Eremophilane-type sesquiterpenes from cultured lichen mycobionts of Sarcographa tricosa

Article abstract of DOI:10.1016/j.phytochem.2012.01.009

Spore-derived mycobionts of the crustose lichen Sarcographa tricosa were cultivated on a malt-yeast extract medium supplemented with 10% sucrose. Chemical investigation of the cultivated colonies led to isolation of three eremophilane-type sesquiterpenes, 3-epi-petasol (1), dihydropetasol (2) and sarcographol (3), together with six known eremophilanes and ergosterol peroxide. These structures were elucidated by spectroscopic and chemical methods. This is the first report of eremophilane-type sesquiterpenes from the cultured mycobionts of lichen.

Full text of DOI:10.1016/j.phytochem.2012.01.009

Phytochemistry 91 (2013) 242–248
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Eremophilane-type sesquiterpenes from cultured lichen mycobionts
of Sarcographa tricosa
Duy Hoang Le a, Yukiko Takenaka a, Nobuo Hamada b, Takao Tanahashi a,
⇑
a Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
b Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennouji-ku, Osaka 543-0026, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 28 January 2012
Spore-derived mycobionts of the crustose lichen Sarcographa tricosa were cultivated on a malt-yeast
extract medium supplemented with 10% sucrose. Chemical investigation of the cultivated colonies led
to isolation of three eremophilane-type sesquiterpenes, 3-epi-petasol (1), dihydropetasol (2) and sarcog-
raphol (3), together with six known eremophilanes and ergosterol peroxide. These structures were elu-
cidated by spectroscopic and chemical methods. This is the first report of eremophilane-type
sesquiterpenes from the cultured mycobionts of lichen.
This paper is dedicated to the memory of
Prof. Dr. Meinhart H. Zenk.
Keywords:
Sarcographa tricosa
Mycobiont
Ó 2012 Elsevier Ltd. All rights reserved.
Lichen
Sesquiterpene
Eremophilane
1. Introduction
of the new sesquiterpenes are described. Detailed spectroscopic
data and further studies on the absolute stereochemistry of two
Lichens are symbiotic organisms of fungi (mycobionts) and
photoautotrophic algal partners, namely, green algae and/or cya-
nobacteria. About 18,500 different lichen taxa have been described
worldwide. Lichens have adapted to extreme ecological conditions,
being dominant at high altitudes and in Arctic boreal and tropical
habitats. Vietnam has a tropical monsoon climate that is favorable
for diverse tropical lichens, but previous studies on the Vietnamese
lichens focused mainly on their taxonomy and not on their chem-
ical constituents (Aptroot and Sparrius, 2006). Lichens produce
many unique compounds, which are considered to have important
biological and ecological functions, such as antimicrobial activity
(Ahmadjian, 1993; Huneck, 1999, 2001). Most of these metabolites
are produced by the fungal partner, in symbiosis or in the aposym-
biotic state. Cultures of isolated lichen mycobionts, however, often
exhibit the ability under osmotically stressed conditions to pro-
duce substances that have never been detected in the lichenized
state (Miyagawa et al., 1994; Tanahashi et al., 1997; Takenaka
et al., 2003, 2010). From our interest in Vietnamese lichens and
the metabolic ability of isolated lichen mycobionts, the spore-
derived mycobionts of the crustose lichen Sarcographa tricosa col-
lected in Vietnam were cultured and three new eremophilane-type
sesquiterpenes together with seven known compounds were iso-
lated. In this paper, the isolation and the structural determination
known sesquiterpenes are also reported.
2. Results and discussion
Specimens of S. tricosa (Ach.) Müll. Arg. were collected from tree
bark in Dong Nai Province, Vietnam, in 2008. The polyspore-
derived mycobionts were cultivated on
a malt-yeast extract
medium supplemented with 10% sucrose at 18 °C in the dark. After
several months, the cultures were harvested and extracted with
n-hexane and Et2O. These extracts were separated by chromato-
graphic procedures to afford three new eremophilane-type sesqui-
terpenes 1–3 together with six known eremophilanes 4–9 and
ergosterol peroxide (10) (Fig. 1).
Among the isolated compounds from the culture, petasol (4)
and dihydrosporogen-AO 1 (5) were the major products. Petasol
(4) was first isolated from Petasites fragrans in its free form
(Sugama et al., 1983), although its esters such as petasin (11)
and S-petasin (12) have since been isolated. Since alkaline hydroly-
sis of the esters failed to yield petasol (4), but afforded isopetasol
(9), the absolute stereostructure was investigated on its esters
but not on petasol (4) itself (Aebi and Djerassi, 1959; Herbst and
Djerassi, 1960; Neuenschwander et al., 1979). Dihydrosporogen-
AO 1 (5) was isolated from the culture broth of fungus Alternaria
citri. Its absolute configuration was determined previously by the
CD spectrum of its dibenzoate derivative (Kono et al., 1989). As
the 1H NMR spectroscopic data of 4 and 5 have not been fully
⇑
Corresponding author. Tel./fax: +81 78 441 7547.
