A benzofuran glycoside and an acetylenic acid from the fungus Laetiporus sulphureus var. miniatus.

Abstract

A new benzofuran glycoside, masutakeside I (1) and a new C10 acetylenic acid, masutakic acid A (2) were isolated from the fruiting bodies of the fungus Laetiporus sulphureus var. miniatus. Their structures were established by spectroscopic and chemical methods. The known compounds 3-6 were also obtained and identified as egonol, demethoxyegonol, egonol glucoside and egonol gentiobioside. Some of these compounds exhibited cytotoxicity against Kato III cells.

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As a part of a research program aimed at the discovery of
biologically active compounds from fungi, we have previ-
ously reported the isolation and structure elucidation of five
lanostane triterpenoids, named versisponic acids A—E and
seven lanostanol glycosides, designated as laetiposides A—G
from the fruit bodies of Laetiporus versisporus (LLOYD) IMAZ.
(Polyporaceae).1,2)We have recently initiated a chemical
study of Laetiporus sulphureus (FR.) MURR. var. miniatus
(JUNGH.) IMAZ. (Polyporaceae), which grows on the needle-
leaf tree and is distributed throughout Japan.3)An earlier
chemical constituent study of this fungus resulted in the iso-
lation of a ceramide and ergosterol.4)The EtOH extract of the
fresh fruiting bodies of the fungus was separated by re-
versed-phase HPLC with 40—70% MeOH to give two new
compounds, masutakeside I (1) and masutakic acid A (2), in
addition to five known compounds. The structures of the
known compounds, egonol (3), demethoxyegonol (4), egonol
glucoside (5), egonol gentiobioside (6),5,6)and 2-(3,4-dihy-
droxyphenyl)-2,3-dihydro-7-hydroxy-3-hydroxymethyl-5-
benzofuranpropanol,7)were determined by a combination of
spectroscopic analysis and comparison with reported data.
Masutakeside I (1) was obtained as an amorphous powder.
The molecular formula was established as C30H36O14based
on the molecular ion at m/z 643.1970 [M?Na]?in the high-
resolution (HR)-FAB-MS, requiring 13 degrees of unsatura-
tion. The IR absorption maxima at 3395, 1605 and 1520
cm?1and the absorbance at 210, 233 and 278nm in the UV
spectrum suggested the presence of aromatic rings. The 13C-
NMR and distortionless enhancement by polarization trans-
fer (DEPT) spectrum revealed signals for all 30 carbons,
consisting of one methoxyl, eight sp2quaternary carbons, six
sp2methines, four sp3methylenes (two oxygenated), and 11
signals due to a pentose unit and a hexose unit (Table 1). The
1H-NMR and
COSY) spectra of 1 exhibited signals for a propanol group at
d 4.25 (1H, m), 3.70 (1H, m), 2.86 (2H, m) and 2.04 (2H,
m), two meta-coupled doublets at d 7.11 (1H, d, J?1.2Hz)
and 6.83 (1H, d, J?1.2Hz), an ABX-type signal set at d 7.56
(1H, d, J?1.6Hz), 7.54 (1H, dd, J?8.0, 1.6Hz), and 6.94
(1H, d, J?8.0Hz), and an independent olefinic proton at d
7.16 (1H, s). An isolated methylene signal at d 6.00 (2H, s),
which correlated with the carbon signal at d 102.0 in the 1H-
1H–1H correlation spectroscopy (1H–1H
detected multiple quantum coherrence spectrum (HMQC),
was suggestive of the presence of a methylenedioxy moiety
in 1. The units described above were connected by the 1H-de-
tected heteronuclear multiple bond correlation spectrum
(HMBC) and by rotating frame nuclear Overhauser and ex-
change spectroscopy (ROESY). Thus, H2-8 (dH2.86) showed
in the HMBC spectrum a two-bond connectivity with C-5
(dC138.5), and three-bond connectivities with C-4 (dC
113.1) and C-6 (dC108.4). Further, H-4 (dH7.11) showed
