Synthesis and biological activity of 4′,8-dihydroxyisoflavon-7-yl D-hexopyranosides

Article abstract of DOI:10.1016/S0008-6215(01)00246-4

Four 4′,8-dihydroxyisoflavon-7-yl hexopyranoside derivatives having an aglycon part of A-76202 were synthesized, and their biological activities were evaluated toward rat liver α-glucosidase. However, the activities were disappointing.

Full text of DOI:10.1016/S0008-6215(01)00246-4

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Carbohydrate Research 335 (2001) 283–289
Note
Synthesis and biological activity of
4%,8-dihydroxyisoflavon-7-yl
D-hexopyranosides
Yukiko Watanabe, Masao Shiozaki,* Reiko Kamegai
Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd., Hiromachi 1-2-58, Shinagawa-ku,
Tokyo 140-8710, Japan
Received 24 May 2001; accepted 30 August 2001
Abstract
Four 4%,8-dihydroxyisoflavon-7-yl hexopyranoside derivatives having an aglycon part of A-76202 were synthesized,
and their biological activities were evaluated toward rat liver a-glucosidase. However, the activities were disappoint-
ing. © 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Glycosidation; Isoflavone; Stereoselective synthesis
Many phenolic isoflavone glycoconjugates
are known in nature, and some of them have
been synthesized. Recently, A-76202 (4%,8-di-
hydroxyisoflavon-7-yl a-D-arabinofuranoside)
was isolated from Rhodococcus sp. SANK
61694,1 and synthesized.2 A-76202 is a strong
inhibitor of a-glucosidase I and II existing in
endoplasmic reticulum, and it also participates
in the processing of secretory, cell membrane,
and virus surface-glycoproteins. Although A-
76202 has strong inhibitory activity for these
enzymes in vitro, its activity is very weak in
vivo. This is presumed difficult for A-76202 to
penetrate through the cell membrane. We
were interested in 4%,8-dihydroxyisoflavon-7-yl
sugar derivatives having the exact aglycon of
A-76202 to overcome this defect. Therefore,
we tried to synthesize 4%,8-dihydroxyisoflavon-
7-yl sugar derivatives such as hexopyranosides
instead of the arabinofuranoside in order to
examine their activities toward a-glucosidase
(Fig. 1).
First, we attempted a synthetic procedure
reported in the synthesis of A-76202 for the
glycosidation reaction of 2,3,4,6-tetra-O-ace-
tyl-a-
D
-glucopyranosyl bromide (1)3 with 4%,8-
diallyloxy-7-hydroxyisoflavone
(2).2
The
reaction of bromide 1 with the lithium salt of
2 prepared in THF by addition of a hexane
solution of BuLi at room temperature was
carried out under various conditions. How-
ever, no glycosidation product was obtained
as only the phenolic isoflavone 2 was recov-
ered. Second, a mixture of bromide 1 and
phenolic isoflavone 2 was treated with 1.2
Fig. 1. A-76202.
* Corresponding author. Tel.: +81-3-34913131; fax: +81-
3-54368570.
E-mail address: shioza@shina.sankyo.co.jp (M. Shiozaki).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00246-4

