Helvetica Chimica Acta – Vol. 91 (2008)
The molecular formula of strobilol E (5) was determined as C15H22O4 by HR-EI-
MS. The 13C-NMR spectrum of 5 gave 15 signals assignable to two sp3 quaternary C-
atoms, two sp2 quaternary C-atoms, four sp3 CH groups, one sp2 CH group, three sp3
CH2 groups, one sp2 CH2 group, and two Me groups. The 1H-NMR (CD3OD) spectrum
(Table 1) of 5 revealed signals due to a vinyl Me group (d(H) 1.77 (s, Me(15)), a Me
group (d(H) 0.90 (d, J ¼ 7.1, Me(14)), an olefinic H-atom (d(H) 5.86 (br. s, HꢀC(4)),
an exo-CH2 group (d(H) 5.03 (t, J ¼ 2.4, CH2(12)), 5.37( t, J ¼ 2.4, CH2(12))), an
oxygenated CH2 group (d(H) 4.13 (dd, J ¼ 13.2, 2.4, CH2(13)), 4.50 (dd, J ¼ 13.2, 2.4,
CH2(13))), and an oxygenated CH group (d(H) 4.07(br. s, HꢀC(2))). In addition, it
showed also signals of three CH groups (d(H) 1.10 – 1.19 (m), 2.25 – 2.80 (overlapped
signal), 1.42 – 1.46 (m)), and two CH2 groups (d(H) 1.02 – 1.11 (m), 1.21 – 1.32 (m), 1.80
(dd, J ¼ 13.8, 3.3), 2.25 – 2.80 (overlapped signal)). Since two out of five unsaturation
degrees were accounted for by the presence of the two C¼C bonds, compound 5 was
inferred to be a tricyclic system. Compound 5 was assumed to be a cadinane-type
sesquitrpenoids, because the 1H- and 13C-NMR spectral data of 5 were similar to those
of strobilols A (1) – D (4) co-occurring in this species. The 1H,1H-COSY spectrum of 5
indicated the two partial structures a and b (Fig. 1). Moreover, the 13C,1H-COSY and
HMBC spectra of 5 clarified the connectivity of each partial structure. HMBC
Correlations of Me(15) with C(2) and C(4), HꢀC(2) with C(4), C(10), and C(15),
HꢀC(4) with C(6) and C(10), and CH2(8) with C(6) were detected, implying a
bicyclo[4.4.0]decane moiety. HMBC correlations from the exo-CH2 group (CH2(12)) to
C(6) and C(13) and from CH2(13) to C(6) and C(7) defined the locations of
tetrahydrofuran ring with the exo-CH2 group (fragment b). Accordingly, the constitu-
tional formula of 5 was deduced as shown in the Formulae above. The relative
configuration of 5 was elucidated by the analysis of NOE difference experiments and
coupling constants. In the 1H-NMR spectrum (C5D5N) of 5, the large coupling constant

Helvetica Chimica Acta – Vol. 91 (2008)
1597
1
Fig. 1. Important H,1H-COSY (bold lines) and HMBC (arrows) corre-
lations observed for 5
of HꢀC(5) (J(5,10) ¼ 11.1), characteristic for a diaxial relationship with HꢀC(10),
demonstrated the trans-juncture of the decalin skeleton. NOEs from HꢀC(9) to
HꢀC(5) suggested that the Me group is equatorial. The b-orientation of the OH group
at C(2) was deduced from an NOE from HꢀC(10) to HꢀC(2). Furthermore, NOEs
from CH2(12) to HꢀC(2) and HꢀC(10) pointed to a cis fusion between the five- and
six-membered ring. A chair-like conformation of the cyclohexane ring consistent with
the results from NOEs experiments is shown in Fig. 2.
Fig. 2. Selected NOE correlations observed for 5
Table 1. 1H-NMR Data of Strobilols E (5) and F (6). At 400 MHz; d in ppm, J in Hz.
