New phenylphenalenones from banana fruit

Article abstract of DOI:10.1016/S0040-4020(01)00749-9

Six new phenylphenalenone derivatives were isolated from unripe banana, Musa acuminata, fruit that had been wounded followed by inoculation with Colletotrichum musae. Absolute configurations of optically active phenylphenalenone derivatives were elucidated by their chemical conversion to L-arabitol pentaacetate. Antifungal activities against C. musae and a biosynthetic pathway of phenylphenalenones are also discussed.

Full text of DOI:10.1016/S0040-4020(01)00749-9

TETRAHEDRON
Pergamon
Tetrahedron 57 62001) 7649±7656
New phenylphenalenones from banana fruit
Tsunashi Kamo,a,b Nobuhiro Hirai,a,b,p Kumiko Iwami,a Daie Fujiokac and Hajime Ohigashia
aDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
bCREST, Japan Science and Technology Corporation &JST), 4-1-8 Hon-cho, Kawaguchi 332-0012, Japan
cKansai Consumer's Cooperative Union, 3-3-3 Tenjinbashi, Kita-ku, Osaka 530-0041, Japan
Received 4 June 2001; accepted 19 July 2001
AbstractÐSix new phenylphenalenone derivatives were isolated from unripe banana, Musa acuminata, fruit that had been wounded
followed by inoculation with Colletotrichum musae. Absolute con®gurations of optically active phenylphenalenone derivatives were
elucidated by their chemical conversion to l-arabitol pentaacetate. Antifungal activities against C. musae and a biosynthetic pathway of
phenylphenalenones are also discussed. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
and the ®rst determination of the absolute con®gurations of
the optically active phenylphenalenone derivatives. An
alternative biosynthetic pathway is also discussed based
on their structures and the results obtained so far.
Unripe fruit of banana, Musa acuminata, produce antifungal
compounds upon infection by Colletotrichum musae.1
These compounds are involved in the defense mechanism
by which the pathogen remains quiescent until the fruit
ripen.2 We have isolated 17 phenylphenalenone derivatives
including 2-640-hydroxyphenyl)-1,8-naphthalic anhydride
61), irenolone 62) and hydroxyanigorufone 63) from
unripe fruit of M. acuminata [AAA] cv. BunÄgulan and
M. balbisiana [BBB] cv. Saba sa Hapon, which were
wounded and then inoculated with conidia of C. musae.3±5
Quantitative analysis showed that these compounds were
not detected in the healthy tissues and accumulated after
wounding and/or inoculation, con®rming that they were
phytoalexins.6 On further analysis of extracts from the
fruit peel, we found six phenylphenalenone derivatives
64±9) as novel compounds. Among the six phenylphenal-
enones, compounds 5, 8 and 9 are optically active. In
addition, known phenylphenalenones from banana, 62)-
1,2-trans-2,3-cis-2,3-dihydro-1,2,3-trihydroxy-4-640-methoxy-
phenyl)phenalene 610), 61)-cis-2,3-dihydro-2,3-dihydroxy-
4-640-hydroxyphenyl)phenalene 611), 61)-cis-2,3-dihydro-
2,3-dihydroxy-4-640-methoxyphenyl)phenalene 612), 62)-
trans-2,3-dihydro-2,3-dihydroxy-9-640-hydroxyphenyl)-
phenalene 613) and 62)-trans-2,3-dihydro-2,3-dihydroxy-9-
phenylphenalene 614) are also optically active, but their
absolute con®gurations have not been elucidated.4,7±10
Determination of their con®gurations is needed to estimate
phenylphenalenone biosynthesis in banana fruit. This paper
describes isolation and identi®cation of the new compounds,
2. Results and discussion
Unripe fruit were wounded and then inoculated with a
suspension of conidia of C. musae. TLC analysis of the
extract from the peel revealed the presence of six unidenti-
®ed spots, and new compounds 4±9 corresponding to the
spots were isolated. Compound 4 showed signals of a 1,2,4-
substituted benzene, a 2,4- or 2,9-substituted phenalene-1-
Keywords: phytoalexins; phenalenes; ozonolysis; absolute con®guration.
Corresponding author. Address: Division of Applied Life Sciences,
p
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502,
Japan. Tel.: 181-75-753-6282; fax: 181-75-753-6284;
e-mail: hirai@kais.kyoto-u.ac.jp
1
one and three hydroxyl groups in the H NMR spectrum,
suggesting that 4 was 2-hydroxyphenalen-1-one substituted
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020601)00 749-9

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T. Kamo et al. / Tetrahedron 57 &2001) 7649±7656
Scheme 1. Determination of absolute con®gurations of compounds 9±14. 6i) CH2N2/MeOH; 6ii) NaBH4/EtOH 6room temperature); 6iii) Ac2O/pyridine;
6iv) 6a) O3/AcOH, 6b) H2O2/AcOH; 6v) NaBH4/MeOH 61008C).
at C-4 or C-9 with a 2,4- or 3,4-dihyroxyphenyl group.
Signals of three protons on the dihydroxyphenyl group
were observed at d 6.88 61H, dd, Jˆ8.1, 2.1 Hz), 7.02
61H, d, Jˆ2.1 Hz), and 7.03 61H, d, Jˆ8.1 Hz) ppm.
These chemical shifts indicated that the phenyl group had
two hydroxyl groups not at C-2 and -4 but at C-3 and -4,
since in a 2,4-dihydroxyphenyl group, a doublet signal of
3-H would have appeared at about d 6.4 ppm due to the
electron-donating effect of 2,4-dihydroxy groups.11 The
position of the dihydroxyphenyl group, C-4 or C-9, was
identi®ed by comparison of the relative intensity of the
[M]1 ion with that of the [M2H]1 ion in the EI mass
spectrum. 4-Phenylphenalenones give lower intensities of
[M2H]1 ions than [M]1 ions, whereas 9-phenylphenale-
nones give higher intensities of [M2H]1 ions than [M]1
ions due to ready formation of stable [M2H]1 ions by
bonding between the 1-oxygen and C-20 followed by
elimination of a hydrogen radical at C-20.4 Compound 4
gave lower intensity 616%) of the [M2H]1 ion at m/z 303
than that 6100%) of an [M]1 ion at m/z 304 in the mass
spectrum, indicating that the side chain was located at
C-4. These results showed that 4 was 4-630,40-dihydroxy-
phenyl)-2-hydroxyphenalen-1-one.
indicating that the latter signal was due to a hydroxyl proton.
The mass spectrum of 6 showed an [M]1 ion at m/z 292,
which was equal to the molecular weight of a 9- or 10-
dihydro derivative of 1. The doublet corresponding to 4-H
68.24 ppm) appeared in a magnetic ®eld lower than those
corresponding to 5-H 67.78 ppm) and 7-H 68.07 ppm) in the
1H NMR spectrum. This would be due to the electron-
attracting effect of the carbonyl group at C-10, indicating
that 6 is the 9-dihydro derivative of 1. Thus, compound 6
was identi®ed as 2-640-hydroxyphenyl)naphthal-8-formyl-1-
carboxylic anhydride. Compound 6 was an optically inactive
hemiacetal, indicating that it was a racemic mixture. Race-
mization could be achieved easily via the aldehyde form.
The 1H NMR spectrum of 7 was similar to that of 6, but the
doublets corresponding to 2-H 68.40ppm) and 4- H
68.33 ppm) appeared in a magnetic ®eld lower than that of
1
5-H 68.12 ppm) in the H NMR spectrum. The molecular
weight of 7 was the same as that of 6. These observations
indicated that 7 was the 10-dihydro derivative of 1, 7-640-
hydroxyphenyl)naphthal-8-formyl-1-carboxylic anhydride.
Compound 7 was also a racemic mixture. In banana fruit,
compounds 6 and 7 might be precursors of 1 since oxidation
of 6 and 7 at C-9 gave 1.