E-mail address: tanahash@kobepharma-u.ac.jp (T. Tanahashi).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2012.01.009

D.H. Le et al. / Phytochemistry 91 (2013) 242–248
243
H
H
1
O
OH
10
8
O
8
7
12
3
5
7
12
11
HO
HO
RO
RO
11
OH
14
13
15
13
2 : 8α-OH, R = H
1
3
6
13 : 8β-OH, R = -COCH=CHS(O)CH3
14 : 8β-OH, R = H
O
OH
O
HO
HO
O
O
4 : R = H
5
11 : R = -COC(CH3)=CHCH3
12 : R = -COCH=CHSCH3
R1
H
H
O
O
O
HO
HO
R2
HO
7 : R1 = H, R2 = CH2OH
8 : R1 = OH, R2 = CH3
15
9
O
O
HO
10
Fig. 1. Structures of isolated compounds 1–10 and related compounds.
reported, a detailed spectroscopic analysis including 2D-NMR
experiments was performed to assign all proton signals of both
compounds and to confirm their relative configurations. Further-
more, the absolute stereochemistry of 4 and 5 was re-examined
by the 1H NMR analyses on the corresponding methylphenylacetic
acid (MPA) esters (Latypov et al., 1996). Preparation of the (R)- and
(S)-MPA esters 4a and 4b from petasol (4) and 5a and 5b from
dihydrosporogen-AO 1 (5) led us to determine that 4 and 5 pos-
sessed 3R and 3R, 8S configurations, respectively (Fig. 2). Conse-
quently, the stereochemistry of petasol (4) was confirmed as 3R,
4R, 5R, 7S, and the absolute configuration of dihydrosporogen-AO
1 (5) was established as 3R, 4R, 5R, 6R, 7S, 8S.
The other known compounds were identified as sporogen-AO 1
(6) (Tirilly et al., 1983; Tanaka et al., 1984), JBIR-27 (7) (Motohashi
et al., 2009), 1b-hydroxypetasol (8) (Sugawara et al., 1993), isopet-
asol (9) (Neuenschwander et al., 1979), and ergosterol peroxide
(10) (Fisch et al., 1973; Takaishi et al., 1991; Sgarbi et al., 1997)
by comparison of their physical and spectroscopic ([
a
]D, UV, IR,
NMR and MS) data with reported information. A detailed analysis
of the 2D-NMR spectra of 8 has shown that the literature assign-
ment resonances of C-13, C-14 and C-15 (Sugawara et al., 1993)
must be interchanged.
Compound 1 was obtained as colorless needles and the molec-
ular formula was established as C15H22O2 by HR-ESIMS. Its UV
spectrum showed a maximum at 240 nm, and its IR spectrum
exhibited absorption bands at 3479 and 1657 cmÀ1, indicating
the presence of hydroxyl and
a,b-unsaturated carbonyl groups.
The 1H NMR spectrum of 1 showed signals for three methyls at d
1.11 (d, J = 7.0 Hz), 1.39 (s), and 1.74 (br s), one olefinic proton at
d 5.80 (d, J = 1.5 Hz), two vinyl protons at d 4.82 (br s) and 4.98
(quint, J = 1.5 Hz), three methines at d 1.52 (qd, J = 7.0, 2.5 Hz),
3.17 (dd, J = 14.0, 4.5 Hz) and 3.95 (q, J = 2.5 Hz), and three sets of
methylene protons at d 1.72–2.83 (Table 1). Analysis of the 13C
NMR and DEPT spectra of 1 indicated 15 carbon signals due to
+0.048
+0.064
+0.023
-0.308
OR
O
+0.173
+0.216
+0.169
+0.183
8
(S)
+0.379
+0.347
-0.026
(R)
(R)
-0.018
CH2
CH3
CH2 -0.024
3
3
RO
RO
O
CH3
CH3
CH3
-0.124
-0.111
-0.047
CH3
CH3-0.015
-0.050
+0.322
-0.433
-0.057
-0.092
-0.391
4a : R = (R)-MPA
4b : R = (S)-MPA
5a : R = (R)-MPA
5b : R = (S)-MPA
Fig. 2. Values of
D dRS (dR–dS) obtained from 4a/4b and 5a/5b.

244
D.H. Le et al. / Phytochemistry 91 (2013) 242–248
Table 1
1H NMR spectroscopic data for 1–4 and 14 (CDCl3, 500 MHz).