5a)
5b)
6c)
6b)
CH2(1)
1.02 – 1.11 (m),
1.45 – 1.63d),
2.59 – 2.64 (m)
4.51 – 4.56 (m)
2.06 (dd, J ¼ 16.1, 13.4), 2.11 (dd, J ¼ 16.1, 13.8),
2.74 (dd, J ¼ 16.1, 6.4) 2.82 (dd, J ¼ 16.1, 3.8)
2.25 – 2.80d)
HꢀC(2) 4.07(br. s)
1.23 – 1.27( m),
1.58 – 1.67d)
1.48 – 1.56d), 1.70 – 1.83d)
or CH2(2)
HꢀC(4) 5.86 (br. s)
HꢀC(5) 2.25 – 2.80d)
6.42 (br. s)
7.15 (br. s)
7.23 (br. s)
2.84 (br. d, J ¼ 11.1)
2.65 (dt, J ¼ 9.7, 2.4)
2.98 (dt, J ¼ 10.6, 2.4)
CH2(8)
1.21 – 1.32 (m),
1.45 – 1.63d),
1.31 – 1.40d),
1.48 – 1.56d),
1.80 (dd, J ¼ 13.8, 3.3) 2.25 (br. d, J ¼ 13.7)
1.97( dd, J ¼ 14.5, 3.3) 2.20 (dd, J ¼ 13.7, 2.5)
HꢀC(9) 1.42 – 1.46 (m)
HꢀC(10) 1.10 – 1.19 (m)
CH2(12) 5.03 (t, J ¼ 2.4),
5.37( t, J ¼ 2.4)
1.72 – 1.77 (m)
1.58 – 1.67d)
1.31 – 1.40d)
1.70 – 1.83d)
1.45 – 1.63d)
5.12 (br. s), 5.71 (br. s) 5.16 (t, J ¼ 2.4),
1.70 – 1.83d)
5.10 (t, J ¼ 2.5),
5.34 (t, J ¼ 2.4)
5.40 (t, J ¼ 2.5)
CH2(13) 4.13 (dd, J ¼ 13.2, 2.4), 4.35 (dd, J ¼ 13.2, 2.3), 4.24 (dt, J ¼ 13.3, 2.4), 4.32 (dt, J ¼ 13.4, 2.2),
4.50 (dd, J ¼ 13.2, 2.4) 4.69 (dd, J ¼ 13.2, 2.3) 4.60 (dt, J ¼ 13.3, 2.4)
4.67( dt, J ¼ 13.4, 2.2)
0.80 (d, J ¼ 6.5)
Me(14)
Me(15)
0.90 (d, J ¼ 7.1)
1.77 (br. s)
0.90 (d, J ¼ 6.3)
2.09 (br. s)
0.90 (d, J ¼ 6.5)
1.85 (br. d, J ¼ 2.4)
1.92 (dd, J ¼ 2.4, 1.2)
a) In CD3OD. b) In C5D5N. c) CDCl3. d) Overlapped signals.

1598
Helvetica Chimica Acta – Vol. 91 (2008)
The absolute configuration of 5 was determined by the CD excitation chirality
method for the allylic benzoate 5a as shown in Fig. 3 [5]. Thus, treatment of 5 with
benzoyl chloride in the presence of 4-(dimethylamino)pyridine (4-DMAP) afforded
the 2-O-benzoate (5a). The 2-O-benzoate (5a) showed a negative Cotton effect
(245 nm (De ꢀ 3.0)), which indicated the absolute configuration (S) for C(2).
Fig. 3. Sign of Cotton effect of p-bromobenzoate derivative 5a
The molecular formula of strobilol F (6) was determined to be C15H22O3 by HR-EI-
1
MS, and implied the presence of one O-atom less than in 5. The H- and 13C-NMR
spectral data of 6 (Tables 1 and 2) indicated many common signals, which were
attributable to two Me groups, an exo-CH2 moiety, and an isolated CH2 group as those
of 5. However, the data lacked the oxygenated CH group at C(2) which had been
observed in 5, and they were accompanied by the appearance of an additional CH2
group (d(H) 1.23 – 1.27( m, 1 H), 1.58 – 1.67( m, 1 H), d(C) 28.5 (t)). In the HMBC
experiments of 6, correlations between Me(15) and C(2) and between HꢀC(4) and
C(2) indicated that 6 was a 2-deoxy-derivative of 5.
Table 2. 13C-NMR Spectral Data of Strobilols E (5), F (6), G (7), and H (8). At 100 MHz; d in ppm.