The 1H NMR spectrum of 5 was similar to that of 4, except
for the absence of a singlet assigned to 3-H and the presence
of two doublets at d 4.79 and 5.34 ppm 6Jˆ3.3 Hz). The
doublets were assigned to vicinal protons in a partial struc-
ture CX±CH6OH)±CH6OH)±CX0, in which X and X0 were
electron-inducting groups, suggesting that 5 was the 2,3-
hydrated derivative of 4. In its mass spectrum, compound
5 did not give a molecular ion, but gave an ion at m/z 304,
which would be a dehydrated ion formed by dehydration at C-
2 and C-3. The structure presumed was supported by an obser-
vation that 5 partly changed to 4 during puri®cation and
preservation. The relative con®guration between C-2 and C-
3 was deduced to be cis, due to the J value. These results
identi®ed 5 as 61)-2,3-cis-4-630,40-dihydroxyphenyl)-2,3-
dihydro-2,3-dihydroxyphenalen-1-one. The determination of
absolute con®gurations of C-2 and C-3 will be discussed later.
1
Compound 8 seemed to be an isomer of 13, since the H
NMR spectrum of 8 showed signals similar to that of 13
except for the J value between 2-H and 3-H. The J value,
3.7 Hz, suggested that 2-H and 3-H were cis in 8 but trans
6Jˆ10.0 Hz) in 13. In the mass spectrum of 8, an [M]1 ion
was observed at m/z 306, similarly to 13. These results
showed that 8 was 62)-cis-2,3-dihydro-2,3-dihydroxy-9-
640-hydroxyphenyl)phenalen-1-one.
The 1H NMR spectrum of 9 was similar to that of 10, except
for the absence of a singlet signal of a methoxy group at d
3.89 ppm, suggesting that 9 was the demethyl form of 10.7
The mass spectrum of 9 showed an [M]1 ion at m/z 308,
consistent with the molecular weight of the demethyl
form of 10. The relative con®gurations of 9 between C-1
and C-2, and C-2 and C-3 were deduced to be trans and
cis, respectively, by the J values 69.8 and 2.7 Hz)
observed, the same as 10. These results indicated that 9
was 62)-1,2-trans-2,3-cis-2,3-dihydro-1,2,3-trihydroxy-4-
640-hydroxyphenyl)-phenalene.
The 1H NMR spectrum of 6 was similar to that of 1, except
for the presence of two singlet signals 61H, respectively) at
d 6.89 and 7.02 ppm.3 The latter signal disappeared follow-
ing addition of deuterated water, while the former did not,

T. Kamo et al. / Tetrahedron 57 &2001) 7649±7656
7651
Scheme 2. A proposed pathway of phenylphenalenone biosynthesis in banana fruit. Compounds 18 and 24 have not been found in banana.
Elucidation of the absolute con®guration of 10 preceded that
of 9, since a larger amount of 10 than of 9 was available and
10 would possess the same absolute con®guration as 9. The
absolute con®gurations at C-1, C-2 and C-3 of 10 could be
determined by conversion of its C-9a±C-1±C-2±C-3±C-3a
moiety to arabitol. We attempted degradation of 10 to
arabitol by oxidative ozonolysis, but this was unsuccessful.
The C-1±C-9a and C-3±C-3a bonds of 10 seemed to be
cleaved with ®ssion of double bonds due to the presence
of electron-donating groups 6±OH) in the vicinity of double
bonds.12 Thus, 10 was derived to 62)-1,2-trans-2,3-cis-
1,2,3-triacetoxy-2,3-dihydro-4-640-methoxyphenyl)phena-
lene 615) to protect the C-1±C-9a and C-3±C-3a bonds from
ozonolysis 6Scheme 1).13 Compound 15 successfully gave
the dimethyl ester of tri-O-acetyl-l-arabinaric acid 616)
by oxidative ozonolysis and subsequent methylation.
Compound 16 was then reduced and acetylated, giving
arabitol pentaacetate 617) consisting of C-9a, C-1, C-2,
phenalen-1-one and 62S,3S)-61)-2,3-dihydro-2,3-dihydroxy-
4-640-methoxyphenyl)phenalen-1-one, respectively. Reduc-
tion of 13 gave 9 and its 3-epimer, revealing that 13 was
62R,3S)-61)-2,3-dihydro-2,3-dihydroxy-9-640-hydroxy-
phenyl)phenalen-1-one. Compound 14 was transformed to
62)-1,2-trans-2,3-cis-2,3-dihydro-1,2,3-trihydroxy-4-phenyl-
phenalene 618) and its 3-epimer by reduction. Acetylation of
18 gave 62)-1,2-trans-2,3-cis-1,2,3-triacetoxy-2,3-dihydro-
4-phenylphenalene 619), which was degraded to give 16.
These results identi®ed the absolute con®guration of the
1,2,3-trihydroxypropane moiety in 18 as 61S,2R,3S), indi-
cating that 14 was 62R,3S)-61)-2,3-dihydro-2,3-dihydroxy-
9-phenylphenalen-1-one. The absolute con®gurations of 5
and 8 were not elucidated due to the small amounts avail-
able for analysis. However, the [a]D25 value of 5ˆ1318 6c
0.03, MeOH), was similar to those of 11 and 12, suggesting
that the absolute con®guration of 5 was 62S,3S).
25
C-3 and C-3a of 10. The [a]D value of 17ˆ2348 6c
Compounds 4±9 were not detected in extracts of unripe
intact fruit peel on HPLC 6data not shown), indicating that
they were induced by wounding and subsequent inoculation,
similarly to other phenylphenalenone derivatives isolated
from banana fruit. No phenylphenalenones other than 1±3
and 2-phenyl-1,8-naphthalic anhydride 620) showed anti-
fungal activities at less than 10 mg on TLC assays.4,5
a-Hydroxyenone and 4-hydroxyphenyl moieties would be
essential for the activities of 4- and 9-phenylphenalenones,
and a naphthalic anhydride moiety would be needed for
those of 2-phenyl-1,8-naphthalic anhydrides. The differ-
ences in the moieties essential for the activity between
the phenylphenalenones and 2-phenyl-1,8-naphthalic
anhydrides may be due to the differences in their antifungal
mechanisms of action. In fact, 1,8-naphthalic anhydride
showed antifungal activity at 0.1 mg on a TLC plate,
suggesting that the lability of the anhydride group is
involved in the antifungal mechanism of 1 and 20.
0.07, CHCl3), was close to that of authentic l-arabitol
pentaacetate, 2318 6c 5.20, CHCl3), prepared from
l-arabitol. This result identi®ed the absolute con®gurations
at the C-1, C-2 and C-3 of 10 as S, R and S, respectively,
indicating that 10 was 61S,2R,3S)-62)-2,3-dihydro-1,2,3-
trihydroxy-4-640-methoxyphenyl)phenalene. The speci®c
optical rotation of 40-O-methyl ether of 9 was the same as
that of 10, and thus the absolute con®guration of 9 was
elucidated as 61S,2R,3S).
The absolute con®gurations of compounds 11 and 12, 13,
and 14 were determined by chemical conversion to 10, 9 and
16, respectively 6Scheme 1). Methylation of 11 gave 12, and
reduction of 12 afforded 10 and its 1-epimer, indicating that
the absolute con®guration at C-1 and C-2 of 11 and 12 was
62S,3S). Thus, compounds 11 and 12 were identi®ed as
62S,3S)-61)-2,3-dihydro-2,3-dihydroxy-4-640-hydroxyphenyl)-

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T. Kamo et al. / Tetrahedron 57 &2001) 7649±7656
Compounds 1±3 and 20 inhibited the spore germination of
C. musae by 50% at 3.5, 3.2, 2.9 and 3.1 ppm, respectively,
in aqueous solution. These strong activities suggested that
these compounds could be candidates for lead compounds
of fungicides.
spectrometers. Mass spectra were obtained with a Jeol
JMS-600H mass spectrometer. UV spectra were determined
with a Shimadzu UV-2200AI spectrometer. Optical rotation
was recorded with a Jasco DIP-1000 polarimeter. Column
chromatography was carried out on Wakogel C-200 6Wako
Pure Chemical Industries) and ODS-AM 120-S50 6YMC).