H
1
2
3
4
14
1ax 2.83 tdd (14.5, 5.0, 1.5)
1eq 2.14 ddd (14.0, 4.0, 2.5) 2.17 ddd (14.5, 5.0, 3.0)
2ax 1.72 tdd (14.5, 4.0, 2.5)
2eq 2.03 ddt (14.5, 5.0, 2.5)
2.29 tdt (14.5, 4.5, 1.5)
2.29 tdt (14.5, 4.5, 2.0)
2.23 ddd (14.5, 4.5, 3.0) 2.34 ddd (15.0, 4.5, 2.5)
m
2.45 tdd (15.0, 4.5, 2.0)
2.26 tdt (14.0, 4.5, 1.5)
2.14 ddd (14.0, 5.0, 2.5)
1.36 dddd (14.5, 12.0, 11.0, 5.0) 1.36
2.07 dtd (12.0, 4.5, 3.0)
3.54 td (11.0, 4.5)
1.45 dddd (15.0, 12.0, 11.0, 2.5) 1.30
m
2.03 dtd (12.0, 4.5, 3.0)
3.53 td (11.0, 4.5)
1.18 dq (11.0, 6.5)
0.92 t (13.5)
1.76 dd (13.5, 5.0)
2.00 dt (13.5, 5.0)
4.52 td (5.0, 2.0)
5.59 dd (5.0, 2.0)
3.73 d (10.0)
2.16 dtd (12.0, 4.5, 2.5)
3.62 td (11.0, 4.5)
1.34 dq (11.0, 6.5)
1.88 t (14.0)
2.02 dd (14.0, 4.5)
3.11 dd (14.0, 4.5)
2.08 dtd (12.0, 4.5, 2.0)
3.53 td (10.5, 4.5)
1.14 dq (10.5, 6.5)
1.32 br t (13.0)
1.68 dd (13.0, 3.0)
2.19 ddd (13.0, 9.5, 3.0)
4.08 br d (9.5)
5.39 t (1.5)
3
4
3.95 q (2.5)
1.52 qd (7.0, 2.5)
1.25
1.57
1.60
m
m
m
6ax 1.83 br t (13.5)
6eq 1.97 dd (13.0, 4.5)
7
3.17 dd (14.0, 4.5)
2.25 br dt (11.5, 3.5)
4.09 br
5.63 dd (5.5, 1.5)
4.83 br s
8
9
12
5.80 d (1.5)
4.82 br s
5.78 d (2.0)
4.82 br s
4.89 br s
4.98 quint (1.5)
1.74 br s
5.03 br s
1.84 br s
3.83 d (10.0)
4.98 quint (1.5)
1.74 br s
4.92 quint (1.5)
1.74 dd (1.5, 1.0)
1.02 d (1.0)
13
14
15
1.34
0.93
s
s
1.39
s
0.96
s
1.18
s
1.11 d (7.0)
1.06 d (6.5)
1.06 d (6.5)
1.08 d (6.5)
1.00 d (6.5)
1H–1H coupling constants (Hz) are in parentheses.
three methyls, three sp3 methylenes, one sp2 methylene, three sp3
methines, one sp2 methine and four quaternary carbons, including
two olefinic and one carbonyl carbons (Table 2). These spectro-
scopic features of 1 closely resembled those of petasol (4) (Sugama
et al., 1983). The only marked difference in their 1H NMR spectra
was that the oxygenated methine proton signal of 1 was observed
at d 3.95 as a quartet (J = 2.5 Hz) instead of a triplet of doublets (d
3.62, J = 11.0, 4.5 Hz) as seen in petasol (4). The 1H–1H COSY and
HMBC experiments indicated that compound 1 possessed the same
eremophilane backbone as petasol (4) and differed from 4 only in
configuration at C-3. The detailed inspection of the coupling con-
stants enabled assignment of the signals for methylene protons
at d 1.72, 1.83, 1.97, 2.03, 2.14 and 2.83 to H-2ax, H-6ax, H-6eq,
H-2eq, H-1eq and H-1ax, respectively. The NOESY correlations
observed between H-1ax/H3-14, H3-14/H3-15, H-4/H-6ax, H-6eq/
H3-14, H-6eq/H3-15 and H-7/H3-14 indicated the b-orientation of
presence of hydroxyl group(s) but the absence of an a,b-unsatu-
rated carbonyl group. The 1H and 13C NMR spectra of 2 (Tables 1
and 2) together with DEPT and HMBC experiments suggested that
the structure of 2 was closely related to 4. Differences between two
isolates were ascribable to the absence of the carbonyl carbon and
the presence of an additional oxygenated methine [dH 4.09 and dC
63.6] in 2. These findings implied that the carbonyl group in 4 was
reduced to a hydroxyl group in 2. The substitution of the hydroxyl
group at C-8 was evidenced from the COSY correlations between
H-7 (d 2.25) and an oxygenated methine at d 4.09 (H-8), and be-
tween H-8 and an olefinic proton at d 5.63 (H-9), as well as HMBC
correlation from H-9 to an oxygenated methine carbon at dC 63.6
(C-8) (Fig. 4).
The relative configuration of 2 was deduced from the coupling
constants and NOESY correlations. The NOESY cross-peaks ob-
served between H-1ax/H3-14, H-3/H3-14, and H3-14/H-7 indicated
that H-1ax, H-3, H3-14 and H-7 were axially oriented on the same
face on the ring system. The coupling constants (J = 11.0 or
11.5 Hz) of H-3/H-2ax, H-3/H-4 and H-6ax/H-7 implied a trans-
diaxial relationship between each pair of protons and indicated
that compound 2 had the same configurations at C-3, C-4, C-5
and C-7 as petasol (4). The coupling constant (J = 3.5 Hz) of H-7/
H-8 was different from that of petasinol (13) (J = 9.6 Hz) (Lin
et al., 1998), indicating a cis relationship of H-7 and H-8 and
furthermore a quasi-equatorial orientation of H-8 (Fig. 4). Finally,
the absolute stereochemistry of 2 was determined by its chemical
correlation with petasol (4). Reduction of petasol (4) with NaBH4-
CeCl3 (Luche, 1978) yielded 2 as a minor product along with its
8-epimer 14 (Fig. 5), whose structure was confirmed by spectro-
scopic means and comparison with the reported data for petasinol
(13) (Lin et al., 1998). Thus, the structure of 2 was established as
shown and designated dihydropetasol.