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Y. Watanabe et al. / Carbohydrate Research 335 (2001) 283–289
Scheme 1. Reagents and conditions: (a) Ag2CO3, pyridine, rt, 18 h, 13%; (b) RhCl3, EtOH, reflux, 1.5 h, 73%; (c) ca. KOH, EtOH,
rt, 4 h, 100%.
equivalents silver trifluoromethanesulfonate,
anomeric form mainly due to the 2-OBz
neighboring group effect. Deprotection of the
two allyl groups of 11 by treatment with
rhodium trichloride gave diphenol 12 in 67%
yield. Debenzoylation of 12 with 0.2 M NaOH
in MeOH gave 14, which was contaminated
with unidentified byproducts. Therefore, the
mixture was acetylated with Ac2O in pyridine
to yield 13 in two steps 60% yield after chro-
matographic purification. Deacetylation of 13
with a catalytic amount of KOH in EtOH
gave 14 in 97% yield (Scheme 3).
1.2 equivalents of bis(cyclopentadienyl)-
,
hafnium dichloride and an excess of 4 A
molecular sieves in dichloromethane at 0–
24 °C under nitrogen according to Suzuki’s
method.4 However, this reaction also did not
proceed. Finally, a glycosidation reaction of 2
with 1 using silver carbonate in pyridine5 was
carried out to yield b-
yield), the recovered 2, and 2,3,4,6-tetra-O-
acetyl- -glucopyranose derived from 1. Al-
D
-glucoside 3 (13%
D
though the yield of this glycosidation reaction
was low, the reaction exclusively produced the
1,2-trans b anomer mainly due to the 2-OAc
neighboring group effect. Deprotection of the
two allyl groups of 3 by treatment with
rhodium trichloride gave diphenol 4 in 73%
yield. Deacetylation of 4 with a catalytic
amount of KOH in EtOH quantitatively
yielded 5 (Scheme 1).
On addition, glycosidation of 2 with 2,3,4,6-
tetra-O-benzoyl-a-
D
-allopyranosyl
bromide
(15), prepared from 1,2,3,4,6-penta-O-ben-
zoyl-b-D-allopyranose according to the proce-
dure described in the formation of
2,3,4,6-tetra-O-acetyl-a-D-allopyranosyl bro-
mide,8 by the same procedure used for the
formation of 3 from 1 and 2 using silver
And also, glycosidation of 2 with 2,3,4,6-
carbonate in pyridine, yielded b- -allopyran-
D
tetra-O-acetyl-a- -galactopyranosyl bromide
D
oside 16 (22% yield), producing only the b
anomeric form also mainly due to the 2-OBz
neighboring group effect. Deprotection of the
two allyl groups of 16 by treatment with
(6),6 by the same procedure as for the forma-
tion of 3 from 1 and 2 using silver carbonate
in pyridine, yielded b-
D
-galactoside 7 (55%
yield), producing only the 1,2-trans
b
anomeric form. This should be also due to the
2-OAc neighboring group effect. Deprotection
of the two allyl groups of 7 by treatment with
rhodium trichloride gave diphenol 8 in 62%
yield. Deacetylation of 8 with a catalytic
amount of KOH in EtOH quantitatively
yielded 9 (Scheme 2).
In addition, glycosidation of 2 with 2,3,4,6-
tetra-O-benzoyl-a-
D
-mannopyranosyl
bro-
mide (10),7 by the same procedure used for the
formation of 3 from 1 and 2 using silver
Scheme 2. Reagents and conditions: (a) 2, Ag2CO3, pyridine,
rt, 18 h, 55%; (b) RhCl3, EtOH, reflux, 1.5 h, 62%; (c) ca.
KOH, EtOH, rt, 4 h, 100%.
carbonate in pyridine, yielded a-
11 (31% yield), exclusively producing the a
D
-mannoside

Y. Watanabe et al. / Carbohydrate Research 335 (2001) 283–289
285
Fig. 2. Inhibition toward rat liver-a-D-glucosidase.
Scheme 3. Reagents and conditions: (a) 2, Ag2CO3, pyridine,
rt, 18 h, 31%; (b) RhCl3, EtOH, reflux, 1.5 h, 67%; (c) (1) 0.2
M NaOH in MeOH, rt, 18 h; (2) Ac2O, pyridine, rt, 1 h, 2
steps 60%; (d) ca. KOH, EtOH, rt, 4 h, 100%.
Fig. 3. Inhibition toward rat liver-a-D-glucosidase.
mg/mL and 5.6 ng/mL, respectively. These
four compounds synthesized and natural A-
76202 have inhibitory activities as shown in
Figs. 2 and 3; however, the activities of these
compounds are much weaker than that of
A-76202. Compound 14, which has an a-man-
nose moiety as the glycan part, showed the
most effective inhibitory activity among these
four compounds except A-76202. Both A-
76202 and 14 have the a-oriented aglycon part
on arabinose and mannose, respectively. This
may reveal that an a-oriented sugar-substrate
may be mimicked as the part recognized by
this a-glucosidase as a matter of course. Ac-
cordingly, compounds 5, 9, and 19 having
b-oriented aglycon moieties may be less active
than 14. Among compounds 5, 9, and 19,
compound 5 was more active than the others.
This may explain that the glucose moiety of 5
may be recognized as a better binding site
Scheme 4. Reagents and conditions: (a) 2, Ag2CO3, pyridine,
rt, 18 h, 22%; (b) RhCl3, EtOH, reflux, 1.5 h, 80%; (c) (1) 0.2
M NaOH in MeOH, rt, 18 h, (2) Ac2O, pyridine, rt, 1 h, 2
steps 72%; (d) ca. KOH, EtOH, rt, 4 h, 100%.
rhodium trichloride gave diphenol 17 in 80%
yield. Debenzoylation of 17 with 0.2 M NaOH
in MeOH gave 19, which was contaminated
with unknown substances. Therefore, the mix-
ture was also acetylated with Ac2O in pyridine
to give 18 in two steps after chromatographic
purification (72% yield). Deacetylation of 18
with a catalytic amount of KOH in EtOH
quantitatively gave 19 (Scheme 4).
The biological activities (IC50 values) to-
ward rat liver microsome a-glucosidase of
compounds 5 (b-D-glucopyranoside), 9 (b-D-
galactopyranoside), 14 (a-D-mannopyran-
oside), 19 (b-D-allopyranoside) and A-76202
were 14.3, \100 mg/mL, 103.1 ng/mL, \100