HPLC was performed with an ODS column 6YMC, AQ-
311, 6£100 mm) at a ¯ow rate 1.0 mL/min with detection
at UV 254 nm.
Absolute con®gurations of 9±14 suggested that these
compounds would be precursors or metabolites of each
other in the biosynthetic pathway of phenylphenalenones.
It has remained unknown how phenylphenalenones are
biosynthesized from or to 3, although biosynthetic pre-
cursors of 3 were shown to be two molecules of cinnamic
acid and one molecule of malonate.6 Luis et al. proposed a
putative pathway in which both 4- and 9-phenylphenal-
enones are biosynthesized from a common intermediate,
9.7 However, the accumulation of 3 in wounded or inocu-
lated unripe fruit did not accompany that of 4-phenylphenal-
enones such as 2, and preceded that of other 4- and
9-phenylphenalenones in unripe fruit wounded followed
by inoculation.6 These results led us to notice an alternative
biosynthetic pathway of phenylphenalenones, in which
4-phenylphenalenones are biosynthesized from 9-phenyl-
phenalenones via 9.6 Here, we discussed the alternative
pathway of phenylphenalenones including methyl ether
derivatives in detail based on the results reported so far
6Scheme 2). Compound 3 could give 2110 by oxidation at
C-3, while 3 would also give 1 by oxidative decarboxylation
as shown in our feeding experiment,6 and 224 by 2-O-
methylation. Compound 21 may be converted to 13 by
saturation at the C-2 double bond, probably giving 8 as a
by-product. Compound 239 could give 24, 20 and 25,9 and
also 14 and 269 from 24, respectively, similarly to 3. The
natural occurrence of phenalenones possessing a phenyl
group at C-4 in banana fruit has not been found,3±5,7±10
suggesting that 14 could not be reduced to 18, while 13 is
converted to 9. Compound 9 is probably oxidized to 11 and
then transformed to 2 by dehydration, since accumulation of
11 was observed prior to that of 2 in wounded and inocu-
lated unripe fruit.6 Compound 2 might be metabolized to 1
at a rate much lower than 3 to 1.6 The 9-substituents of
9-substituted phenalenones isolated from banana fruit are
phenyl or 40-hydroxyphenyl groups, while the 4-substituents
of 4-substituted phenalenones are 40-hydroxyphenyl or
40-methoxyphenyl groups.3±5,7±10 This suggests that ®rst
methylation of the 40-hydroxyl group occurs at 9 to give
10, and 10 might be transformed to 12, 278 and 28,5 the
same as 9 to 1. This pathway is not as simple as the Luis'
pathway, but agrees with the results of the changes in the
phenylphenalenone contents, the natural occurrence and the
feeding experiments.6 According to the pathway, 3 would be
a major phytoalexin for banana fruit, and 1 and 2 seem to be
minor phytoalexins which are formed by wounding
followed by inoculation.6 Structural diversity of phyto-
alexins might be one of the strategies by which plants
protect themselves from severe damage.
3.2. Plant materials and fungi
M. acuminata [AAA] cv. Bungulan cultivated on Negulos
Island, the Philippines, was used for the experiments.
Unripe fruits, which were not treated with fungicides,
were imported by Alter Trade Japan, Inc., Tokyo, Japan.
C. musae 6Berk. and Curt.) Arx. strains no. 1679 and 5501
were obtained from the Department of Scienti®c and
Industrial Research, Mount Albert Research Centre,
Auckland, New Zealand, and maintained on potato±
sucrose±agar medium in the dark at 238C. Suspensions of
conidia were prepared from 5- to 9-day-old cultures. 10mL
of sterile water was poured onto the surface of the media,
which was then rubbed with a glass bar. The suspension was
®ltered with paper 6Crecia Corp., Wipers S-200) and diluted
to 6.0£106 conidia/mL with sterile water.
Ä
3.3. Treatment and incubation
Unripe fruit were washed with water and wiped with 70%
EtOH prior to experiments. Fruit were then wounded by
rubbing with sandpaper 6G-60) and soaked in a suspension
of conidia of C. musae strain no. 1679 for 10sec. Treated
fruit were placed in plastic boxes 6width 24 cm£depth
31 cm£height 10cm) and kept in the dark at 17 8C for 4±9
days. Wet cotton was placed in the boxes to maintain high
humidity.
3.4. Isolation of compounds 4, 6, 7 and 9
The fruit peel 62.5 kg) was cut into 1£1 cm pieces after
incubation, and extracted with 3.6 L of EtOAc for 3 days
at room temperature. The EtOAc layer was concentrated to
500 mL, washed with H2O 6150mL £3), dried over Na2SO4,
and evaporated to dryness, yielding crude extract 65.2 g).
The extract was subjected to silica gel column chroma-
tography 6800 g) using mixtures of toluene±EtOAc as the
eluent to give the materials eluted with 50 and 100% EtOAc.
The 50% EtOAc eluate 60.2 g) was subjected to silica
gel column chromatography 625 g) using mixtures of
n-hexane±EtOAc±MeOH 618:2:1, 16:4:1, 10:10:1 and
0:2:1), each 80 mL, as the eluent. The eluate was collected
in fractions of 15 mL to give fractions no. 12±14. The
materials from fraction no. 12 were subjected to silica
gel column chromatography 620g) using mixtures of
n-hexane±EtOAc±MeOH 614:6:1, 12:8:1, and 10:10:1),
each 80, 40 and 50 mL, as the eluent. The eluate was
collected in fractions of 5 mL, and fractions no. 24 and 25
were puri®ed by preparative HPLC with a linear gradient,
MeOH±H2O 62:3 to 11:9), over 20min. The materials
eluted at tR 19.2 and 20.8 min were collected to give 7
60.2 mg) as a white powder and 4 60.2 mg) as an orange
powder, respectively. The materials from fractions no. 13
3. Experimental
3.1. General
1H and 13C NMR spectra were recorded with TMS as an
internal standard using Bruker AC300 and ARX500

T. Kamo et al. / Tetrahedron 57 &2001) 7649±7656
7653
and 14 obtained from the second silica gel column
chromatography were subjected to silica gel column
chromatography 65 g) using mixtures of n-hexane±
EtOAc±MeOH 614:6:1 and 12:8:1), each 20mL, as the
eluent. The eluate was collected in fractions of 3 mL, and
fraction no. 9 was puri®ed by preparative HPLC with
elution with MeOH±H2O 623:27). Collection of the material
eluted at tR 27.6 min yielded 6 60.3 mg) as a white powder.
The 100% EtOAc eluate 60.2 g) was subjected to silica
gel column chromatography 615 g) using mixtures of
n-hexane±EtOAc±MeOH 614:6:1, 12:8:1, 10:10:1 and
0:20:1), each 70 mL, as the eluent. The eluate was collected
in fractions of 30mL, and the materials from fractions no. 7
and 8 were puri®ed by preparative HPLC with elution with
MeOH±H2O 649:51), and collection of the material eluted at
tR 15.0min yielded 9 60.3 mg) as white powder.
by preparative HPLC with CH3CN±H2O 61:3) as the eluent.
The material eluted at tR 16.4 min was collected to give 5
60.3 mg) as a white powder. Fraction no. 9 was puri®ed by
preparative HPLC with CH3CN±H2O 61:3) as the eluent.
