Phenyl ethers from cultured lichen mycobionts of Graphis scripta var. serpentina and G. rikuzensis.

Article abstract of DOI:10.1248/cpb.51.794

Spore-derived mycobionts of the lichen Graphis scripta var. serpentina and G. rikuzensis were cultivated on a malt-yeast extract medium supplemented with 10% sucrose and their metabolites were investigated. 3,3'-Dihydroxy-5,5'-dimethyldiphenyl ether was isolated from the cultures of the mycobionts of G. scripta var. serpentina, while a new phenyl ether, rikuzenol, along with two known diphenyl ethers, violaceol-I and violaceol-II, were isolated from those of G. rikuzensis. The structure of the new compound was determined by spectroscopic methods. Violaceol-I was chemically synthesized and interconversion between violaceol-I and violaceol-II was proven.

Full text of DOI:10.1248/cpb.51.794

794
Chem. Pharm. Bull. 51(7) 794—797 (2003)
Vol. 51, No. 7
Phenyl Ethers from Cultured Lichen Mycobionts of Graphis scripta var.
serpentina and G. rikuzensis
a
b
Yukiko TAKENAKA, Takao TANAHASHI, ,a Naotaka NAGAKURA,a and Nobuo HAMADA
*
a Kobe Pharmaceutical University; 4–19–1 Motoyamakita-machi, Higashinada-ku, Kobe 658–8558, Japan: and b Osaka
City Institute of Public Health and Environmental Sciences; 8–34 Tojo-cho, Tennouji-ku, Osaka 543–0026, Japan.
Received January 14, 2003; accepted March 24, 2003
Spore-derived mycobionts of the lichen Graphis scripta var. serpentina and G. rikuzensis were cultivated on a
malt-yeast extract medium supplemented with 10% sucrose and their metabolites were investigated. 3,3؅-Dihy-
droxy-5,5؅-dimethyldiphenyl ether was isolated from the cultures of the mycobionts of G. scripta var. serpentina,
while a new phenyl ether, rikuzenol, along with two known diphenyl ethers, violaceol-I and violaceol-II, were iso-
lated from those of G. rikuzensis. The structure of the new compound was determined by spectroscopic methods.
Violaceol-I was chemically synthesized and interconversion between violaceol-I and violaceol-II was proven.
Key words Graphis scripta var. serpentina; Graphis rikuzensis; lichen; isolated mycobiont; phenyl ether
Lichens, symbiotic associations of mycobiont and photo-
biont partners, produce a variety of characteristic secondary
metabolites.1) Some lichen substances have been found to ex-
hibit a wide range of potentially useful biological activities,
e.g., antibiotic and anticancer activities, and a monoamine
oxidase inhibitory effect.2) On the other hand, our recent
studies have demonstrated that cultures of spore-derived
lichen mycobionts are capable of producing certain lichen
substances or novel metabolites under osmotically stressed
conditions.3,4) It was pointed out that cultures of lichen myco-
bionts could be new sources of bioactive compounds. In a
continuation of our studies on cultured lichen mycobionts,5)
we cultivated spore-derived mycobionts of Graphis scripta
(L.) ACH. var. serpentina MEYER and G. rikuzensis (VAIN.)
NAK. and isolated four phenolics from their cultures. In this
paper, we describe the isolation and characterization of these
compounds.
Polyspore-derived mycobionts of Graphis scripta var. ser-
pentina and G. rikuzensis collected in Hidakokufu, Gifu Pre-
fecture, Japan and Aizuwakamatsu, Fukushima Prefecture,
Japan, respectively, were cultured on conventional malt-yeast
extract medium supplemented with 10% sucrose at 18 °C in
the dark. After 8—10 months, the cultivated colonies were
harvested and extracted with acetone.
Chart 1
methylated compounds, 2a and 3a. Compounds 2 and 3 have
already been obtained as an inseparable mixture from Emeri-
cella violacea.7) It was, however, found in our study that 2
and 3 could be separated by HPLC, but isomerized after
standing in MeOH. This surprising finding prompted us to
synthesize violaceol-I in order to confirm the unexpected
phenomenon.