5a)
6b)
7a)
8b)
HꢀC(1) or CH2(1)
HꢀC(2) or CH2(2)
C(3)
38.6 (t)
41.9 (t)
43.7( t)
127.9 (d)
129.3 (d)
135.9 (s)
128.6 (d)
134.4 (s)
77.5 (s)
72.0 (d)
139.3 (s)
125.9 (d)
48.6 (d)
78.1 (s)
107.2 (s)
43.6 (t)
28.5 (t)
135.9 (s)
145.0 (d)
45.9 (d)
77.0 (s)
104.8 (s)
40.3 (t)
202.3 (s)
137.0 (s)
149.3 (d)
48.8 (d)
77.8 (s)
106.9 (s)
43.2 (t)
HꢀC(4)
HꢀC(5) or C(5)
C(6)
C(7)
CH2(8)
103.1 (s)
37.1 (t)
HꢀC(9)
35.1 (d)
43.6 (d)
149.9 (s)
111.5 (t)
68.6 (t)
33.6 (d)
42.9 (d)
146.7( s)
112.1 (t)
66.5 (t)
35.7( d)
45.2 (d)
149.6 (s)
111.7( t)
68.6 (t)
31.4 (d)
137.6 (s)
150.7( s)
108.7( t)
67.3 (t)
HꢀC(10) or C(10)
C(11)
CH2(12)
CH2(13)
Me(14)
Me(15)
19.9 (q)
20.1 (q)
18.0 (q)
15.7( q)
19.3 (q)
16.5 (q)
22.9 (q)
21.0 (q)
a) In CD3OD. b) In CDCl3.
The molecular formula C15H20O4 for strobilol G (7) was established by HR-EI-MS,
indicating that 7 had two H-atoms less than 5. The 1H-NMR spectrum of 7 was similar
to that of 5, but it lacked the HꢀC(2) group present in 5. In the 13C-NMR spectrum of
7, the signal for C(2) that had been observed in 5 also disappeared, and new signals

Helvetica Chimica Acta – Vol. 91 (2008)
1599
assigned to an a,b-unsaturated C¼O C-atom system were observed (d(C) 202.3 (s),
137.0 (s), 149.3 (d)). The presence of the a,b-unsaturated ketone was also supported by
the 1670 cmꢀ1 absorption in the IR spectrum, and by the 234 nm (log e ¼ 3.6) band in
the UV spectrum. From these results, the structural difference between 5 and 7 was
explainable by replacement of the HOꢀCH(2) group of 5 with a O¼C(2) group for 7.
Furthermore, these assumptions were confirmed by 13C,1H-COSY and HMBC
correlations between Me(15) and C(2) and between HꢀC(4) and C(2).
The molecular formula of strobilol H (8) was determined to be C15H18O3 by HR-EI-
MS. The IR spectrum of 8 revealed the presence of OH (3430 cmꢀ1) and aromatic
groups (1506 cmꢀ1). The UV spectrum of 8 also showed an aromatic maximum at
1
239 nm (log e ¼ 3.2), indicating the presence of an aromatic system. The H-NMR
spectrum (CDCl3) of 8 showed three new aromatic signals at d(H) 7.10 (dd, J ¼ 7.9, 2.1,
HꢀC(2)), 7.11 (d, J ¼ 7.9, HꢀC(1)), and 7.38 (br. s, HꢀC(4)), suggesting a 1,2,4-
trisubstituted aromatic spin system. The downfield shift observed for Me(15) (d(H)
2.33) was indicative of an aromatization of the cyclohexene ring in compound 8.
Furthermore, signals at d(H) 3.12 – 3.17( m, HꢀC(9)), 1.93 (dd, J ¼ 13.9, 5.0, CH2(8)),
2.24 (dd, J ¼ 13.9, 6.6, CH2(8)), 4.29 (dt, J ¼ 12.8, 2.4, CH2(13)), 4.54 (dt, J ¼ 12.8, 2.4,
CH2(13)), 5.24 (t, J ¼ 2.4, CH2(12)), and 5.58 (t, J ¼ 2.4, CH2(12)) revealed the presence
of one benzylic CH group, two CH2 groups and one exo-CH2 group. 13C-NMR and
DEPT data of 8 showed six aromatic C-atoms, two Me groups: one benzylic and one
aliphatic; one benzylic CH group, three quaternary C-atoms, two CH2 groups, one exo-
CH2 group. These data suggested a strobilol derivative. The complete attribution for
the observed signals was accomplished based on the 13C,1H-COSY and HMBC
correlations (Fig. 4). HMBC correlations between Me(15) and C(2), C(3), and C(4),
between HꢀC(1) and C(3) and C(5), and between HꢀC(4) and C(2) and C(6) allowed
us to fully determine the aromatic moiety. The benzylic function linked at C(5) and
C(10) was supported by the correlations between Me(14) and C(10), between HꢀC(9)
and C(5). The linkage of tetrahydrofurane moiety was determined through the
correlations from CH2(12) to C(6), from CH2(13) to C(6) and C(7). These HMBC
correlations allowed us to determine the structure of 8.