The material eluted at tR 21.6 min was collected to give 8
60.6 mg) as a white powder. Fractions no. 10±12 were again
puri®ed by ODS column chromatography 617 g) using a
mixture of CH3CN±H2O 63:7) as the eluent. The eluate
was collected in fractions of 15 mL, and fractions no. 9
and 10were concentrated to give 13 66.7 mg). Fourth, the
materials from fraction no. 11 were subjected to silica
gel column chromatography 625 g) using a mixture of
n-hexane±CHCl3±MeOH 611:9:1) as the eluent. The eluate
was collected in fractions of 30mL, and fraction no. 4 was
then subjected to ODS column chromatography 617 g) using
a mixture of MeOH±H2O 63:2) as the eluent. The eluate was
collected in fractions of 15 mL, and fractions no. 10and 11
were puri®ed by preparative HPLC with CH3CN±H2O
67:13) as the eluent. The material eluted at tR 12.6 min
was collected to give 10 63.1 mg) as colorless needles.
3.5. Isolation of compounds 5, 8 and 10±14
The fruit peel 63.3 kg) was extracted with 4 L of EtOAc,
yielding crude extract 66.0g) in the manner described
above. The extract was subjected to silica gel column
chromatography 6250g) using mixtures of toluene±EtOAc
69:1, 4:1, 1:1 and 0:1), each 750 mL, as the eluent. The
eluate was collected in fractions of 250mL to give fractions
no. 6, 7, 9±11. First, the materials from fractions no. 6 and 7
were subjected to silica gel column chromatography using
625 g) mixtures of n-hexane±CHCl3±MeOH 610:10:1) as
the eluent. The eluate was collected in fractions of 10mL,
and fractions no. 8±10were then subjected to ODS column
chromatography 617 g) using a mixture of MeOH±H2O
63:2) as the eluent. The eluate was collected in fractions
of 15 mL, and fractions no. 7±10were puri®ed by prepara-
tive HPLC with CH3CN±H2O 62:3) as the eluent. The
material eluted at tR 14.9 min was collected to give 12
630.4 mg). The materials from fraction no. 8 were subjected
to silica gel column chromatography 625 g) using mixtures
of n-hexane±CHCl3±MeOH 68:12:1) as the eluent. The
eluate was collected in fractions of 10mL, and fractions
no. 5±8 were then subjected to ODS column chroma-
tography 617 g) using a mixture of MeOH±H2O 63:2) as
the eluent. The eluate was collected in fractions of 5 mL,
and fractions no. 13±30were puri®ed by preparative HPLC
with CH3CN±H2O 62:3) as the eluent. The material eluted at
tR 12.8 min was collected to give 14 68.0mg) as a white
powder. Second, the materials from fraction no. 9 were
subjected to silica gel column chromatography 625 g)
using mixtures of n-hexane±CHCl3±MeOH 68:12:1) as
the eluent. The eluate was collected in fractions of 10mL,
and fractions no. 17±21 were then subjected to ODS column
chromatography 617 g) using a mixture of CH3CN±H2O
67:13) as the eluent. The eluate was collected in fractions
of 10mL, and fractions no. 9 and 10 were puri®ed by
preparative HPLC with CH3CN±H2O 68:17) as the eluent.
The material eluted at tR 11.4 min was collected to give 11
611.5 mg) as a white powder. Third, the materials from
fraction no. 10were subjected to silica gel column chroma-
tography 625 g) using a mixture of n-hexane±CHCl3±
MeOH 65:15:1) as the eluent. The eluate was collected in
fractions of 50mL, and fraction no. 4 was then subjected to
ODS column chromatography 617 g) using a mixture of
MeOH±H2O 63:2) as the eluent. The eluate was collected
in fractions of 8 mL, and fractions no. 7 and 8 were puri®ed
3.5.1. 4--30,40-Dihydroxyphenyl)-2-hydroxyphenalen-1-
one -4). UV lmax 6MeOH) nm 61): 243 613,000), 283
611,000), 328 64000), 338 64000), 440 64300); EIMS
6probe) 70eV m/z 6%): 304 [M]1 6100), 303 [M2H]1
616), 287 649), 286 650); HREIMS m/z 6M1): calculated
for C19H12O4, 304.0735; found 304.0731; NMR dH
6300 MHz, acetone-d6): 6.88 61H, dd, Jˆ8.1, 2.1 Hz,
60-H), 7.02 61H, d, Jˆ2.1 Hz, 20-H), 7.03 61H, d, Jˆ
8.1 Hz, 50-H), 7.29 61H, s, 3-H), 7.62 61H, d, Jˆ8.5 Hz,
5-H), 7.89 61H, dd, Jˆ8.0, 7.4 Hz, 8-H), 8.08 61H, d, Jˆ
8.5 Hz, 6-H), 8.09 61H, s, 2,30 or 40-OH), 8.25 61H, s, 2,30or
40-OH), 8.26 61H, s, 2,30 or 40-OH), 8.43 61H, dd, Jˆ8.0,
1.2 Hz, 7-H), 8.69 61H, dd, Jˆ7.4, 1.2 Hz, 9-H).
3.5.2. -1)-2,3-cis-4--30,40-Dihydroxyphenyl)-2,3-dihydro-
2,3-dihydroxyphenalen-1-one -5). UV lmax 6MeOH) nm
61): 268 623,000), 282 613,000); EIMS 6probe) 70 eV m/z
6%): 304 [M2H2O]1 6100), 303 [M2H2O±H]1 615), 287
647), 286 644), 258 620); HREIMS m/z 6M2H2O1): calcu-
lated for C19H12O4, 304.0735; found 304.0724; NMR dH
6500 MHz, acetone-d6): 4.79 61H, d, Jˆ3.3 Hz, 2-H), 5.34
0
61H, d, Jˆ3.3 Hz, 3-H), 6.94 61H, d, Jˆ8.0Hz, 5 -H), 7.01
61H, dd, Jˆ8.0, 2.0 Hz, 60-H), 7.18 61H, d, Jˆ2.0Hz, 2 -H),
0
7.61 61H, d, Jˆ8.5 Hz, 5-H), 7.68 61H, dd, Jˆ8.1, 7.0Hz,
8-H), 8.04 61H, d, Jˆ8.5 Hz, 6-H), 8.13 61H, dd, Jˆ7.0,
1.1 Hz, 9-H), 8.69 61H, dd, Jˆ8.1, 1.1 Hz, 7-H).
3.5.3. 2--40-Hydroxyphenyl)naphthal-8-formyl-1-carb-
oxylic anhydride -6). UV lmax 6MeOH) nm 61): 227
619,000), 245 614,000), 290 65300), 303 64700), 332
64100); EIMS 6probe) 70 eV m/z 6%): 292 [M]1 6100),
275 [M2OH]1 611), 264 663), 247 668); HREIMS m/z
6M1): calculated for C18H12O4, 292.0735; found 292.0742;
NMR dH 6300 MHz, acetone-d6): 6.89 61H, s, 9-H), 6.89
62H, d, Jˆ8.6 Hz, 30,50-H), 7.02 61H, brs, 9-OH), 7.30
62H, d, Jˆ8.6 Hz, 20,60-H), 7.59 61H, d, Jˆ8.5 Hz, 3-H),
7.67 61H, dd, Jˆ8.2, 6.5 Hz, 6-H), 7.78 61H, dd, Jˆ6.5,
1.0Hz, 5- H), 8.07 61H, dd, Jˆ8.2, 1.0Hz, 7- H), 8.24 61H,
d, Jˆ8.5 Hz, 4-H), 8.47 61H, brs, 40-OH).