Compound 5 prepared from methyl gallate8) was reduced
with LiAlH4 to afford 6. Subsequent hydrogenolysis of 6
gave 7. On the other hand, bromoveratraldehyde was treated
with NaBH4 and the resulting alcohol 8 was protected as a
tetrahydropyranyl (THP) ether to yield 9. Ullmann reaction9)
of 7 and 9 afforded 10, which was hydrogenated over Pd–C
to 2a. The methyl ether 2a was deprotected with BBr3 (Chart
2). The phenolic product, which should exclusively be 2, was
analyzed by HPLC to be found to be a mixture of 2 and 3 in
the ratio of 18 : 11. This result confirmed that diphenyl ether
2 could be isomerized to 3.
Subsequent purification of the extract from the cultured
mycobionts of G. scripta var. serpentina by preparative TLC
1
and preparative HPLC afforded compound 1. The H- and
13C-NMR spectra of 1, together with its structural formula
C14H14O3, suggested a symmetrical diphenyl ether structure
for 1. Compound 1 was identified as 3,3Ј-dihydroxy-5,5Ј-di-
methyldiphenyl ether, which has been isolated from Emeri-
cella falconensis.6) This is the first instance of isolation of
this compound from cultured mycobionts of lichens.
A new compound 4, named rikuzenol, was obtained as
colorless oil. The HR-EI mass spectrum of 4 exhibited a
peak at m/z 382.1058 [M]ϩ, indicating a molecular formula
of C21H18O7. It showed UV maxima at 208, 230sh, 253sh,
1
and 280 nm. Its H-NMR spectrum exhibited signals for
Fractionation of the extract from cultured mycobionts of
G. rikuzensis by a combination of chromatographic proce-
dures afforded three compounds, 2, 3 and 4.
Compounds 2 and 3 were found to be isomers with a mol-
ecular formula C14H14O5. The 1H- and 13C-NMR spectral fea-
tures suggested 2 and 3 to be violaceol-I and violaceol-II, re-
spectively, both of which are dimeric ethers consisting of
3,4,5-trihydroxytoluene units. Further structural confirmation
was given by detailed 2D-NMR spectral analyses of their
three methyl groups at d 2.01, 2.06 and 2.21 (each s), two
aromatic protons at d 6.59 and 6.60 (each br s), and a pair of
meta-coupled aromatic protons at d 6.47 and 6.75 (each m).
The 13C-NMR spectrum of 4 showed three methyl signals
and eighteen aromatic carbon signals, of which four were CH
carbons and fourteen quaternary sp2 carbons. These spectral
features, together with its molecular formula and co-occur-
rence of violaceols-I and II, suggested that the compound
To whom correspondence should be addressed. e-mail: tanahash@kobepharma-u.ac.jp
© 2003 Pharmaceutical Society of Japan

July 2003
795
Chart 2
Table 1. 1H- and 13C-NMR Spectral Data for Compounds 2, 3 and 4 in CD3OD
2
3
4
C
dH
dC
dH
dC
dH
dC
1
2
3
4
5
146.6b,y)
134.9
148.3b,y)
132.8
147.1b,x)
111.3c,x)
129.8
107.9c,y)
21.2
130.1
151.6
109.7
136.7
109.7
151.6
21.4
143.9d)
136.3e,x)
147.3f,x)
114.8g,x)
128.0h,x)
124.2i,x)
19.18j)
147.5b,x)
112.5c,x)
129.0
6.40a,x)
6.15a,y)
m
m
6.30a,x)
5.90a,y)
m
m
6.59a,x) br s
2.06b,x) br s
6
111.6c,y)
21.0
Me
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
Me
1Љ
2Љ
3Љ
4Љ
5Љ
6Љ
Me
2.12 br s
2.05 br s
146.6b,y)
134.9
143.4d)
137.4e,y)
147.5f,y)
114.5g,y)
128.3h,y)
124.0i,y)
19.21j)
147.5b,x)
112.5c,x)
129.0
6.27 br s
6.27 br s
2.21 br s
6.40a,x)
6.15a,y)
m
6.60a,y) br s
2.01b,y) br s
m
111.6c,y)
21.0
2.12 br s
150.1k,w)
136.3
151.0k,z)
112.8l,z)
135.0
6.47c,z)
6.75c,w)
m
m
115.7l,w)
21.2
2.21 br s
a—l) Values with the same superscript may be interchangeable. x—w) The carbon signals are correlated to the proton signals with the same superscript by means of
HMBC and HMQC.