The HR-FAB-MS data of strobilol I (9) indicated that the molecular formula of 9
1
was C25H40O6. The H- (Table 3) and 13C-NMR (Table 4) spectra of 9 were similar to
those of strobilol A (1), except for the presence of additional signals at d(H) 0.87( t, J ¼
6.4, Me(10’)), 1.25 – 1.29 (overlapped signal), 1.54 – 1.67(overlapped signal), 2.44 – 2.49
(m, CH2(2’)), d(C) 22.6 (t), 24.7( t), 29.0 (t), 29.2 (t), 29.4 (t), 31.8 (t), 34.6 (t), 14.1 (q),
172.6 (s). The signals assignable to a HꢀC(4) group, which were observed at d(H) 4.60
(d, J ¼ 10.8) in 1, were observed at d(H) 6.00 (d, J ¼ 10.9) in 9. The presence of the
Fig. 4. HMBC (arrows) Correlations observed for 8

1600
Helvetica Chimica Acta – Vol. 91 (2008)
C¼O resonance in the 13C-NMR spectrum could be accounted for a saturated C10 fatty
acid ester. A combination of HMQC and HMBC experiments thus permitted the
assignment of a cadinane sesquiterpene skeleton esterified with decanoic acid.
Furthermore, the HMBC correlation between HꢀC(4) and C(1’) and the substantial
downfield shift for HꢀC(4) revealed the location of the decanoyloxy moiety was at
C(4) (Fig. 5). Thus, the structure of 9 was determined to be the 4-decanoyl ester
derivative of strobilol A (1).
Fig. 5. HMBC (arrows) Correlations observed for 9
The molecular formula of strobilol J (10), C25H40O5, was determined by HR-FAB-
1
MS measurement. The H- (Table 3) and 13C-NMR (Table 4) data for 10 were very
similar to those of strobilol B (2), except for the lower HꢀC(4) chemical shift (d(H)
6.01 of 10 and d(H) 4.90 of 2). The NMR spectral data of 10 indicated additional
signals due to a decanoyl moiety (d(H) 0.87( t, J ¼ 7.0, Me(10’)), 1.20 – 1.34 (overlapped
signal), 1.59 – 1.64 (overlapped signal), 2.35 (t, J ¼ 7.6, CH (2’)) and d(C) 14.1 (q), 22.6
2
(t), 24.9 (t), 29.2 (t), 29.4 (t), 31.8 (t), 34.8 (t), 175.5 (s)). The identification of the 4-
Table 3. 1H-NMR Data of Strobilols I (9), J (10), and K (11). At 400 MHz; d in ppm, J in Hz.
9a)
10a)
11b)
CH2(1)
1.54 – 1.67c),
1.68 – 1.73 (m),
2.26 – 2.29 (m)
5.82 (br. d, J ¼ 6.6)
6.01 (d, J ¼ 6.6)
2.05 (dd, J ¼ 12.5, 6.6)
1.20 – 1.35c),
1.58 – 1.65c),
2.21 (ddd, J ¼ 15.8, 7.5, 4.6)
1.78 – 1.84c)
HꢀC(2)
HꢀC(4)
HꢀC(5)
CH2(8)
3.07( d, J ¼ 4.6)
3.59 (t, J ¼ 3.0)
5.72 (d, J ¼ 12.3)
2.27( t, J ¼ 12.3)
1.35 – 1.39 (m),
1.78 – 1.84c)
6.00 (d, J ¼ 10.9)
2.40 (dd, J ¼ 12.0, 10.9)
1.10 – 1.22c),
1.80 (dd, J ¼ 12.5, 3.8)
1.89 (dd, J ¼ 13.9, 3.8)
HꢀC(9)
HꢀC(10)
CH2(12)
1.48 – 1.52 (m)
1.50 – 1.54 (m)
1.47– 1.53 c)
1.47– 1.53 c)
1.10 – 1.22c)
1.20 – 1.35c)
5.06 (br. s), 5.44 (br. s)
5.14 (t, J ¼ 2.3),
5.22 (t, J ¼ 2.3)
4.19 (dt, J ¼ 12.5, 2.3),
4.62 (dt, J ¼ 12.5, 2.3)
0.88 (d, J ¼ 6.6)
1.64 (br. s)
5.11 (t, J ¼ 2.7),
5.56 (t, J ¼ 2.7)
4.17( dt, J ¼ 12.5, 2.7),
4.46 (dt, J ¼ 12.5, 2.7)
0.88 (d, J ¼ 7.2)
1.15 (s)
CH2(13)
4.13 (dt, J ¼ 12.5, 2.2),
4.55 (dt, J ¼ 12.5, 2.2)
0.88 (d, J ¼ 6.4)
1.25 (s)
Me(14)
Me(15)
CH2(2’)
CH2(3’)
2.44 – 2.49 (m)
2.35 (t, J ¼ 7.6)
2.35 – 2.39 (m)
1.54 – 1.67c)
1.59 – 1.64c)
1.58 – 1.65c)
CH2(4’)ꢀCH2(9’)
1.25 – 1.29c)
0.87( t, J ¼ 6.4)
1.20 – 1.34c)
0.87( t, J ¼ 7.0)
1.24 – 1.32c)
0.90 (t, J ¼ 6.0)
Me(10’)
a) In CDCl3. b) In CD3OD. c) Overlapped signals.