3.5.4. 7--40-Hydroxyphenyl)naphthal-8-formyl-1-carb-
oxylic anhydride -7). UV lmax 6MeOH) nm 61): 228

7654
T. Kamo et al. / Tetrahedron 57 &2001) 7649±7656
617,000), 245 611,000), 274 66700), 308 63300), 319
63400), 340 62800); EIMS 6probe) 70 eV m/z 6%): 292
[M]1 6100), 275 [M2OH]1 637), 264 658), 247 663);
HREIMS m/z 6M1): calculated for C18H12O4, 292.0735;
found 292.0732; NMR dH 6acetone-d6, 300 MHz): 6.67
61H, s, 9-H), 6.99 62H, d, Jˆ8.7 Hz, 30,50-H), 7.05 61H, brs,
1-OH), 7.49 62H, d, Jˆ8.7 Hz, 20,60-H), 7.61 61H, d,
Jˆ8.5 Hz, 6-H), 7.76 61H, dd, Jˆ8.3, 7.2 Hz, 3-H), 8.12
61H, d, Jˆ8.5 Hz, 5-H), 8.33 61H, dd, Jˆ8.3, 1.1 Hz, 4-H),
8.4061H, dd, Jˆ7.2, 1.1 Hz, 2-H), 8.63 61H, brs, 40-OH).
30,50-H), 7.54 62H, d, Jˆ8.6 Hz, 20,60-H), 7.61 61H, d, Jˆ
8.5 Hz, 5-H), 147.67 61H, dd, Jˆ8.1, 7.1 Hz, 8-H), 8.05
61H, d, Jˆ8.5 Hz, 6-H), 8.13 61H, d, Jˆ7.1 Hz, 9-H), 8.26
61H, d, Jˆ8.1 Hz, 7-H), 8.55 61H, brs, 40-OH); NMR dC
6125 MHz, acetone-d6): 71.8 6C-3), 78.2 6C-2), 116.4
6C-30,50), 126.6 6C-9), 126.9 6C-8), 129.5 6C-6), 130.1
0
6C-9a), 131.06C-5), 131.4 6C-3a), 132.1 6C-2 ,60), 132.2
6C-9b), 132.5 6C-10), 134.06C-6a), 135.4 6C-7), 142.7
6C-4), 158.5 6C-40), 199.5 6C-1). See the literature4 for the
other spectral data; the assignment of signals in the 1H NMR
spectrum reported in the literature were partially in error.
Ethereal diazomethane was added to 11 65.0mg) dissolved
in 2 mL of MeOH, and the solution was left at room
temperature for 2 h. The solvent was evaporated, and the
residue was subjected to ODS column chromatography
617 g) using a mixture of MeOH±H2O 63:2) as the eluent.
The eluate was collected in fractions of 15 mL, and fractions
no. 9 and 10were evaporated to give 12 61.7 mg).
3.5.5. -2)-2,3-cis-2,3-Dihydro-2,3-dihydroxy-9--40-hydroxy-
25
phenyl)phenalen-1-one -8). [a]D ˆ21068 6c 0.06,
MeOH); UV lmax 6MeOH) nm 61): 246 623,000), 287
69800), 320 66100); EIMS 6probe) 70 eV m/z 6%): 306
[M]1 66), 288 [M2H2O]1 659), 287 [M2H2O±H]1 6100),
271 620); HREIMS m/z 6M1): calculated for C19H14O4,
306.0892; found 306.0869; NMR dH 6500 MHz, acetone-
d6): 4.28 61H, d, Jˆ4.0Hz, 2-O H), 4.47 61H, d, Jˆ3.0Hz,
3-OH), 4.91 61H, dd, Jˆ4.0, 3.7 Hz, 2-H), 5.24 61H, dd,
Jˆ3.7, 3.0Hz, 3- H), 6.87 62H, d, Jˆ8.6 Hz, 30,50-H), 7.20
62H, d, Jˆ8.6 Hz, 20,60-H), 7.51 61H, d, Jˆ8.4 Hz, 8-H),
7.63 61H, dd, Jˆ8.2, 7.0Hz, 5- H), 7.74 61H, d, Jˆ7.0Hz,
4-H), 8.03 61H, d, Jˆ8.2 Hz, 6-H), 8.18 61H, d, Jˆ8.4 Hz,
7-H), 8.38 61H, s, 40-OH).
25
1
[a]D ˆ1658 6c 0.17, CHCl3); the H NMR spectrum was
identical to that of natural 12.
3.5.9. -2S,3S)--1)-2,3-Dihydro-2,3-dihydroxy-4--40-meth-
25
oxyphenyl)phenalen-1-one -12). [a]D ˆ1658 6c 0.70,
CHCl3); NMR dH 6500 MHz, CDCl3): 2.96 61H, s, 3-OH),
3.88 63H, s, 40-OCH3), 4.23 61H, s, 2-OH), 4.72 61H, d,
Jˆ3.1 Hz, 2-H), 5.3061H, d, Jˆ3.1 Hz, 3-H), 7.02 62H, d,
Jˆ8.7 Hz, 30,50-H), 7.57 61H, d, Jˆ8.6 Hz, 5-H), 7.6062H,
d, Jˆ8.7 Hz, 20,60-H), 7.63 61H, dd, Jˆ8.0, 7.1 Hz, 8-H),
7.96 61H, d, Jˆ8.6 Hz, 6-H), 8.15 61H, d, Jˆ8.0Hz, 7- H),
8.23 61H, d, Jˆ7.1 Hz, 9-H); NMR dC 6125 MHz, CDCl3):
55.4 6OCH3), 69.9 6C-3), 76.2 6C-2), 113.9 6C-30,50), 125.8
6C-8), 126.6 6C-9), 127.4 6C-6a), 127.9 6C-3a), 128.9 6C-6),
129.9 6C-5), 130.7 6C-20,60), 131.06C-9b), 132.06C-1 0),
132.7 6C-9a), 135.06C-7), 142.6 6C-4), 159.5 6C-4 0),
198.2 6C-1). See the literature4,9 for the other spectral
3.5.6. -1S,2R,3S)--2)-2,3-Dihydro-1,2,3-trihydroxy-4--40-
25
hydroxyphenyl)phenalene -9). [a]D ˆ258 6c 0.49,
MeOH); UV lmax 6MeOH) nm 61): 231 624,000), 259
615,000), 290 66800); EIMS 6probe) 70 eV m/z 6%): 308
[M]1 646), 290[M 2H2O]1 653), 273 6100), 272 693);
HREIMS m/z 6M1): calculated for C19H16O4, 308.1048;
found 308.1041; NMR dH 6300 MHz, acetone-d6): 2.46
61H, s, 1-OH), 2.54 61H, d, 3-OH), 2.82 61H, d, 2-OH),
3.85 61H, dd, Jˆ9.7, 3.2 Hz, 2-H), 5.02 61H, d, Jˆ3.2 Hz,
3-H), 5.28 61H, d, Jˆ9.7 Hz, 1-H), 6.94 62H, d, Jˆ8.7 Hz,
30,50-H), 7.47 61H, d, Jˆ8.1 Hz, 5-H), 7.49 62H, d, Jˆ
8.7 Hz, 20,60-H), 7.58 61H, dd, Jˆ8.5, 7.6 Hz, 8-H), 7.84
61H, d, Jˆ8.1 Hz, 6-H), 7.84 61H, d, Jˆ7.6 Hz, 7-H), 7.90
61H, d, Jˆ8.5 Hz, 9-H); NMR dC 6125 MHz, acetone-d6):
70.0 6C-3), 70.5 6C-1), 76.1 6C-2), 116.1 6C-30,50), 124.3
6C-9), 126.8 6C-8), 127.8 6C-7), 129.3 6C-6), 129.5 6C-9b),
129.9 6C-5), 132.4 6C-20,60), 132.4 6C-3a), 133.4 6C-10), 133.7
6C-6a), 139.3 6C-9a), 141.4 6C-4), 158.1 6C-40). Ethereal
diazomethane was added to 9 64.9 mg) dissolved in 2 mL of
MeOH, and the solution was left at room temperature for 2 h.