consisted of three units of 3,4,5-trihydroxytoluene, which diphenyl derivative A or dibenzofuran B (Fig. 2) was con-
were connected to each other through a carbon–carbon bond nected with another unit of trihydroxytoluene through an
as well as two ether linkages. The heteronuclear multiple- ether linkage(s).
1
bond correlation (HMBC) experiments (Fig. 1) and H–1H
The structure was finally confirmed by detailed 2D-NMR
shift correlation spectroscopy (1H–1H COSY) of 4 allowed us studies on a methylated compound of 4 (Fig. 1). Methylation
to assign all of the H- and 13C-NMR signals in each unit of of 4 with Me2SO4 gave pentamethyl ether 4a, indicating the
1
3,4,5-trihydroxytoluene (Table 1). The corresponding signals presence of five hydroxyl groups in 4. Duplicated HMBC in-
(C-1—C-6, 5-Me to C-1Ј—C-6Ј, 5Ј-Me) due to two units teractions from an aromatic proton (H-4/H-4Ј) to two carbon
with a single aromatic proton resonated at nearly identical signals attached to a methoxyl group (C-2 and C-3/C-2Ј and
frequencies. The signals for C-6 and C-6Ј were observed as C-3Ј) and to a signal for a methyl group (5-Me/5Ј-Me) indi-
quaternary carbons at d 124.2 and 124.0 in the 13C-NMR cated a partial structure p-1, and further HMBC cross peaks
spectrum of 4, while that for C-6 of violaceol-I appeared at d between OMe (d 3.89) and C-3Љ, between H-4Љ and C-2Љ, C-
111.6. These findings suggested that two units of 3,4,5-trihy- 3Љ, and between H-6Љ and C-1Љ, C-2Љ revealed a partial struc-
droxytoluene were linked through a carbon–carbon bond be- ture p-2 (Fig. 2). Two partial structures could be depicted
tween C-6 and C-6Ј. On the other hand, the 13C-NMR spec- as a single structure 4a. The proposed structure was further
tral features of the residual unit (C-1Љ—C-6Љ, 5Љ-Me) with supported by nuclear Overhauser enhancement spectroscopy
two aromatic protons differed remarkably from those of the (NOESY) experiments (Fig. 1). Consequently, the structure
former units and showed an asymmetrical character. Accord- of the new compound rikuzenol was represented by 4.
ingly, 4 could be assumed to have a structure in which
Phenyl ether derivatives represented by violaceols-I and II

796
Vol. 51, No. 7
100 g, agar 15 g, H2O 1 l, pH 7) at 18 °C in the dark. After cultivation for
8—10 months, the colonies and slants were harvested and freeze-dried.
Isolation of Compounds
Graphis scripta var. serpentina: The harvested colonies (56 test tubes, dry
weight 14.11 g) were extracted with acetone at room temperature, and the
combined extracts were concentrated under reduced pressure to give a
residue (446 mg). The extracts were repeatedly subjected to preparative TLC
(toluene–acetone 4 : 1) and preparative HPLC (mBondasphere 5mC18-100 Å,
MeCN–H2O, 9 : 11), giving rise to 1 (2.9 mg). 1: UV lmax nm: 207, 232sh,
278. UV maxima were measured by HPLC equipped with a photodiode
1
array detector, using MeCN–H2O (1 : 1) as a solvent. H-NMR: as in ref 6.