long-range correlation to C-3a (dC131.6) and C-7a (dC
143.0). In turn, the C-2 (dC156.2), C-3a and C-7a carbons
displayed HMBC correlation with H-3 (dH7.16). A nuclear
Overhauser effect (NOE) interaction was observed between
H-6 (dH6.83) and the methoxyl group (dH3.95). Hence, a
benzofuran unit having a propanol at C-5 and a methoxyl
group at C-7 was evident. The long-range correlation from
H2-7? (dH6.00) to C-3? (dC148.7) and C-4? (dC148.5) fur-
ther confirmed the involvement of C-3? and C-4? in a methyl-
enedioxy ether linkage. Key NOE interactions between H-3
(dH7.16) and H-2? (dH7.56) and H-6? (dH7.54) led to the
assignment of the aglycone moiety of 1, 5-(3-hydroxy-
propyl)-7-methoxy-2-(3,4-methylenedioxyphenyl) benzofu-
ran [egonol (3)].5,6)Acid hydrolysis of 1 afforded 3, besides
D-glucose and D-xylose confirmed by specific rotation using
chiral detection in HPLC analysis.8)Furthermore, the cou-
pling constants observed for the anomeric protons at d 5.02
(1H, d, J?7.4Hz) and d 4.82 (1H, d, J?8.0Hz) in the 1H-
NMR spectrum of 1 indicated a b-glycosidic linkage for both
the glucopyranose and xylopyranose units. The sugar se-
quence was established by the glycosylation shift rule,9)and
March 2001 Notes
Chem. Pharm. Bull. 49(3) 327—329 (2001)327
∗ To whom correspondence should be addressed. e-mail: yosikawa@ph.bunri-u.ac.jp © 2001 Pharmaceutical Society of Japan
A Benzofuran Glycoside and an Acetylenic Acid from the Fungus
Laetiporus sulphureus var. miniatus
Kazuko YOSHIKAWA,*,aShinya BANDO,aShigenobu ARIHARA,aEiko MATSUMURA,band
Satoshi KATAYAMAb
Faculty of Pharmaceutical Sciences, Tokushima Bunri University,aYamashiro-Cho, Tokushima 770–8514, Japan and
Osaka University of Pharmaceutical Sciences,b4–20–1 Nasahara, Takatsuki, Osaka 569–1041, Japan.
Received October 23, 2000; accepted December 5, 2000
A new benzofuran glycoside, masutakeside I (1) and a new C10acetylenic acid, masutakic acid A (2) were
isolated from the fruiting bodies of the fungus Laetiporus sulphureus var. miniatus. Their structures were estab-
lished by spectroscopic and chemical methods. The known compounds 3—6 were also obtained and identified as
egonol, demethoxyegonol, egonol glucoside and egonol gentiobioside. Some of these compounds exhibited cyto-
toxicity against Kato III cells.
Key words
Laetiporus sulphureus var. miniatus; masutakeside I; masutakic acid A; Polyporaceae
Chart 1

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HMBC experiments. The downfield-shifted 13C-NMR reso-
nance observed at d 70.0 among the sugar units could be as-
signed to C-6 of glucose. Further, long-range correlations
were observed between H-1 (dH4.82) of glucose and C-10
(dC68.9) of the aglycon, and H-1 (dH5.02) of xylose and C-
6 (dC70.0) of glucose, indicating the presence of a pri-
meverose moiety (–Glc–
as egonol primeveroside.
Masutakic acid A (2) was obtained as an amorphous pow-
der, and the molecular formula was established as C10H16O4
on the basis of HR-CI-MS [m/z 201.1149 (M?H)?, D?2.2
mmu]. The nmaxat 2220cm?1in the IR spectrum of 2 was
consistent with the presence of a triple bond. This was sup-
ported by the DEPT spectrum, which revealed the presence
of one methyl group, four methylenes, two oxymethines [dC
76.6 (d), and dC65.6 (d)], one acetylenic functionality [dC
85.6 (s), and dC80.8 (s)], in addition to a carboxyl group [dC
175.0 (s)]. The 1H-NMR and COSY spectra showed the pres-
ence of a 1,2-diol group at d 5.50 (1H, dd, J?4.4, 1.6Hz),
5.04 (1H, d, J?4.4Hz), and a pentyl group (H2-6—H3-10).