The solvent was evaporated, and the residue was subjected to
ODS column chromatography 617 g) using a mixture of
MeOH±H2O 63:2) as the eluent. The eluate was collected in
fractions of 15 mL, and fractions no. 8±10were evaporated to
1
data; the assignment of signals in the H NMR spectrum
reported in the literature were partially in error. Compound
12 639 mg) in 12 mL of EtOH was stirred at room tempera-
ture for 2 h after addition of NaBH4 620mg). The solution
was acidi®ed to pH 3 with 1N HCl, and then extracted with
EtOAc 615 mL£3). The organic layer was washed with H2O
610mL £3), dried over Na2SO4, ®ltered and evaporated to
dryness. The residue was subjected to ODS column
chromatography 617 g) using a mixture of MeOH±H2O
63:2) as the eluent. The eluate was collected in fractions
of 15 mL, and fractions no. 9±12 and 14±17 were evapo-
rated to give 10 623.1 mg) and 1-epimer of 10 68.4 mg),
25
respectively. Compound 10. [a]D ˆ2358 6c 2.31,
25
1
CHCl3); the H NMR spectrum was identical to that of
natural 10. 1-Epimer of 10. [a]D ˆ21218 6c 0.84,
25
give 10 63.9 mg). [a]D ˆ 2348 6c 0.39, CHCl3); the 1H NMR
CHCl3); NMR dH 6500 MHz, CDCl3): 3.50±3.70 63H, brs,
1,2, and 3-OH), 4.08 61H, dd, Jˆ2.1, 2.1 Hz, 2-H), 5.05 61H,
d, Jˆ2.1 Hz, 1 or 3-H), 5.08 61H, d, Jˆ2.1 Hz, 1 or 3-H),
6.97 62H, d, Jˆ8.7 Hz, 30,50-H), 7.47 61H, d, Jˆ8.4 Hz,
5-H), 7.48 62H, d, Jˆ8.7 Hz, 20,60-H), 7.52 61H, dd, Jˆ
8.0, 6.7 Hz, 8-H), 7.6061H, d, Jˆ6.7 Hz, 7-H), 7.88 61H,
d, Jˆ8.0Hz, 9- H), 7.88 61H, d, Jˆ8.4 Hz, 6-H).
spectrum was identical to that of natural 10.
3.5.7.
-1S,2R,3S)--2)-2,3-Dihydro-1,2,3-trihydroxy-4-
-40-methoxyphenyl)phenalene -10). Mp 92±968C;
[a]D ˆ2358 6c 0.31, CHCl3). See the literature7 for the
25
other spectral data.
3.5.8.
-2S,3S)--1)-2,3-Dihydro-2,3-dihydroxy-4--40-
3.5.10.
-2R,3S)--2)-2,3-Dihydro-2,3-dihydroxy-9--40-
25
25
hydroxyphenyl)phenalen-1-one -11). [a]D ˆ1568 6c
1.15, MeOH); NMR dH 6500 MHz, acetone-d6): 4.5061H,
brs, 2-OH), 4.58 61H, brs, 3-OH), 4.79 61H, d, Jˆ3.1 Hz,
2-H), 5.28 61H, d, Jˆ3.1 Hz, 3-H), 6.98 62H, d, Jˆ8.6 Hz,
hydroxyphenyl)phenalen-1-one -13). [a]D ˆ21378 6c
0.56, MeOH). See the literature10 for the other spectral
data. Compound 13 65.5 mg) in 2 mL of EtOH was stirred
at room temperature for 2 h after addition of NaBH4 62 mg).

T. Kamo et al. / Tetrahedron 57 &2001) 7649±7656
7655
The solution was acidi®ed to pH 3 with 1N HCl, and then
extracted with EtOAc 65 mL£3). The organic layer was
washed with H2O 63 mL£3), dried over Na2SO4, ®ltered
and evaporated to dryness. The residue was subjected to
ODS column chromatography 617 g) using a mixture of
MeOH±H2O 611:9) as the eluent. The eluate was collected
in fractions of 15 mL. Fractions no. 4±7 were evaporated
and then puri®ed by preparative HPLC with MeOH±H2O
61:1) as the eluent. The materials eluted at tR 11.6 min and tR
14.4 min were collected to give 9 60.6 mg) and 3-epimer of
the solution, 0.5 mL of 30% hydrogen peroxide was then
added, and the mixture was stirred overnight at room
temperature and for 1.5 h at 1008C. The solution was
concentrated, dissolved in MeOH 61 mL), and then ethereal
diazomethane was added. The solvent was evaporated, and
the residue was subjected to silica gel column chroma-
tography 63 g) using a mixture of n-hexane±EtOAc 63:1)
as the eluent. The eluate was collected in fractions of
5 mL, and fractions no. 5 and 6 were puri®ed by preparative
HPLC with MeOH±H2O 62:3) as the eluent. The material
eluted at tR 11.2 min was collected to give 16 as a colorless
25
9 63.7 mg), respectively. Compound 9. [a]D ˆ268 6c 0.06,
25
1
MeOH); the H NMR spectrum was identical to that of
25
oil 60.5 mg). [a]D ˆ2318 6c 0.05, CHCl3); EIMS 6probe)
natural 9. 3-Epimer of 9. [a]D ˆ1578 6c 0.37, MeOH);
70eV m/z 6%): 303 [M2OCH3]1 66), 275 [M2CO2CH3]1
630), 261 633), 203 650), 173 6100); HREIMS m/z
6M2CO2CH31): calculated for C11H15O8, 275.0767; found
275.0764; NMR dH 6500 MHz, CDCl3): 2.08 63H, s,
OCOCH3), 2.13 63H, s, OCOCH3), 2.19 63H, s, OCOCH3),
3.74 63H, s, COOCH3), 3.75 63H, s, COOCH3), 5.13 61H, d,
Jˆ8.6 Hz, 4-H), 5.48 61H, d, Jˆ 2.8 Hz, 2-H), 5.77 61H, dd,
Jˆ8.6, 2.8 Hz, 3-H); NMR dC 6125 MHz, CDCl3): 20.3±20.5
6COCH3£3), 53.1 6CO2CH3), 53.2 6CO2CH3), 68.9 6C-3),
69.4 6C-2 or 4), 69.6 6C-2 or 4), 167.06 CO2CH3), 167.1
6CO2CH3), 169.3±169.8 6COCH3£3).
NMR dH 6500 MHz, acetone-d6): 4.44 61H, dd, Jˆ2.5,
1.8 Hz, 2-H), 4.95 61H, d, Jˆ1.8 Hz, 1 or 3-H), 4.98 61H,
d, Jˆ2.5 Hz, 1 or 3-H), 6.93 62H, d, Jˆ8.7 Hz, 30,50-H),
7.46 61H, d, Jˆ8.5 Hz, 5-H), 7.5061H, dd, Jˆ8.2, 7.0Hz,
8-H), 7.51 62H, d, Jˆ8.7 Hz, 20,60-H), 7.57 61H, d, Jˆ
7.0Hz, 7- H), 7.87 61H, d, Jˆ8.2 Hz, 9-H), 7.89 61H, d,
Jˆ8.5 Hz, 6-H), 8.39 61H, brs, 40-OH).
3.5.11. -2R,3S)--2)-2,3-Dihydro-2,3-dihydroxy-9-phenyl-
25
phenalen-1-one -14). [a]D ˆ21018 6c 0.80, CHCl3);
NMR dC 6CDCl3, 125 MHz): 74.4 6C-3), 79.3 6C-2),
125.06C-6a), 125.06C-4), 127.06C-5), 127.6 6C-4
0),
3.5.14. l-Arabitol pentaacetate -17). NaBH4 614 mg) was
added to MeOH 60.5 mL) solution of 16 62.2 mg), and the
mixture was stirred for 2 h at 1008C. H2O was added to the
solution cooled to room temperature, which was then
acidi®ed to pH 3 with 1N HCl, and concentrated to dryness.