13C-NMR (CD3OD): d 21.6 (5-Me), 104.3 (C-2), 111.8, 112.0 (C-4, C-6),
141.6 (C-5), 159.6, 159.7 (C-1, C-3). HR-EI-MS m/z 230.0938 [M]ϩ (Calcd
for C14H14O3, 230.0944).
Fig. 1. HMBC Correlations (Bold Arrows) and NOEs (Dotted Arrows)
Observed for Compounds 4 and 4a
G. rikuzensis: The harvested colonies (81 test tubes, dry weight 11.34 g)
were extracted with acetone at room temperature, and the combined extracts
were concentrated under reduced pressure to give a residue (377 mg). The
slants were collected and extracted in the same way to give a residue (401
mg). The extracts were repeatedly purified by preparative TLC (toluene–ace-
tone 4 : 1) and preparative HPLC (mBondasphere 5mC18-100 Å, MeCN–
H2O, 2 : 3 or 7 : 13) to give 2 (8.4 mg), 3 (9.4 mg), and 4 (3.4 mg). Com-
pounds 2 and 3 were separated as single compounds, but isomerized to give
a mixture of 2 and 3 after standing in MeOH. 2 (violaceol-I): colorless oil.
UV lmax nm: 207, 231sh, 280.* 1H- and 13C-NMR: Table 1. 3 (violaceol-II):
colorless oil. UV lmax nm: 207, 251sh, 274.* 1H- and 13C-NMR: Table 1.
*UV maxima were measured by an HPLC equipped with a photodiode array
detector, using MeCN–H2O (2 : 3) as a solvent. HR-EI-MS m/z 262.0843
[M]ϩ (Calcd for C14H14O5, 262.0842).
4: Colorless oil. UV lmax nm: 208, 230sh, 253sh, 280. UV maxima were
measured by an HPLC equipped with a photodiode array detector, using
MeCN–H2O (2 : 3) as a solvent. 1H- and 13C-NMR: Table 1. HR-EI-MS m/z
382.1058 [M]ϩ (Calcd for C21H18O7, 382.1053).
Methylation of 2 and 3 K2CO3 (110 mg) and Me2SO4 (0.1 ml) were
added to a mixture of 2 and 3 (10.3 mg) in acetone (10 ml) and the resulting
solution was heated under reflux for 6.5 h. After removal of K2CO3 by filtra-
tion, the reaction mixture was diluted with aq. NaHCO3 solution and ex-
tracted with CHCl3. The washed and dried organic layer was concentrated in
vacuo and the residue was purified by preparative TLC (Et2O) and prepara-
tive HPLC (mBondasphere 5mC18-100 Å, MeCN–H2O, 11 : 9) to yield 2a
Fig. 2
(4.6 mg) and 3a (3.3 mg).
EtOH
2a: Colorless oil. UV l
nm (log e): 206.5 (4.73), 232sh (4.29), 271
have so far been isolated as fungal metabolites, but not from
natural thalli of lichens, although orcinol, a unit of 3,3Ј-dihy-
droxy-5,5Ј-dimethyldiphenyl ether, is known as a lichen sub-
stance. This is the first instance of the isolation of phenyl
ethers from lichen mycobionts. It is of great interest from the
viewpoint of their physiological and biological significances
that 1, 2 and 3 were found to exhibit antimicrobial activity. It
might be postulated that the metabolic pathways of the
diphenyl ether derivatives could be expressed for survival of
the mycobiont in pre-lichenized conditions.
max
(3.27). IR nmKaBxr cmϪ1: 1578, 1506, 820. 1H-NMR (CDCl3): d: 2.24 (6H, br s,
5-Me, 5Ј-Me), 3.85 (6H, s, 2-OMe, 2Ј-OMe), 3.87 (6H, s, 3-OMe, 3Ј-OMe),
6.31 (2H, m, H-6, H-6Ј), 6.50 (2H, m, H-4, H-4Ј). NOE: 3-OMe ↔H-4. HR-
EI-MS m/z 318.1459 [M]ϩ (Calcd for C18H22O5, 318.1468).