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Y. Watanabe et al. / Carbohydrate Research 335 (2001) 283–289
stirred for 18 h at rt, filtered, and concentrated
in vacuo to give a mixture. This mixture was
diluted with EtOAc, washed with aq 0.1 M
HCl, water, satd aq NaHCO3, and brine,
dried over MgSO4, filtered, concentrated in
vacuo, and chromatographed on a silica gel
column. Elution with 1:1 hexane–EtOAc gave
3 (175 mg, 13%) as a gum. [h]2D4 −17.3° (c
0.79, CHCl3). IR wmax(CHCl3) 1758, 1646,
1607, 1511 cm−1. 1H NMR (CDCl3): l 2.05 (3
H, s), 2.07 (3 H, s), 2.09 (3 H, s), 2.10 (3 H, s),
3.92 (1 H, m), 4.20 (1 H, dd, J 2.2, 12.5 Hz),
4.31 (1 H, dd, J 5.1, 12.5 Hz), 4.57 (1 H, ddt,
J 1.5, 5.1, 11.7 Hz), 4.58 (2 H, dt, J 1.5, 5.1
Hz), 4.67 (1 H, ddt, J 1.5, 5.1, 11.7 Hz),
5.17–5.46 (8 H, m, containing 1 H, d, J 7.3
Hz, at 5.18 ppm), 6.03–6.16 (2 H, m), 6.99 (2
H, d, J 8.8 Hz), 8.00 (1 H, d, J 8.8 Hz), 8.01
(1 H, s). FABMS (positive-ion): m/z 703
[M+Na]+, 681 [M+H]+. HRFABMS,
Calcd for C35H37O14: 681.2183; Found:
681.2180.
than those of either the galactose or allose
moiety by this a-glucosidase. Nonetheless, the
activities of these four compounds were
disappointing.
Thus, 4%,8-dihydroxyisoflavon-7-yl
D
-gly-
cosides (5, 9, 14 and 19), which were
synthesized by the glycosidation reaction of
glycosyl bromides (1, 6, 10 and 15) and
4%,8-diallyloxy-7-hydroxyisoflavone (2) using
silver carbonate in pyridine and successive
deprotection of the hydroxy groups, were less
active than A-76202 toward rat liver
microsome a- -glucosidase.
D
1. Experimental
Melting points were determined on a
Yanagimoto micro melting-point apparatus
and are uncorrected. Optical rotations were
obtained by the use of a JASCO P-1030 polar-
1
imeter. H NMR (400 MHz) spectra were
recorded with JEOL JNM-GSX 400 spec-
trometers using tetramethylsilane as the inter-
nal standard. IR absorption spectra were
determined with an IR A-2 spectrophotome-
ter, and mass spectra were obtained with a
JMS-700 mass spectrometer. Elemental analy-
ses were performed by the Institute of Science
and Technology, Inc., Tokyo. Separation of
the compounds by column chromatography
was carried out with Silica Gel 60 (230–400
mesh ASTM, E. Merck) under a slightly ele-
vated pressure (1.2–1.5 atm) for rapid elution.
The quantity of silica gel used was 50–100
times the weight of material charged on the