The residual white powder was partly dissolved in EtOH
65 mL), and the EtOH layer was concentrated to dryness,
dissolved in pyridine 61 mL), and stirred at room temperature
for 18 h after adding acetic anhydride 60.5 mL). The mixture
was fractionated between EtOAc and H2O after stopping the
reaction by adding H2O. The organic layer was dried over
Na2SO4, ®ltered, and concentrated to dryness. The residue
was subjected to silica gel column chromatography 61.5 g)
using a mixture of n-hexane±EtOAc 63:1) as the eluent. The
eluate was collected in fractions of 2 mL, and fractions no. 6±
10were evaporated to give 17 60.7 mg) as a colorless oil.
127.9 6C-6), 128.06C-3 ,50), 128.7 6C-20,60), 129.8 6C-8),
130.0 6C-3a), 132.4 6C-9a), 133.6 6C-7), 133.8 6C-9b), 140.8
6C-10), 142.8 6C-9), 197.8 6C-1). See the literature11 for the
other spectral data.
0
3.5.12. -1S,2R,3S)--1)-1,2,3-Triacetoxy-2,3-dihydro-4-
-40-methoxyphenyl)phenalene -15). Compound 10
615.5 mg) was dissolved in pyridine 60.5 mL), and stirred
at room temperature for 48 h after addition of acetic
anhydride 60.3 mL). The mixture was fractionated between
EtOAc and H2O, and the organic layer 620mL) was washed
with H2O 610mL £3), dried over Na2SO4, ®ltered and
concentrated to dryness, giving 15 620.6 mg) as a yellow
25
oil. [a]D ˆ11418 6c 0.42, CHCl3); UV lmax 6MeOH) nm
61): 230 666,000), 243 618,000), 287 611,000), 302 67600);
EIMS 6probe) 70eV m/z 6%): 448 [M]1 621), 345 618), 329
635), 303 618), 287 6100); HREIMS m/z 6M1): calculated
for C26H24O7, 448.1522; found 448.1520; NMR dH
6CDCl3, 500 MHz): 1.96 63H, s, 3-OCOCH3), 2.00 63H, s,
1-OCOCH3), 2.25 63H, s, 2-OCOCH3), 3.85 63H, s,
40-OCH3), 5.42 61H, dd, Jˆ10.2, 3.0 Hz, 2-H), 6.57 61H,
d, Jˆ3.0Hz, 3- H), 6.75 61H, d, Jˆ10.2 Hz, 1-H), 6.96 62H,
d, Jˆ8.5 Hz, 30,50-H), 7.2062H, d, Jˆ8.5 Hz, 20,60-H), 7.41
61H, d, Jˆ7.2 Hz, 9-H), 7.44 61H, d, Jˆ8.4 Hz, 5-H), 7.54
61H, dd, Jˆ8.1, 7.2 Hz, 8-H), 7.87 61H, d, Jˆ8.1 Hz, 7-H),
7.91 61H, d, Jˆ8.4 Hz, 6-H); NMR dC 6CDCl3, 125 MHz):
20.8±21.1 6±OCOCH3£3), 55.3 6OCH3-40), 67.7 6C-3),
69.1 6C-1), 72.3 6C-2), 113.8 6C-30,50), 123.9 6C-9), 125.8
6C-8), 126.2 6C-3a), 128.1 6C-7), 128.3 6C-9b), 129.3 6C-6),
25
[a]D ˆ2348 6c 0.07, CHCl3); EIMS 6probe) 70eV m/z 6%):
289 [M2CH2OAc]1 632), 217 [M2C2H36OAc)2]1 664), 187
677), 145 [M2C3H46OAc)3]1 689), 115 6100); HREIMS m/z
6M2CH2OAc1): calculated for C12H17O8, 289.0923; found
289.0923; NMR dH 6500 MHz, CDCl3): 2.04±2.13 615H,
s£5, COCH3£5), 3.95 61H, dd, Jˆ11.7, 7.0Hz, 1 or 5- H),
4.15 61H, dd, Jˆ12.5, 5.0Hz, 1 or 5- H), 4.24 61H, dd, Jˆ
12.5, 2.7 Hz, 1 or 5-H), 4.28 61H, dd, Jˆ11.7, 5.0Hz, 1 or
5-H), 5.15±5.42 63H, m, 2-4-H); NMR dC 6125 MHz,
CDCl3): 20.5±20.8 6COCH3£5), 61.8 6C-1 or 5), 62.06C-1
or 5), 68.1 6C-2), 68.1 6C-4), 68.4 6C-3), 169.6±170.6
6COCH3£5). l-Arabitol 620mg; Aldrich) was dissolved in
pyridine 62 mL), and stirred for 24 h at room temperature
after adding acetic anhydride 61 mL). H2O was added to the
mixture, and followed by fractionation between EtOAc and
H2O. The organic layer 620mL) was washed with H 2O
610mL £3), dried over Na2SO4, ®ltered and concentrated to
dryness, giving l-arabitol pentaacetate as a colorless oil
648 mg).
0
129.3 6C-5), 130.3 6C-20,60), 131.6 6C-9a), 132.06C-1 ),
132.4 6C-6a), 141.2 6C-4), 159.3 6C-40), 169.2±171.0
6±OCOCH3£3).
3.5.13. Dimethyl ester of tri-O-acetyl-l-arabinaric acid
-16). Compound 15 620.4 mg) was dissolved in AcOH
63 mL), and O3 gas from an ozone generator 6Japan Ozone
Co. Ltd, Model O-3-2) was passed through the solution for
24 h at room temperature. After N2 gas was passed through
3.5.15. -1R,2S,3R)--2)-2,3-Dihydro-1,2,3-trihydroxy-4-
phenylphenalene -18). Compound 14 627.5 mg) in 6 mL
of EtOH was stirred at room temperature for 1.5 h after

7656
T. Kamo et al. / Tetrahedron 57 &2001) 7649±7656
addition of NaBH4 610mg). The solution was acidi®ed to
pH 3 with 1N HCl, and then extracted with EtOAc 620mL).
The organic layer was washed with H2O 610mL £3), dried
over Na2SO4, ®ltered and evaporated to dryness. The
residue was subjected to ODS column chromatography
617 g) using a mixture of MeOH±H2O 63:2) as the eluent.
The eluate was collected in fractions of 15 mL, and fraction
no. 9 was evaporated to give 18 66.0mg) as colorless
needles and 3-epimer of 18 615.1 mg) as a white powder.
3.6. Antifungal assay on TLC
EtOAc solutions containing 0.01±10 mg of test compound
were spotted 65 mm in diameter) onto silica gel TLC plates
6Merck, Silica gel 60F 254). Conidia of C. musae strain no.
5501 were suspended in a Czapek-Dox medium at a density
of 6.0£106 conidia/mL, and sprayed onto the TLC plates to
completely wet the surface. The thin layers were incubated
in a moist chamber at 238C for 2 days in the dark. Zones
of antifungal activity were detected as ¯at spots lacking
mycelia.