EtOH
3a: Colorless crystals, mp. 92 °C (MeOH). UV l
(4.81), 234sh (4.01), 269 (3.29). IR n
nm (log e): 206.5
max
cmϪ1: 1601, 1506, 818. H-NMR
KBr
max
1
(CDCl3): d: 2.16 (3H, br s, 5-Me), 2.37 (3H, br s, 4Ј-Me), 3.75 (6H, s, 2Ј-
OMe, 6Ј-OMe), 3.85 (3H, s, 3-OMe), 3.97 (3H, s, 2-OMe), 5.96 (1H, m, H-
6), 6.36 (1H, m, H-4), 6.45 (2H, br s, H-3Ј, H-5Ј). NOE: 3-OMe ↔H-4; 5-
Me ↔H-4, H-6; H-3Ј, H-5Ј↔ 4Ј-Me, 2Ј-OMe, 6Ј-OMe. HR-EI-MS m/z
318.1459 [M]ϩ (Calcd for C18H22O5, 318.1468).
Synthesis of Violaceol-I (2) Compound 5 was prepared from methyl
gallate according to ref. 8. LiAlH4 (280 mg) was added to a solution of 5
(2 g) in anhydrous ether (40 ml) and the mixture was stirred at 60 °C for
15 min and at room temperature for 4 h. The reaction mixture was poured
into water and then acidified with diluted HCl. The aqueous solution was ex-
tracted with ether and the ethereal solution was washed with water and satu-
rated brine, dried, and evaporated to give 6 as quantitative yield. 6: 1H-NMR
(CDCl3): d: 1.83 (1H, br s, CH2OH), 3.85, 3.86 (each 3H, s, OMeϫ2), 4.57
(2H, br s, 1-CH2OH), 5.12 (2H, br s, 5-OCH2Ar), 6.60, 6.62 (each 1H, br s,
H-2, H-6), 7.3—7.4 (5H, m, ArH). EI-MS m/z: 274 [M]ϩ.
Experimental
Melting points were measured on a Yanaco micro melting point apparatus
and are reported uncorrected. The UV spectra were recorded on a Shimadzu
UV-240 spectrophotometer and the IR spectra on a Shimadzu FTIR-8200 in-
frared spectrophotometer. HR-EI-MS were obtained with a Hitachi M-4100
mass spectrometer. The NMR experiments were performed with Varian
VXR-500, Varian Gemini-300 and Varian Gemini-200 spectrometers, with
tetramethylsilane as internal standard. HPLC was performed using a Waters
system (600E Multisolvent Delivery System, 486 Tunable Absorbance De-
tector). Thin-layer chromatography was performed on precoated Kieselgel
60F254 plates (Merck) and spots were visualized under UV light.
Plant Material Specimens of Graphis scripta var. serpentina and G.
rikuzensis were collected from the bark of trees in Hidakokufu, Gifu Prefec-
ture, Japan (500 m alt.) and Aizuwakamatsu, Fukushima Prefecture, Japan
(400 m alt.), respectively. The voucher specimens were identified by Prof. M.
Nakanishi of Hiroshima University, Japan and were deposited at Osaka City
Institute of Public Health and Environmental Sciences with the registration
No. NH9881341 (G. scripta var. serpentina) and No. NH9810144 (G.
rikuzensis). Mycobionts of Graphis spp. were obtained from the spores dis-
charged from apothecia of a thallus, and were cultivated in test tubes con-
taining modified MY10 medium (malt extract 10 g, yeast extract 4 g, sucrose
A solution of 6 (1.87 g) in MeOH (30 ml) was hydrogenated over 5% pal-
ladium-charcoal (2.2 g) at room temperature and atmospheric pressure and
then the filtered solution was evaporated. The residue was purified on silica
1
gel column chromatography (CHCl3) to afford 7 (1.09 g, 95%). 7: H-NMR
(CDCl3): d: 2.24 (3H, s, 1-Me), 3.81, 3.83 (each 3H, s, OMeϫ2), 6.00 (1H,
br s, OH) 6.27, 6.42 (each 1H, br s, H-2, H-6). EI-MS m/z: 168 [M]ϩ, 153
[MϪMe]ϩ.