Helvetica Chimica Acta – Vol. 91 (2008)
1601
Table 4. 13C-NMR Data of Strobilols I (9), J (10), and K (11). At 100 MHz; d in ppm.
9a)
10a)
11b)
CH2(1)
HꢀC(2)
C(3)
HꢀC(4)
HꢀC(5)
C(6)
29.0 (t)
28.6 (t)
34.4 (t)
75.6 (d)
75.5 (s)
75.5 (d)
46.1 (d)
79.8 (s)
107.0 (s)
42.3 (t)
36.5 (d)
38.6 (d)
149.2 (s)
112.9 (t)
69.4 (t)
20.9 (q)
26.5 (q)
175.8 (s)
36.5 (t)
33.1 (t)
60.5 (d)
59.7( s)
74.3 (d)
42.2 (d)
76.9 (s)
104.4 (s)
39.4 (t)
35.5 (d)
39.4 (d)
147.2 (s)
109.5 (t)
68.0 (t)
19.1 (q)
19.6 (q)
172.6 (s)
34.6 (t)
24.7( t)
128.5 (d)
131.5 (s)
72.2 (d)
48.8 (d)
77.2 (s)
104.7( s)
40.7( t)
34.7( d)
41.6 (d)
147.2 (s)
109.8 (t)
67.7 (t)
18.6 (q)
19.7( q)
175.5 (s)
34.8 (t)
24.9 (t)
C(7)
CH2(8)
HꢀC(9)
HꢀC(10)
C(11)
CH2(12)
CH2(13)
Me(14)
Me(15)
C(1’)
CH2(2’)
CH2(3’)
CH2(4’) – CH2(8’)
CH2(9’)
Me(10’)
c
d
e
)
)
)
)
c
22.6 (t)
14.1 (q)
24.5 (t)
15.2 (q)
14.1 (q)
a) CDCl3. b) CD3OD. c) d(C) 22.6 (t), 29.0 (t), 29.2 (t), 29.4 (t), 31.8 (t). d) d(C) 29.2 (t), 29.4 (t), 31.8 (t).
e) d(C) 31.1 (t), 31.2 (t), 31.4 (t), 33.8 (t).
substituent in 10 as the 4-decanoyloxy group was deduced from data of HMBC
correlations between HꢀC(4) and C(1’). Thus, the structure of 10 was determined to
be 4-decanoyloxystrobilol B.
The molecular formula of strobilol K (11), C25H42O7, was determined by HR-FAB-
MS measurement, suggesting a decanoyl (C10H19O) derivative of strobilol C (3). The
1H- (Table 3) and 13C-NMR (Table 4) data for 11 were very similar to those of 3, except
for the signals of a decanoyl moiety (d(H) 0.90 (t, J ¼ 6.0, Me(10’)), 1.24 – 1.32
(overlapped signal), 1.58 – 1.65 (overlapped signal), 2.35 – 2.39 (m, CH2(2’)), and d(C)
15.2 (q), 24.5 (t), 31.1 (t), 31.2 (t), 31.4 (t), 33.1 (t), 33.8 (t), 36.5 (t), 175.8 (s)). The
esterification site was determined by a HMBC correlation between HꢀC(4) to the
ester C¼O group at d(C) 175.8. From these results, the structure of 11 was determined
to be 4-decanoyloxystrobilol C.
The absolute configurations of strobilols F – K (6 – 11, resp.) are considered to be
the same as that of 5 lead on the biosynthetic considerations.
Strobilols A – K (1 – 11, resp.) were screened for cytotoxicity and found weakly
active or inactive.
Strobilols are members of a novel series of fungal metabolites consisting of
cadinane sesquiterpenoids possessing 6,6,5-fused tricycles. It is noteworthy that there
has been only one report of a cadinane sesquiterpenoid possessing a 6,6,5-fused
tricycle: a hydrated panal derivative which was considered to be formed from cadinane
sesquiterpene, panal, in wet CHCl3. Panal was originally isolated from luminous

1602
Helvetica Chimica Acta – Vol. 91 (2008)
mushroom Panellus stipticus. In addition, the panal derivative has not been reported as
an isolated natural product [6].