Compound 3, named sarcographol, was obtained as a crystalline
solid, which was shown to have a molecular formula of C15H24O3
by HR-ESIMS. Its IR spectrum exhibited absorption bands at
3441, 3375 and 1659 cmÀ1, characteristic of a hydroxyl group(s)
and a double bond. The 1H and 13C NMR spectra of 3 (Tables 1
and 2) closely resembled those of 2 except that the double bond
C-11/C-12 was replaced by an oxygenated quaternary carbon at
dC 81.9 (C-11) and an oxygenated methylene at dH 3.73 and 3.83
(each d, J = 10.0 Hz, H2-12), and dC 77.9 (C-12). The HMBC correla-
tions from H-7 (dH 2.00) to C-11, from H2-12 to C-7 (dC 44.9), C-8
(dC 74.4) and C-11, and from H3-13 (dH 1.34) to C-7, C-11 and C-
12 suggested that an ether linkage between C-8 and C-12 forms
a tetrahydrofuran ring with methyl and hydroxyl groups at C-11
as in cyclodebneyol (15) (Burden et al., 1986). Furthermore, the
H-1ax, H-6eq, H-7, H3-14 and H3-15 and the
a-orientation of H-
4, H-6ax, the isopropenyl group as in petasol (4) (Fig. 3). The
1H–1H coupling constant (J = 2.5 Hz) of H-3 with H-2ax, H-2eq
and H-4 implied an equatorial orientation of H-3 and thus an axial
orientation of the hydroxyl group on the upward face of the ring
system. Accordingly, compound 1 was formulated as shown and
designated as 3-epi-petasol.
Compound 2 was isolated as a colorless crystalline solid. The
HR-ESIMS established a molecular formula of C15H24O2, that is,
two mass units more than that of petasol (4). The IR spectrum
showed absorption bands at 3341, 1450, 1022 and 884 cmÀ1, and
the UV spectrum exhibited an end absorption, indicating the
Table 2
13C NMR spectroscopic data for 1–5 and 14 (CDCl3, 125 MHz).
C
1
2
3
4
5
14
30.3
36.6
72.0
50.8
39.1
41.3
47.3
68.7
123.1
146.3
144.6
112.6
19.4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
27.3
33.8
71.4
46.4
39.4
42.2
50.5
30.5
36.2
71.9
50.8
39.3
35.4
42.0
63.6
121.0
147.9
146.3
111.9
22.8
30.7
35.7
72.0
50.1
38.4
36.2
44.9
74.4
31.0
35.1
71.0
50.2
39.8
41.7
50.3
198.8
124.4
167.9
143.4
114.4
20.0
30.2
35.7
71.9
43.6
38.8
67.9
65.6
65.3
119.5
140.4
142.1
113.4
19.6
199.0
124.1
170.2
143.7
114.3
20.0
118.6
148.0
81.9
77.9
20.4
18.1
11.1
18.9
11.9
18.1
17.3
17.6
11.3
18.8
10.4
10.8
10.4

D.H. Le et al. / Phytochemistry 91 (2013) 242–248
245
OH
H
1
H
3
H
O
H
14CH3
H31C5
H
9
H
H
HO
7
H
H
COSY
O
HMBC
NOE
CH3
H
Fig. 3. Key COSY, HMBC and NOESY correlations of 1.
H
H
H
3
OH
H
14
HO
H3C
CH3
H
9
H
H
H
HO
COSY
HMBC
NOE
H
H
7
OH
H
CH3
Fig. 4. Key COSY, HMBC and NOESY correlations of 2.
O
OH
OH
NaBH4-CeCl3
+
HO
HO
HO
4
2
14
Fig. 5. Reduction of 4 to 2 and 14.
H
H
1
H
3
H
CH3
HO
H3C
O
10
H
9
H
H
H
H
O
8
HO
7
6
OH
H
COSY
HMBC
NOE
H
OH
13
H3C
11
12
H
H
Fig. 6. Key COSY, HMBC and NOESY correlations of 3.
molecular formula of 3 and chemical shifts of 13C NMR spectrum
indicated the location of a hydroxyl group at C-3.
established a quasi-equatorial orientation of H-8 and a cis-fused
tetrahydrofuran ring. Furthermore, the NOESY spectrum showed
significant cross peaks of H-6eq/H3-13 and H-6ax/H3-13, demon-
strating that H3-13 was in close proximity to H2-6, hence the con-
figuration of C-11 was assigned as S⁄. Accordingly, the structure
of sarcographol was thereby elucidated as 3.