NaBH4 (37 mg) was added to a solution of 5-bromoveratraldehyde (500
mg) in ethanol (5 ml). After standing at room temperature for 30 min, the so-
lution was poured into water and extracted with CHCl3. The CHCl3 layer
1
was dried and evaporated to give 8 (474 mg, 94%). 8: H-NMR (CDCl3): d
2.40 (1H, br s, CH2OH), 3.82, 3.85 (each 3H, s, OMeϫ2), 4.57 (2H, br s, 1-

July 2003
797
CH2OH), 6.85, 7.08 (each 1H, br s, H-2, 6) HR-EI-MS m/z 245.9908 [M]ϩ
(Calcd for C9H1179BrO3, 245.9892), 247.9877 [M]ϩ (Calcd for C9H1181BrO3,
247.9872).
solution of 4 (3.4 mg) in acetone (10 ml) and the resulting solution was
heated under reflux for 6.5 h. The reaction mixture was worked up in the
same way as for 2a and 3a and the crude product was purified by prep. TLC
(Et2O) and prep. HPLC (mBondasphere 5mC18-100 Å, MeCN–H2O, 7 : 3) to
p-TsOH·H2O (p-toluenesulfonic acid monohydrate) (1.9 g) was added to
a solution of 8 (474 mg) and DHP (3,4-dihydro-2H-pyran) (420 mg) in dry
CH2Cl2 (5 ml), and the mixture was stirred at room temperature for 2 h. The
reaction mixture was diluted with CHCl3, and then washed with saturated
NaHCO3 solution and saturated brine. The organic layer was dried and evap-
orated, and then the residue was subjected to prep. TLC (CHCl3) to give 9
(376 mg, 59%). 9: 1H-NMR (CDCl3): d: 1.5—2.0 (6H, m, THP-CH2ϫ3),
3.70, 3.90 (each 1H, m, THP-OCH2), 3.84, 3.87 (each 3H, s, OMeϫ2) 4.41,
4.70 (each 1H, d, Jϭ12.0 Hz, 1-CH2O), 4.69 (1H, br t, Jϭ5.0 Hz, THP-
OCH), 6.85, 7.08 (each 1H, br s, H-2, H-6) HR-EI-MS m/z 330.0471 [M]ϩ
(Calcd for C14H1979BrO4, 330.0468), 332.0454 [M]ϩ (Calcd for
C14H1981BrO4, 332.0447).
Finely powdered anhydrous K2CO3 (250 mg) and CuO (50 mg) were
added with stirring to a solution of 7 (185 mg) and 9 (221 mg) in anhydrous
pyridine (5 ml) and then the mixture was heated for 6 h on an oil bath under
N2 atmosphere. The temperature of the bath was kept at 150 °C during the
reaction. After the reaction ended, the mixture was poured into ether. This
ethereal solution was washed successively with 3% NaOH, 5% citric acid
solution, and saturated brine, dried over anhydrous MgSO4, and then the sol-
vent was evaporated off. The residual substance was purified by prep. TLC
(CHCl3) to give an oily product 10 (47.5 mg, 17% yield from 9). 10: 1H-
NMR (CDCl3): d: 1.5—1.9 (6H, m, THP-CH2ϫ3), 2.23 (3H, br s, 5-Me),
3.5, 3.9 (each 1H, m, THP-OCH2), 3.85, 3.87ϫ2, 3.90 (each 3H, s, OMeϫ4)
4.38, 4.66 (each 1H, d, Jϭ12.0 Hz, 5Ј-CH2O), 4.65 (1H, br t, Jϭ5.0 Hz,
THP-OCH), 6.30, 6.49, 6.51, 6.72 (each 1H, br s, H-4, H-4Ј, H-6, H-6Ј).