Experimental Part
General. Column chromatography (CC): silica gel (SiO2; 200 – 300 mesh), and ODS (RP-18). TLC:
Kieselgel F254 (0.25 mm; Merck); detection by splaying with 10% vanillin in H2SO4, follwed by heating at
1208. Optical rotations: Horiba model SEPA-300 polarimeter. UV Spectra: Shimadzu UV mini-1240
spectrophotometer. CD Spectra: Jasco J-20A spectrophotometer. IR spectra: JASCO J-20A spectro-
.
photometer, KBr pellets; n in cmꢀ1 1H- and 13C-NMR Spectra: JEOL EX-400 spectrometer, at 400/
100 MHz, resp.; d in ppm rel. to Me4Si as internal standard; coupling constants J in Hz. EI-MS: JEOL
JMS-700 instrument spectrometer; in m/z (rel. %).
Fungus and Cultivation. The producing strain Strobilurus ohshimae NBRC 30370 was purchased
from biological resource center, National Institute of Technology and Evaluation, Chiba, Japan. For
fermentation, the fungal strain was grown in three 500 ml-Sakaguchi flasks containing 100 ml of a
medium consisting of 40 g of malt extract, 40 g of glucose, and 1.0 g peptone per 1 l of H2O. The
inoculated flask was incubated at 258 for 4 weeks on a rotary shaker. The culture broth (5.0 l) was
separated from the mycelia by filtration. The filtrate was extracted with AcOEt. The org. layer was
concentrated in vacuo to give an oily residue (2.6 g). The residue was subjected to SiO2 CC with mixture
of hexane/AcOEt, and mixture of AcOEt/MeOH to give Fr. 1 – 13. Fr. 5 (40% AcOEt eluate, 165.2 mg)
was further chromatographed on SiO2 by eluting with CHCl3 and increasing volume of AcOEt to afford
40 – 50% AcOEt eluates (99 mg).
These fractions were combined and rechromatographed on ODS with H2O/MeOH (10% stepwise
gradient) to yield crude strobilol J (10), which was finally purified by SiO2 flash-CC with hexane/AcOEt
(1:1, v/v) to obtain 6.9 mg of strobilol J (10, 11.2 mg). Fr. 6 (50% AcOEt eluate, 177.9 mg) was further
chromatographed on SiO2 with mixture of CHCl3/AcOEt (10% stepwise gradient). The 30% AcOEt
eluate (107mg) was further purified by ODS CC with mixture of H2O/MeOH (10% stepwise gradient),
followed by SiO2 CC with mixture of CHCl3/AcOEt to obtain strobilols H (8, 4.3 mg) and I (9, 10.4 mg).
Fr. 8 and 9 (70 – 80% AcOEt eluates, 221.5 mg) were combined and chromatographed (SiO2, CHCl3/
AcOEt stepwise; then ODS, H2O/MeOH, stepwise). 50 – 80% MeOH eluates were combined and
further subjected to CC (SiO2, CHCl3/MeOH 20 :1) to obtain strobilols E (5, 21.3 mg), F (6, 22.8 mg), G
(7, 6.6 mg), and K (11, 12.2 mg).
Strobilol E (¼(3aS,5R,5aS,7S,9aS,9bR)-1,2,4,5,5a,6,7,9a-Octahydro-5,8-dimethyl-1-methylidene-
naphtho[2,1-b]furan-3a,7,9b-triol; 5). Colorless oil. [a]2D0 ¼ þ19.0 (c ¼ 0.47, MeOH). IR (KBr): 3443,
2955, 1031. 1H- and 13C-NMR: see Tables 1 and 2. EI-MS: 266 (5, Mþ), 230 (11), 167(16), 149 (64), 119
(26), 85 (82), 83 (100). HR-EI-MS: 266.1519 (Mþ, C15H22O4þ ; calc. 266.1518).
Strobilol F (¼(3aS,5R,5aS,9aS,9bR)-1,2,4,5,5a,6,7,9a-Octahydro-5,8-dimethyl-1-methylidenenaph-
tho[2,1-b]furan-3a,9b-diol; 6). Colorless oil. [a]2D0 ¼ ꢀ10.6 (c ¼ 0.24, MeOH). IR (KBr): 3409, 2929,
1031. 1H- and 13C-NMR: see Tables 1 and 2. EI-MS: 250 (12, Mþ), 246 (71), 177 (92), 159 (100), 135 (95),
121 (43), 109 (80). HR-EI-MS: 250.1573 (Mþ, C15H22O3þ ; calc.250.1569).