The absolute stereochemistry of 1 and 3 could not be chemically
determined owing to the minute amount of the isolated com-
pounds. Compounds 1–9 isolated in this study could be expected
to have a close biosynthetic relationship. Compounds 1–3 were dif-
ferent from 4 only in the oxidation pattern. Since the absolute con-
figuration of 4 was determined, it is reasonable to assign the same
absolute configurations for the chiral centers C-4, C-5 and C-7
within molecules 1–4 on the basis of biogenetic considerations.
The relative configuration of 3 could be deduced from analysis of
1H–1H coupling constants and NOESY correlations (Fig. 6). The cou-
pling constants J2ax,3 (11.0 Hz), J3,4 (11.0 Hz) and J6ax,7 (13.5 Hz) as
well as NOESY correlations of H-1ax/H3-14, H-3/H3-14, H3-14/H3-
15 and H-7/H3-14 indicated that the relative configurations at
C-3, C-4, C-5 and C-7 of 3 were consistent with those of 2. Moreover,
from the coupling constants J7,8 (5.0 Hz) and J8,9 (5.0 Hz), and the
magnitudes of allylic coupling J9,1ax (2.0 Hz) and homoallylic
coupling J8,1ax (2.0 Hz), the dihedral angles between H-1ax and
the C1-C10-C9 plane (Garbisch, 1964), and between H-8 and the
plane formed by C8-C9-C10 (Barfield and Sternhell, 1972), were
estimated as ca. 90° and ca. 140°, respectively. These findings

246
D.H. Le et al. / Phytochemistry 91 (2013) 242–248
The Et2O extract was subjected to silica gel CC and eluted by the
3. Concluding remarks
solvent system n-hexane-Et2O with increasing Et2O ratios to obtain
four fractions, fr-I (0–10% Et2O, 170 mg), fr-II(20–30% Et2O, 135 mg),
fr-III (50–70% Et2O, 284 mg) and fr-IV (70–100% Et2O, 245 mg). Fr-I
was a mixture of fatty compounds (168.9 mg). Fr-II was purified
by preparative TLC (toluene–EtOAc, 3:2, 1:1), giving 1 (2.1 mg) and
10 (10.5 mg). Fr-III was separated by preparative TLC (toluene–
EtOAc, 1:1, 1:2) to yield 4 (83.7 mg), 5 (119.8 mg) and 9 (8.0 mg,
colorless crystalline solid, mp. 120–121 °C (n-hexane-CHCl3);
Diverse eremophilane-type sesquiterpenes have been isolated
from plants (Lin et al., 1998; Liu et al., 2008; Sugama et al., 1983)
and fungi (Tanaka et al., 1984; Tirilly et al., 1983; Kono et al.,
1989; Sugawara et al., 1993; Motohashi et al., 2009), and exhibited
a variety of biological activities, such as anti-HIV (Singh et al.,
1999), antibacterial (Mohamed and Ahmed, 2005), and cytotoxic
activities (Beattie et al., 2011; Liu et al., 2008; Motohashi et al.,
2009). This type of sesquiterpenes has never been isolated from
lichen thalli or lichen mycobionts. This investigation of cultured li-
chen mycobionts of S. tricosa yielded three new eremophilane-type
sesquiterpenes 1–3 along with six known eremophilanes 4–9 and
ergosterol peroxide (10). This is the first instance of isolation of
eremophilane-type sesquiterpenes from lichen mycobionts in cul-
ture. It is also noteworthy that the metabolic ability was expressed
only in the isolated mycobionts.
[a]
20 + 106 (c 0.25, CHCl3)). Purification of fr-IV by preparative
D
TLC (n-hexane-EtOAc, 1:4; toluene-EtOAc-AcOH, 5:5:0.2) afforded
2 (6.9 mg), 3 (2.6 mg), 7 (2.3 mg, colorless solid, [
0.13, MeOH)) and 8 (4.9 mg).
a]
22 + 166 (c
D
4.3.1. 3-Epi-petasol (1)
Colorless needles, mp. 130–131 °C (n-hexane-CHCl3);
EtOH
[
a
]
23 + 114 (c 0.17, CHCl3); UV kmax
nm (loge): 240 (4.02);
D
KBr
IR mmax
cmÀ1: 3479, 2937, 1657, 1444, 1250, 1208, 949, 900;
for 1H NMR and 13C NMR spectroscopic data, see Tables 1 and 2;
4. Experimental
HR-ESIMS m/z: 235.1691 (calcd for C15H23O2: 235.1699 [M+H]+).
4.1. General
4.3.2. Dihydropetasol (2)
Colorless crystalline solid, mp. 138–140 °C (n-hexane-CHCl3);
D
1022, 884; for 1H NMR and 13C NMR spectroscopic data, see Tables
1 and 2; HR-ESIMS m/z: 259.1669 (calcd for C15H24O2Na: 259.1675
[M+Na]+).
Melting points were measured on a Yanaco micro melting point
apparatus and are uncorrected. UV spectra were recorded on a Shi-
madzu UV-240 spectrophotometer and IR spectra were obtained
with a Shimadzu FTIR-8200 infrared spectrophotometer. Optical
rotations were measured on a Jasco DIP-370 digital polarimeter.