HR-EI-MS m/z 418.1981 [M]ϩ (Calcd for C23H30O7, 418.1993).
yield 4a (2.4 mg). 4a: Colorless crystals, mp. 171—172 °C (MeOH). [a]D28
EtOH
ϩ19° (cϭ0.21, CHCl3). UV l
nm (log e): 207 (4.83), 250.5sh (4.20),
max
KBr
285sh (3.54). IR n
cmϪ1: 1605, 1589, 826. 1H-NMR (CDCl3): d: 2.16
max
(3H, s, 5-Me), 2.19 (3H, br s, 5Ј-Me), 2.28 (3H, br s, 5Љ-Me), 3.68 (3H, s, 2-
OMe), 3.72 (3H, br s, 2Ј-OMe), 3.84 (3H, br s, 3-OMe), 3.85 (3H, br s, 3Ј-
OMe), 3.89 (3H, br s, 3Љ-OMe), 6.48 (1H, d, Jϭ2.0 Hz, H-4Љ), 6.66 (1H, br s,
H-4), 6.68 (1H, br s, H-4Ј), 6.75 (1H, m, H-6Љ). 13C-NMR (CDCl3): d: 19.7,
19.8 (5-Me, 5Ј-Me), 21.4 (5Љ-Me), 56.0 (3Љ-OMe), 56.1ϫ2 (3-OMe, 3Ј-
OMe), 61.1 (2-OMe), 61.3 (2Ј-OMe), 108.3 (C-4Љ), 110.68 (C-4), 110.73
(C-4Ј), 115.2 (C-6Љ), 124.66 (C-6), 124.71 (C-6Ј), 131.3 (C-5Ј), 131.6 (C-5),
132.8 (C-5Љ), 137.5 (C-2Љ), 140.1 (C-2Ј), 140.3 (C-2), 147.5, 148.3, 149.9
(C-1, 1Ј, 1Љ), 151.8 (C-3Љ), 153.1 (C-3), 153.3 (C-3Ј). HR-EI-MS m/z
452.1817 [M]ϩ (Calcd for C26H28O7, 452.1836).
Acknowledgements This research was financially supported by Grant-
in-Aid for Scientific Research (C) (No. 11672129 and No. 13836009) from
the Ministry of Education, Culture, Sports, Science and Technology of Japan
and the Kobe Pharmaceutical University Collaboration Fund. We are grate-
ful to Prof. Nakanishi (Hiroshima University, Japan) for identification of the
voucher specimen. Thanks are also due to Dr. M. Sugiura (Kobe Pharmaceu-
tical University) for the 1H- and 13C-NMR spectra, and to Dr. K. Saiki (Kobe
Pharmaceutical University) for the mass spectra measurements.
References
1) Huneck S., Yoshimura, I., “Identification of Lichen Substances,”
Springer Verlag, Berlin, Heidelberg, 1996.
A solution of 10 (47.5 mg) in EtOAc was hydrogenated over 5% palla-
dium-charcoal (0.6 g) at room temperature and atmospheric pressure and
then the filtered solution was evaporated. The residue was subjected to prep.
TLC (Et2O) to afford 2a (13 mg, 36%). 1H-NMR (CDCl3): d: 2.24 (6H, br s,
5-Me, 5Ј-Me), 3.85, 3.87 (each 6H, s, OMeϫ4), 6.30, 6.49 (each 2H, br s,
H-4, H-4Ј, H-6, H-6Ј). EI-MS m/z: 318 [M]ϩ.
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BBr3 (2 drops) was added to a solution of 2a (13 mg) in anhydrous
CH2Cl2 and the mixture stirred at room temperature for 1.5 h under a N2 at-
mosphere. The reaction mixture was evaporated to give 2 (11 mg). The prod-
uct was unchanged in MeOH or MeCN–H2O for 12 h. After separation by
prep. HPLC (mBondasphere 5mC18-100 Å, MeCN–H2O), the product was
dissolved in MeOH and set aside for 6 h to yield a mixture of 2 and 3 at a
ratio of 18 : 11. The separation process with an HPLC column seemed to be
requisite for the rearrangement of 2 and 3.
Methylation of 4 K2CO3 (55 mg) and Me2SO4 (0.05 ml) were added to a