Strobilol G (¼(3aS,5R,5aS,9aS,9bR)-1,2,4,5,5a,6,9a,9b-Octahydro-3a,9b-dihydroxy-5,8-dimethyl-1-
methylidenenaphtho[2,1-b]furan-7(3aH)-one; 7). Colorless oil. [a]2D0 ¼ þ29.0 (c ¼ 0.10, MeOH). UV
(MeOH): 234 (3.6). IR (KBr): 3443, 2983, 1670, 1031. 1H- and 13C-NMR: see Tables 2 and 5. EI-MS: 264
(48, Mþ), 246 (50), 216 (29), 191 (31), 172 (67), 171 (63), 143 (58), 109 (63), 91 (100). HR-EI-MS:
264.1316 (Mþ, C15H20Oþ4 ; calc. 264.1362).
Strobilol H (¼(3aS,5R)-1,2,4,5-Tetrahydro-5,8-dimethyl-1-methylidenenaphtho[2,1-b]furan-3a,9b-
diol; 8). Colorless oil. [a]2D0 ¼ þ39.3 (c ¼ 0.22, MeOH). UV (MeOH): 239 (3.2). IR (KBr): 3430,
2935, 1506. 1H- and 13C-NMR: see Tables 2 and 5. EI-MS: 246 (29, Mþ), 228 (18), 210 (19), 186 (39), 172
(72), 159 (100), 128 (40), 115 (41), 91 (95). HR-EI-MS: 246.1256 (Mþ, C15H18O3þ ; calc. 246.1256).
Strobilol I (¼(3aS,5R,5aS,6aS,7aS,8R,8aS,8bR)-Dodecahydro-3a,8b-dihydroxy-5,7a-dimethyl-1-
methylideneoxireno[6,7]naphtho[2,1-b]furan-8-yl Decanoate; 9). Colorless oil. [a]2D0 ¼ þ54.0 (c ¼ 0.05,

Helvetica Chimica Acta – Vol. 91 (2008)
1603
Table 5. 1H-NMR Data of Strobilols G (7) and H (8). At 400 MHz; d in ppm, J in Hz.
7a)
7b)
8c)
8b)
CH2(1)
2.09 (dd, J ¼ 16.4, 13.9), 2.11 (dd, J ¼ 15.9, 13.5), 7.11 (d, J ¼ 7.9)
7.20 (d, J ¼ 8.0)
or HꢀC(1) 2.58 – 2.67d)
2.82 (dd, J ¼ 15.9, 3.9)
C(2)
7.10 (dd, J ¼ 7.9, 2.1) 7.13 (dd, J ¼ 8.0, 2.4)
or HꢀC(2)
HꢀC(4)
HꢀC(5)
CH2(8)
7.28 (br. s)
7.58 (br. s)
2.98 (dd, J ¼ 10.7, 2.4)
7.38 (br. s)
7.88 (br. s)
2.58 – 2.67d)
1.34 (dd, J ¼ 14.0, 11.6), 1.51 (dd, J ¼ 13.7, 11.9), 1.93 (dd, J ¼ 13.9, 5.0), 2.25 (dd, J ¼ 13.9, 6.7),
1.84 (dd, J ¼ 14.0, 3.2) 2.19 (dd, J ¼ 13.7, 3.5) 2.24 (dd, J ¼ 13.9, 6.6) 2.61 (dd, J ¼ 13.9, 5.0)
HꢀC(9)
1.58 – 1.65d)
1.69 – 1.89d)
1.69 – 1.89d)
3.12 – 3.17( m)
3.22 – 3.28 (m)
HꢀC(10) 1.58 – 1.65d)
CH2(12)
5.10 (t, J ¼ 2.6),
5.22 (t, J ¼ 2.6)
5.11 (t, J ¼ 2.5),
5.24 (t, J ¼ 2.4),
5.58 (t, J ¼ 2.4)
5.24 (t, J ¼ 2.5),
5.84 (t, J ¼ 2.5)
5.40 (t, J ¼ 2.5)
CH2(13)
4.18 (dt, J ¼ 13.5, 2.6), 4.32 (dt, J ¼ 13.4, 2.4), 4.29 (dt, J ¼ 12.8, 2.4), 4.40 (dt, J ¼ 12.8, 2.2),
4.55 (dt, J ¼ 13.5, 2.6)
0.89 (d, J ¼ 6.2)
1.78 (br. s)
4.67( dt, J ¼ 13.4, 2.4)
0.81 (d, J ¼ 7.2)
4.54 (dt, J ¼ 12.8, 2.4) 4.65 (dt, J ¼ 12.8, 2.2)
Me(14)
Me(15)
1.36 (d, J ¼ 7.2)
2.33 (s)
1.48 (d, J ¼ 7.2)
2.27( s)
1.49 (br. d, J ¼ 2.4)
a) In CD3OD. b) In C5D5N. c) In CDCl3. d) Overlapped signals.