HR-EIMS data were obtained with a Hitachi M-4100 mass spec-
trometer. The NMR experiments were performed with Varian
VXR-500, Varian UNITY INOVA and Gemini-300 spectrometers,
with TMS as internal standard. HPLC was performed using a
Waters system (600E Multisolvent Delivery System, 2487 Dual k
Absorbance Detector). Silica gel 60 (Merck) was used for column
chromatography (CC). Thin-layer chromatography (TLC) was per-
formed on precoated Kieselgel 60F254 plates (Merck) and spots
were visualized under UV light or sprayed with phosphomolybdic
acid solution (H2O 500 ml, H3PO4 85% 7.5 ml, concentrated H2SO4
25 ml, H3(PMo12O40)ÁnH2O 12.1 g), then heated.
KBr
[
a
]
21 + 208 (c 0.38, CHCl3); IR mmax
cmÀ1: 3341, 2934, 1450,
4.3.3. Sarcographol (3)
Colorless crystalline solid, mp. 224–225 °C (n-hexane-CHCl3);
KBr
[a
]
23 + 102 (c 0.11, CHCl3); IR mmax
cmÀ1: 3441, 3375, 2953,
D
2878, 1659, 1443, 1378, 1262, 1120, 1036, 1012, 918, 881; for 1H
NMR and 13C NMR spectroscopic data, see Tables 1 and 2; HR-
ESIMS m/z: 253.1800 (calcd for C15H25O3: 253.1805 [M+H]+).
4.3.4. Petasol (4)
EtOH
Colorless oil; [
a]
20 + 130 (c 0.82, CHCl3); UV kmax
nm (log
D
KBr
e
): 236.5 (4.12); IR mmax cmÀ1: 3433, 2971, 2942, 1669, 1625,
1444, 1374, 1330, 1252, 1216, 1195, 1070, 1038, 898; for 1H
NMR and 13C NMR spectroscopic data, see Tables 1 and 2; NOESY:
H-1ax/H-3, H-1ax/H3-14, H-1eq/H-9, H-3/H3-14, H-4/H-6ax,
H-6eq/H3-15, H-7/H3-14, H3-14/H3-15; HMBC: H2-1 ? C-2, 5, 9;
H2-2 ? C-3; H-4 ? C-3, 14, 15; H2-6 ? C-4, 5, 7, 8, 10, 11, 14;
H-7 ? C-6, 8, 11, 12, 13; H-9 ? C-1, 5, 7; H2-12 ? C-7, 11, 13;
H3-13 ? C-7, 11, 12; H3-14 ? C-4, 5, 6, 10; H3-15 ? C-3, 4, 5;
HR-ESIMS m/z: 235.1695 (calcd for C15H23O2: 235.1699 [M+H]+).
4.2. Plant material
Specimens of S. tricosa (Ach.) Müll. Arg. were collected from tree
bark in Ma Da forest, Dong Nai Province, Vietnam (94 m alt.), in
September 2008 by D.H.L. The voucher specimens were identified
by Prof. H. Miyawaki, Saga University, Japan, and was deposited
in Saga University, Japan (registration No. LHD068). Mycobionts
were obtained from the spores discharged from apothecia of a thal-
lus, and were cultivated in test tubes containing modified MY10
medium (malt extract 10 g, yeast extract 4 g, sucrose 100 g, agar
15 g, H2O 1 l, pH 7) at 18 °C in the dark. After cultivation for 4–
8 months, the colonies were harvested.
4.3.5. Dihydrosporogen-AO 1 (5)
Colorless needles, mp. 124–125 °C (n-hexane-CH2Cl2);
KBr
[a]
24 + 104 (c 0.84, MeOH); IR mmax cmÀ1: 3348, 2968, 2935,
D
2872, 1675, 1648, 1455, 1380, 1264, 1138, 1037, 958, 917, 882,
841; 1H NMR (CDCl3, 500 MHz): d 1.01 (3H, s, H3-14), 1.16 (3H,
d, J = 6.5 Hz, H3-15), 1.33 (1H, dddd, J = 14.0, 11.5, 10.5, 5.0 Hz, H-
2ax), 1.62 (1H, dq, J = 10.5, 6.5 Hz, H-4), 1.87 (3H, br s, H3-13),
2.04 (1H, dtd, J = 11.5, 5.0, 2.5 Hz, H-2eq), 2.17 (1H, ddd, J = 14.0,
5.0, 2.5 Hz, H-1eq), 2.29 (1H, tddd, J = 14.0, 5.0, 3.0, 2.0 Hz, H-
1ax), 3.08 (1H, s, H-6), 3.52 (1H, td, J = 10.5, 5.0 Hz, H-3), 4.52
(1H, t, J = 3.0 Hz, H-8), 5.06 (1H, quint, J = 1.5 Hz, H-12), 5.17 (1H,
br s, H-12), 5.25 (1H, br t, J = 2.0 Hz, H-9); 13C NMR: Table 2;
NOESY: H-1ax/H3-14, H-1eq/H-9, H-3/H3-14, H-3/H3-15, H-6/H3-
14, H-6/H3-15, H-8/H3-13, H3-14/H3-15; HMBC: H2-1 ? C-2;
H2-2 ? C-3; H-4 ? C-3, 5, 14, 15; H-6 ? C-5, 7, 10, 11, 14;
H2-12 ? C-7, 11, 13; H3-13 ? C-7, 11, 12; H3-14 ? C-4, 5, 6, 10;
H3-15 ? C-3, 4, 5; HR-ESIMS m/z: 273.1463 (calcd for C15H22O3Na:
273.1468 [M+Na]+).