HMBC correlations between H-2 (dH5.04) and C-1 (dC
175.0), and C-4 (dC80.8), and between H2-6 (dH2.11) and
C-5 (dC85.6), C-4 (dC80.8), and C-7 (dC28.6) indicated
that the acetylene group could be placed between C-3 and C-
6. Accordingly, 2 was formulated as 2,3-dihydroxy-dec-4-yn-
oic acid. To the best of our knowledge, this is only the sec-
ond report of the isolation of an acetylenic compound con-
6Xyl). Accordingly, 1 was formulated
taining a C¤C–CH(OH)–CH(OH)–COOH unit from a fun-
gal source.10)
Compounds 1 and 3—6 were tested for cytotoxic activities
against Kato III cells.11,12)Compounds 3, 4 and 5 showed in
vitro cytotoxicity with IC50values of 28.8, 27.5, and 24.9
mg/ml, respectively (positive control, hinokitiol 0.6mg/ml).
Experimental
Optical rotations were taken on a JASCO DIP-1000 polarimeter. IR and
UV spectra were recorded on JASCO FT-IR 5300 and Hitachi U-3000 spec-
trometers, respectively. NMR spectra were recorded on a Varian UNITY 600
spectrometer in C5D5N solution. NMR experiments included 1H–1H COSY,
DEPT, HMQC, HMBC, and ROESY. Coupling constants (J values) are
given in Hertz (Hz). HR-FAB-MS and HR-CI-MS were measured on a
JEOL AX-500 mass spectrometer. Kieselgel 60 (230—400 mesh, Merck)
was used for column chromatography, and silica gel 60F-254 (Merck) for
TLC.
Plant Material
The fruiting bodies of Laetiporus sulphureus var.
miniatus were collected in Tokushima prefecture, in autumn 1998. The
voucher specimen (TB3019) is deposited in the Herbarium of the Faculty of
Pharmaceutical Sciences, Tokushima Bunri University.
Extraction and Isolation
The fresh fruiting bodies (0.55kg) were ex-
tracted with 70% EtOH at room temperature for 6 weeks. The ethanolic ex-
tract (57.5g) was partitioned between H2O and EtOAc. The water layer was
passed through an Amberlite XAD-2 column. After the column was washed
with water, the adsorbed materials were eluted with 100% MeOH. The
MeOH eluate (5.0g) was chromatographed on HPLC (Develosil Lop ODS,
40—100% MeOH) to give 20 fractions. Fractions 13 and 14 were subjected
to HPLC (YMC, ODS S-5, 37—40% CH3OH) to give egonol gentiobioside
(6, 7.4mg) and masutakic acid A (2, 7mg), respectively. Fractions 17, 18
and 25 were purified by preparative HPLC (YMC, ODS S-5, 58—60%
CH3OH) to afford egonol glucoside (5, 10mg) from fr. 17, masutakeside I
(1, 40mg) from fr. 18, and demethoxyegonol (4, 10mg) and 2-(3,4-dihy-
droxyphenyl)-2,3-dihydro-7-hydroxy-3-hydroxymethyl-5-benzofuranpropanol
(10mg) from fr. 25. Fraction 26 was subjected to HPLC (YMC, ODS S-5,
72% CH3OH) to give egonol (3, 7.4mg).
Masutakeside I (1): An amorphous powder, [a]D
MeOH). UV (MeOH) lmax(loge) 210 (4.49), 233 (4.46), 278 (4.17) nm. FT-
IR (film) nmax3395, 1605, 1520cm?1. FAB-MS m/z: 643 [M?Na]?, 659
[M?K]?. HR-FAB-MS m/z: 643.1970 (Calcd for M??Na, C30H36O14Na:
643.2003).
Masutakic Acid A (2): An amorphous powder, [a]D
MeOH). FT-IR (film) lmax3346, 2220, 1728, 1105cm?1. CI-MS m/z: 201
[M?H]?, 183 [M?H?H2O]?. HR-CI-MS m/z: 201.1149 (Calcd for
M??H, C10H17O4: 201.1127).