MeOH). IR (KBr): 3444, 2923, 1733, 1000. 1H- and 13C-NMR: see Tables 3 and 4. FAB-MS (pos.): 459
([M þ Na]þ). HR-FAB-MS (pos.): 459.2719 ([M þ Na]þ, C25H40NaOþ6 ; calc. 459.2723).
Strobilol J (¼(3aS,5R,5aS,9R,9aS,9bR)-1,2,3a,4,5,5a,6,9,9a,9b-Decahydro-3a,9b-dihydroxy-5,8-di-
methyl-1-methylidenenaphtho[2,1-b]furan-9-yl Decanoate; 10). Colorless oil. [a]2D0 ¼ ꢀ49.3 (c ¼ 0.15,
MeOH). IR (KBr): 3467, 2854, 1733, 1033.1H- and 13C-NMR: see Tables 3 and 4. FAB-MS (neg.): 419
([M ꢀ H]ꢀ). HR-FAB-MS (neg.): 419.2796 ([M ꢀ H]ꢀ, C25H39Oꢀ5 ; calc. 419.2797).
Strobilol K (¼(3aS,5R,5aS,7R,8S,9R,9aS,9bR)-Dodecahydro-3a,7,8,9b-tetrahydroxy-5,8-dimethyl-1-
methylidenenaphtho[2,1-b]furan-9-yl Decanoate; 11). Colorless oil. [a]2D0 ¼ þ5.35 (c ¼ 0.64, MeOH). IR
1
(KBr): 3400, 2854, 1733, 1049. H- and 13C-NMR: see Tables 3 and 4. FAB-MS (neg.): 453 [M ꢀ H]ꢀ).
HR-FAB-MS (neg.): 453.2860 ([M ꢀ H]ꢀ, C25H41O7ꢀ ; calc. 453.2852).
Esterification of Strobilol E (5). To a suspension of 5 (3 mg) in pyridine (5 ml) was added p-
bromobenzoylchloride (10 mg) and 4-dimethylaminopyridine (3 mg), and this mixture was stirred
overnight at r.t. The mixture was filtered and purified on SiO2 CC to afford the benzoate 5a (1.5 mg). UV
1
(MeOH): 245 (2.9). CD: De245 ꢀ 3.0 (c ¼ 0.2, MeOH). H-NMR (400 MHz, CDCl3): 0.92 (d, J ¼ 6.5,
Me(14)); 1.24 – 1.43 (m, 3 H, CH2(1), CH2(8), HꢀC(10)); 1.50 – 1.53 (m, HꢀC(9)); 1.75 (br. s, Me(15));
1.98 (dd, J ¼ 14.1, 3.9, CH2(8)); 2.43 – 2.47( m, 2 H, CH2(1), HꢀC(5)); 4.23 (dt, J ¼ 13.2, 2.3, CH2(13));
4.54 (dt, J ¼ 13.2, 2.3, CH2(13)); 5.13 (t, J ¼ 2.5, CH2(12)); 5.49 (t, J ¼ 2.5, CH2(12)); 5.63 – 5.69 (m,
HꢀC(2)); 6.00 (br. s, HꢀC(4)); 7.59 (d, J ¼ 8.5, 2 arom. H); 7.91 (d, J ¼ 8.5, 2 arom. H). EI-MS: 448 (10,
Mþ), 436 (10), 264 (20), 248 (56), 230 (62), 185 (98), 161 (93), 119 (100).
REFERENCES
[1] G. Chihara, Dev. Biol. Stand. 1992, 77, 191.
´
[2] L. Barros, M.-J. Ferreira, B. Queiros, I. C. F. R. Ferreira, P. Baptista, Food Chem. 2007, 103, 413.
[3] F. Hiramatsu, T. Murayama, T. Koseki, Y. Shiono, Phytochemistry 2007, 68, 1267.
[4] Y. Shiono, F. Hiramatsu, T. Murayama, T. Koseki, T. Funakoshi, K. Ueda, H. Yasuda, Z.
Naturforsch., B 2007, 62, 1585.
[5] N. C. Gonnella, K. Nakanishi, V. S. Martin, K. B. Sharpless, J. Am. Chem. Soc. 1982, 104, 3775.
[6] H. Nakamura, Y. Kishi, O. Shimomura, Tetrahedron 1988, 44, 1597.
Received January 10, 2008