4.3. Extraction and isolation
The harvested colonies (285 test tubes, dry weight 185.5 g)
were extracted with n-hexane (2 Â 800 ml) and Et2O (3 Â 400 ml)
at room temperature, and the combined extracts were concen-
trated under reduced pressure to give n-hexane (0.23 g) and Et2O
(0.92 g) extracts, respectively. The n-hexane extract was separated
by preparative TLC (Et2O-EtOAc, 10:1; toluene-EtOAc, 6:4, 1:1 or
1:2) and by preparative HPLC (Sunfire, n-hexane-2-propanol,
9:1), giving 1 (1.2 mg), 4 (49.3 mg), 5 (12.6 mg), 6 (9.3 mg, color-
less oil, [
a]
22 + 221 (c 0.80, MeOH)), 10 (30.3 mg, colorless crystal-
D
22
line solid, mp. 174–175 °C (CHCl3); [
and fatty compounds (58.2 mg).
a]
À 34.0 (c 1.07, CHCl3))
D

D.H. Le et al. / Phytochemistry 91 (2013) 242–248
247
4.3.6. 1b-Hydroxypetasol (8)
(CDCl3, 300 MHz): d 0.962 (3H, d, J = 6.6 Hz, H3-15), 1.013 (3H, s,
H3-14), 1.192 (1H, m, H-2ax), 1.460 (3H, br s, H3-13), 1.822 (1H,
m, H-2eq), 1.940 (1H, dq, J = 10.5, 6.6 Hz, H-4), 2.109 (1H, br d,
J = 14.4 Hz, H-1eq), 2.255 (1H, br t, J = 14.4 Hz, H-1ax), 2.894 (1H,
s, H-6), 3.401, 3.415 (each 3H, s, 3-MPA-OCH3 and 8-MPA-OCH3)
4.552, 4.677 (each 1H, br s, H2-12), 4.751, 4.816 (each 1H, s, 3-
MPA-CH and 8-MPA-CH), 4.805 (1H, td, J = 10.5, 4.5 Hz, H-3),
5.156 (1H, br s, H-9), 5.830 (1H, br t, J = 2.4 Hz, H-8), 7.285–7.440
(10H, m, 3-MPA-Ph and 8-MPA-Ph); HR-ESIMS m/z: 569.2512
(calcd for C33H38O7Na: 569.2517 [M+Na]+).
EtOH
Colorless oil. [
a
]
D
23 + 47.7 (c 0.42, MeOH). UV kmax
nm
): 233.0 (3.98); IR mmax cmÀ1: 3401, 2946, 1665, 1028. 1H-
KBr
(log
e
NMR (CDCl3, 500 MHz): d 1.11 (3H, d, J = 7.0 Hz, H3-15), 1.35 (1H,
m, H-4), 1.38 (3H, s, H3-14), 1.65 (1H, ddd, J = 14.5, 11.0, 3.0 Hz,
H-2ax), 1.73 (3H, dd, J = 1.0, 0.5 Hz, H3-13), 1.92 (1H, t,
J = 14.0 Hz, H-6ax), 2.03 (1H, dd, J = 13.0, 5.0 Hz, H-6eq), 2.35
(1H, ddd, J = 14.5, 4.5, 3.0 Hz, H-2eq), 3.22 (1H, dd, J = 14.0,
5.0 Hz, H-7), 4.03 (1H, td, J = 11.0, 4.5 Hz, H-3), 4.48 (1H, t,
J = 3.0 Hz, H-1), 4.84 (1H, br t, J = 1.0 Hz, H-12), 5.00 (1H, quint,
J = 1.0 Hz, H-12), 5.87 (1H, s, H-9). 13C-NMR (CDCl3, 125 MHz): d
10.4 (C-15), 19.5 (C-14), 20.0 (C-13), 39.2 (C-5), 41.6 (C-2), 43.1
(C-6), 50.0 (C-4), 50.8 (C-7), 67.2 (C-3), 73.7 (C-1), 114.6 (C-12),
126.8 (C-9), 143.1 (C-11), 165.4 (C-10), 199.5 (C-8). HR-ESIMS m/
z: 251.1643 (calcd for C15H23O3: 251.1648 [M+H]+).
4.6. Reduction of 4
To a solution of 4 (7.5 mg) in MeOH (0.4 ml) was added
CeCl3Á7H2O (12.0 mg) and then NaBH4 (1.2 mg), and the mixture
was stirred at room temperature for 1 h. The reaction mixture
was diluted with H2O and extracted with CHCl3 (3 Â 10 ml). The
CHCl3 layer was dried over Na2SO4 and concentrated in vacuo.