Acid Hydrolysis of 1
A solution of compound 1 (10mg) in 5%
H2SO4–dioxane (1:1) was heated at 100°C for 2h. The reaction mixture
was diluted with H2O, and then neutralized with Amberlite IRA-35 and
evaporated in vacuo to dryness. The reaction mixture was diluted with H2O,
and extracted with EtOAc. The EtOAc layer was purified by column chro-
matography on silica gel by elution with CH2Cl2–MeOH (30:1) to afford 3
(3mg), which was confirmed by 1H-NMR data and by co-HPLC (ODS, 65%
MeOH) with an authentic sample. The identification of and the D or L con-
figurations of the sugars were determined using refraction index (RI) detec-
tion (Waters 410) and chiral detection (Shodex OR-1) by HPLC {Shodex
RSpak NH2P-50 4E column, CH3CN–H2O–H3PO4(95:5:1), 1ml/min,
47°C} by comparison with authentic sugar (10mmol of D-Glc and D-Xyl).
The sugar portion from compound 1 gave peaks for D-(?)-Xyl at 9.10min
and D-(?)-Glc at 20.7min.
25?19.9° (c?2.6,
25?13.2° (c?0.7,
Acknowledgment We are grateful to Mr. T. Ohashi, mycologist,
Tokushima Prefectural Tomioka Nishi High School for confirming the iden-
tity of the fungus.
References
1)Yoshikawa K., Matsumoto K., Shigenobu A., J. Nat. Prod., 62, 543—
545 (1999).
2) Yoshikawa K., Matsumoto K., Mine C., Bando S., Shigenobu A.,
Chem. Pharm. Bull., 48, 1418—1421 (2000).
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328 Vol. 49, No. 3
Table 1.
1H- and 13C-NMR Data for Compounds 1 and 2 in Pyridine-d5
12
Position Position
13C
1H, mult (J inHz)
13C
1H, mult (J inHz)
2
3
3a
4
5
6
7
7a
8
9
156.2 s
101.4 d
131.6 s
113.1 d
138.5 s
108.4 d
145.4 s
143.0 s
32.8 t
32.5 t
68.9 t
1
2
3
4
5
6
7
8
9
175.0 s
76.6 d
65.6 d
80.8 s
85.6 s
19.0 t
28.6 t
31.1 t
22.3 t
14.0 q
7.16 s 5.04 (d, 4.4)
5.50 (dd, 4.4,1.6)
7.11 (d, 1.2)
6.83 (d, 1.2)2.11 (dt, 1.6, 7.2)
1.34 (quint, 7.2)
1.21 (quint, 7.2)
1.08 (sext, 7.2)
1.21 (t, 7.2)
2.86 m
2.04 m
3.70 m
4.25 m
10
10
1?
2?
3?
4?
5?
6?
7?
125.2 s
105.8 d
148.7 s
148.5 s
109.1 d
119.5 d
102.0 t
56.1 q
104.8 d
75.3 d
79.1 d
72.6 d
77.2 d
70.0 t
7.56 (d, 1.6)
6.94 (d, 8.0)
7.54 (dd, 8.0, 1.6)
6.00 s
3.95 s
4.82 (d, 8.0)
4.05 (dd, 8.8, 8.0)
4.22 (dd, 9.4, 8.8)
4.24 (dd, 9.4, 8.5)
4.09 (ddd, 8.5, 5.5, 1.9)
4.31 (dd, 11.4, 5.5)
4.86 (dd, 11.4, 1.9)
5.02 (d, 7.4)
4.06 (dd, 8.7, 7.4)
4.16 (dd, 8.7, 8.5)
4.24 (ddd, 9.7, 8.5, 2.7)
3.69 (dd, 11.3, 9.7)
4.35 (dd, 11.3, 2.7)
MeO
Glc 1
2
3
4
5
6
Xyl 1 106.1 d
75.3 d
79.1 d
72.6 d
67.2 t
2
3
4
5

Page 3

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