25
Compound 18. Mp 173±1778C; [a]D ˆ248 6c 0.60,
CHCl3); UV lmax 6EtOH) nm 61): 232 648,000), 243
650,000), 278 69100), 290 611,000), 302 67300); EIMS
6probe) 70eV m/z 6%): 292 [M]1 649), 274 [M2H2O]1
642), 257 693), 256 6100), 228 627), 215 632), 202 624);
HREIMS m/z 6M1): calculated for C19H16O3, 292.1099;
found 292.1105; NMR dH 6500 MHz, CDCl3): 2.79 61H,
brs, OH), 3.05 61H, brs, OH), 3.33 61H, brs, OH), 3.75
61H, dd, Jˆ9.7, 2.8 Hz, 2-H), 4.94 61H, d, Jˆ2.8 Hz,
3-H), 5.22 61H, d, Jˆ9.7 Hz, 1-H), 7.31±7.52 65H, m,
20,30,40,50,60-H), 7.44 61H, d, Jˆ8.4 Hz, 5-H), 7.51 61H,
dd, Jˆ8.2, 7.3 Hz, 8-H), 7.56 61H, d, Jˆ7.3 Hz, 9-H),
7.79 61H, d, Jˆ8.2 Hz, 7-H), 7.87 61H, d, Jˆ8.4 Hz, 6-H);
NMR dC 6125 MHz, CDCl3): 68.2 6C-3), 700.1 6C-2), 74.9
6C-2), 123.06C-7), 126.1 6C-8), 127.4 6C-4 ), 127.5 6C-9),
127.5 6C-9b), 128.1 6C-20,60), 128.6 6C-5), 128.8 6C-6),
129.7 6C-30,50), 129.8 6C-3a), 132.7 6C-6a), 135.7 6C-9a),
3.7. Antifungal assay in aqueous solution
Conidia of C. musae strain no. 1679 were suspended in
sterile H2O at a density of 1.0£104 conidia/mL. A DMSO
solution 625 mL) of test compound and Tween 2062.5 mL)
were added to 5 mL of the conidia-suspension at ®nal
concentrations of 0.3±30 ppm. Tissue culture ¯asks
6Iwaki, 75 cm2, canted neck) containing the suspensions
were placed at 238C in the dark. After 24 h, 100 conidia
were observed under a microscope 6Olympus, IM). The
inhibition ratio was de®ned as [6A2B)/A]£100, where
Aˆthe number of conidia that germinated when H2O
containing 0.05% Tween 20 and 0.5% DMSO was used,
and Bˆthe number of conidia that germinated when test
compound was used. The number A was 62±77.
25
140.4 6C-10), 140.7 6C-4). 3-Epimer of 18. [a]D ˆ1358 6c
0.75, CHCl3); NMR dH 6500 MHz, CDCl3): 2.11 61H, d,
Jˆ5.4 Hz, 1, 2, or 3-OH), 2.86 61H, d, Jˆ5.4 Hz, 1, 2, or
3-OH), 3.19 61H, d, Jˆ5.4 Hz, 1, 2, or 3-OH), 4.28 61H,
ddd, Jˆ5.6, 5.4, 5.4 Hz, 2-H), 5.03 61H, dd, Jˆ5.6, 5.4 Hz,
1 or 3-H), 5.23 61H, dd, Jˆ5.4, 5.4 Hz, 1 or 3-H), 7.41±7.55
65H, m, 20-60-H), 7.47 61H, d, Jˆ8.4 Hz, 5-H), 7.57 61H, dd,
Jˆ8.4, 7.0Hz, 8- H), 7.73 61H, d, Jˆ7.0Hz, 6- H), 7.87 61H,
d, Jˆ8.4 Hz, 7-H), 7.9061H, d, Jˆ8.4 Hz, 9-H).
References
1. Muirhead, I. F.; Deverall, B. J. Physiol. Plant Pathol. 1981,
19, 77±84.
2. Brown, A. E.; Swinburne, T. R. Phytopath. Z. 1980, 99, 70 ±
80.
3. Hirai, N.; Ishida, H.; Ohigashi, H. Phytochemistry 1994, 37,
383±385.
3.5.16. -1R,2S,3R)--1)-1,2,3-Triacetoxy-2,3-dihydro-4-
phenylphenalene -19). Compound 18 65.9 mg) was
dissolved in pyridine 60.5 mL), and stirred at room tempera-
ture for 48 h after addition of acetic anhydride 60.3 mL).
The mixture was concentrated to dryness, giving 19
4. Kamo, T.; Kato, N.; Hirai, N.; Tsuda, M.; Fujioka, D.;
Ohigashi, H. Biosci. Biotechnol. Biochem. 1998, 62, 95±101.
5. Kamo, T.; Kato, N.; Hirai, N.; Tsuda, M.; Fujioka, D.;
Ohigashi, H. Phytochemistry 1998, 49, 1617±1621.
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Biosci. Biotechnol. Biochem. 2000, 64, 2089±2098.
7. Luis, J. G.; Fletcher, W. Q.; Echeverri, F.; Grillo, T. A.;
25
68.5 mg) as a colorless oil. [a]D ˆ11728 6c 0.85,
CHCl3); UV lmax 6EtOH) nm 61): 229 661,000), 278
68400), 288 69400), 301 66300); EIMS 6probe) 70 eV m/z
6%): 418 [M]1 65), 359 [M2OAc]1 64), 358 614), 315 625),
299 623), 273 623), 257 6100); HREIMS m/z 6M1):
calculated for C25H22O6, 418.1416; found 418.1419; NMR
dH 6500 MHz, CDCl3): 1.93 63H, s, 3-OCOCH3), 1.98 63H,
s, 2-OCOCH3), 2.25 63H, s, 1-OCOCH3), 5.44 61H, dd,
Jˆ10.1, 3.1 Hz, 2-H), 6.53 61H, d, Jˆ3.1 Hz, 3-H), 6.72
61H, d, Jˆ10.1 Hz, 1-H), 7.24±7.45 65H, m, 20,30,40,50,60-
H), 7.42 61H, d, Jˆ7.3 Hz, 9-H), 7.47 61H, d, Jˆ8.4 Hz,
5-H), 7.56 61H, dd, Jˆ8.2, 7.3 Hz, 8-H), 7.88 61H, d, Jˆ
8.2 Hz, 7-H), 7.94 61H, d, Jˆ8.4 Hz, 6-H); NMR dC 6125
MHz, CDCl3): 20.7±21.1 6±OCOCH3£3), 67.4 6C-3), 69.1
6C-1), 72.1 6C-2), 124.06C-9b), 126.06C-8), 126.2 6C-3a),
127.8 6C-7), 128.2 6C-9), 128.2 6C-40), 128.3 6C-20,60),
128.8 6C-6), 129.1 6C-30,50), 129.3 6C-5), 131.7 6C-9a),
132.6 6C-6a), 139.8 6C-10), 141.5 6C-4), 169.1±171.06±
OCOCH3£3). Ozonolysis of 19 68.5 mg) gave 16 60.3 mg)
in the same manner described above for preparation of 16
Â
Perales, A.; Gonzalez, J. A. Tetrahedron 1995, 51, 4117±
4130.
8. Luis, J. G.; Fletcher, W. Q.; Echeverri, F.; Grillo, T. A.
Tetrahedron 1994, 50, 10963±10970.
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M. P.; Perales, A. Nat. Prod. Lett. 1995, 6, 23±30.
10. Luis, J. G.; QuinÄones, W.; Echeverri, F.; Grillo, T. A.; Kishi,
M. P.; Garcia-Garcia, F.; Torres, F.; Cardona, G. Phyto-
chemistry 1996, 41, 753±757.
11. Jackman, L. M.; Sternhell, S. Application of Nuclear Magnetic
Resonance Spectroscopy in Organic Chemistry; 2nd ed.;
Pergamon: Oxford, 1969; pp 201±204.
12. Young, W. G.; McKinnis, A. C.; Irving, D. W.; Roberts, J. D.
J. Am. Chem. Soc. 1946, 68, 293±296.
13. Aoki, K.; Kato, M.; Suzuki, M.; Hayakawa, Y.; Nakamura, H.;
Yamada, K.; Hirata, Y. J. Chem. Soc., Chem. Commun. 1973,
618±619.
from 15. [a]D ˆ2318 6c 0.03, CHCl3); the 1H NMR spec-
25
trum was identical to that prepared from 15.