The crude product was subjected to preparative TLC (n-hexane–
4.4. Preparation of (R)- and (S)-MPA esters of 4
To a solution of 4 (6.9 mg) in dry CH2Cl2 (1 ml) were added (R)-
MPA (24.5 mg), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
(39.6 mg) and a catalytic amount of 4-pyrrolidinopyridine, and
the mixture was stirred at room temperature for 15 h. The reaction
mixture was poured into 1 N HCl (10 ml) and extracted with CHCl3
(3 Â 10 ml). The CHCl3 layer was dried over MgSO4 and concen-
trated in vacuo. The residue was purified by preparative TLC
(CHCl3–EtOAc, 9:1) to yield 4a (6.4 mg). Compound 4 (8.4 mg)
was treated with (S)-MPA (29.8 mg) as described above to yield
4b (8.6 mg). 4a: 1H NMR (CDCl3, 300 MHz): d 0.504 (3H, d,
J = 6.9 Hz, H3-15), 1.149 (3H, s, H3-14), 1.478 (1H, m, H-2ax),
1.490 (1H, m, H-4), 1.712 (3H, br s, H3-13), 1.809 (1H, t,
J = 13.5 Hz, H-6ax), 1.930 (1H, dd, J = 13.5, 4.8 Hz, H-6eq), 2.194
(1H, dtd, J = 12.3, 4.5, 2.4 Hz, H-2eq), 2.335 (1H, ddd, J = 15.3, 4.2,
2.4 Hz, H-1eq), 2.484 (1H, tdd, J = 15.3, 4.5, 1.5 Hz, H-1ax), 3.068
(1H, dd, J = 13.5, 4.8 Hz, H-7), 3.410 (3H, s, MPA-OCH3), 4.744
(1H, s, MPA-CH), 4.787 (1H, br s, H-12), 4.859 (1H, td, J = 10.8,
4.5 Hz, H-3), 4.966 (1H, quint, J = 1.5 Hz, H-12), 5.764 (1H, d,
J = 1.5 Hz, H-9), 7.329–7.454 (5H, m, MPA-Ph); HR-ESIMS m/z:
383.2220 (calcd for C24H31O4: 383.2224 [M+H]+). 4b: 1H NMR
(CDCl3, 300 MHz): d 0.895 (3H, d, J = 6.9 Hz, H3-15), 1.199 (3H, s,
H3-14), 1.306 (1H, m, H-2ax), 1.601 (1H, dq, J = 10.8, 4.5 Hz, H-4),
1.727 (3H, br s, H3-13), 1.866 (1H, t, J = 14.1 Hz, H-6ax), 1.978
(1H, m, H-2eq), 2.022 (1H, dd, J = 14.1, 4.8 Hz, H-6eq), 2.271 (1H,
ddd, J = 15.0, 4.5, 2.7 Hz, H-1eq), 2.436 (1H, tdd, J = 15.0, 4.8,
1.5 Hz, H-1ax), 3.093 (1H, dd, J = 14.1, 4.8 Hz, H-7), 3.425 (3H, s,
MPA-OCH3), 4.773 (1H, s, MPA-CH), 4.811 (1H, br s, H-12), 4.914
(1H, td, J = 10.8, 4.5 Hz, H-3), 4.984 (1H, quint, J = 1.0 Hz, H-12),
5.741 (1H, d, J = 1.5 Hz, H-9), 7.198–7.448 (5H, m, MPA-Ph); HR-
ESIMS m/z: 383.2219 (calcd for C24H31O4: 383.2224 [M+H]+).
Et2O–EtOAc, 1:1:0.3) to give
(1.3 mg). Compound 2 was identified with the isolated compound
from the culture (1H NMR, [
]D). 14 (8-epi-dihydropetasol): Color-
less crystalline solid, mp. 126–127 °C (CHCl3); [
2 (0.2 mg), 14 (2.8 mg) and 4
a
26
a]
À 36 (c 0.26,
D
CHCl3); for 1H NMR and 13C NMR spectroscopic data, see Tables 1
and 2; NOESY: H-1ax/H3-14, H-1eq/H-9, H-3/H3-14, H-4/H-6ax,
H-6eq/H3-15, H-7/H3-14; HMBC: H2-1 ? C-2, 5, 9, 10; H2-2 ?
C-3; H-4 ? C-3, 5, 15; H2-6 ? C-4, 5, 7, 8, 11, 14; H-7 ? C-8; H-
9 ? C-1, 5, 7; H2-12 ? C-7, 11, 13; H3-13 ? C-7, 11, 12; H3-
14 ? C-4, 5, 6, 10; H3-15 ? C-3, 4, 5; HR-ESIMS m/z: 259.1663
(calcd for C15H24O2Na: 259.1675 [M+Na]+).
Acknowledgements
We are grateful to the Vietnamese Government (Project 322,
Ministry of Education and Training) for the scholarship to D.H.
Le. We thank Prof. H. Miyawaki (Saga University, Japan) for identi-
fication of the lichen specimens. Thanks are also due to Drs. M.
Sugiura and C. Tode (Kobe Pharmaceutical University) for 1H and
13C NMR spectra, and to Dr. A. Takeuchi (Kobe Pharmaceutical Uni-
versity) for mass spectral measurements.
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