column. Detection involved spraying the chro-
matogram with a solution of 17% H2SO4 in
water (w/w) containing ammonium molybdate
(2.3%) and cerium(IV) sulfate (0.9%), and
heating the plate for several minutes at ca.
180 °C. THF was distilled from sodium–ben-
zophenone ketyl. DMF and pyridine were
4%,8-Dihydroxyisofla6on-7-yl 2,3,4,6-tetra-
O-acetyl-i-D-glucopyranoside (4).—A solu-
tion of 3 (67.5 mg, 0.099 mmol) in anhyd
EtOH (3 mL) containing RhCl3 hydrate (13.1
mg, 0.050 mmol) was refluxed for 1.5 h under
N2. The mixture was concentrated in vacuo to
give a residue, which was chromatographed
on a silica gel column. Elution with 3:7 hex-
ane–EtOAc gave 4 (43.5 mg, 73%). [h]D24
−25.4° (c 0.84, CHCl3). IR wmax(CHCl3) 3595,
3482, 1758, 1613, 1579 cm−1
.
1H NMR
(CDCl3): l 2.07 (6 H, s), 2.12 (3 H, s), 2.14 (3
H, s), 3.91 (1 H, ddd, J 2.2, 5.1, 10.3 Hz), 4.21
(1 H, dd, J 2.2, 12.5 Hz), 4.33 (1 H, dd, J 5.9,
12.5 Hz), 5.09 (1 H, d, J 7.3 Hz), 5.19 (1 H, t,
J 9.5 Hz), 5.29–5.38 (2 H, m), 5.88 (1 H, s,
OH), 6.44 (1 H, s, OH), 6.87 (2 H, d, J 8.8
Hz), 7.07 (1 H, d, J 8.8 Hz), 7.39 (2 H, d, J 8.8
Hz), 7.80 (1 H, d, J 8.8 Hz), 8.02 (1 H, s).
FABMS (positive-ion): m/z 601 [M+H]+.
HRFABMS, Calcd for C29H29O14: 601.1557;
Found: 601.1559.
,
dried by storage over 4 A molecular sieves.
4%,8-Diallyloxyisofla6on-7-yl 2,3,4,6-tetra-
O-acetyl-i-D-glucopyranoside (3).—To a solu-
tion of 4%,8-diallyloxy-7-hydroxyisoflavone (2)
(700 mg, 2.00 mmol) and 2,3,4,6-tetra-O-ace-
tyl-a-D-glucopyranosyl bromide (1) (1.30 g,
3.16 mmol) in pyridine (40 mL) was added
Ag2CO3 (2.00 g, 7.25 mmol). The mixture was
4%,8-Dihydroxyisofla6on-7-yl
i-D-glucopy-
ranoside (5).—A solution of 4 (22.8 mg, 0.038
mmol) in EtOH (3 mL) containing KOH (4
mg) was stirred for 4 h at rt. The solution was
concentrated in vacuo to give 5 (20.4 mg,
100%) as a yellow powder; mp 238.5–
241.0 °C. [h]2D4 −55.9° (c 0.55, MeOH). IR

Y. Watanabe et al. / Carbohydrate Research 335 (2001) 283–289
287
−52.4° (c 0.43, MeOH). IR wmax(KBr) 3394
wmax(KBr) 3201 (broad), 1666, 1595, 1559
1
(broad), 1558 cm−1. H NMR (CD3OD): l
1
cm−1. H NMR (CD3OD): l 3.40–3.58 (4 H,
3.59 (1 H, m), 3.70 (1 H, m), 3.76–3.91 (4 H,
m), 4.74 (1 H, d, J 7.3 Hz), 6.72 (2 H, d, J 8.8
Hz), 7.16–7.25 (4 H, m), 8.17 (1 H, s).
FABMS (positive-ion): m/z 433 [M+H]+.
HRFABMS, Calcd for C21H21O10: 433.1135;
Found: 433.1153.
m), 3.73 (1 H, dd, J 5.1, 11.7 Hz), 3.93 (1 H,
dd, J 2.2, 10.3 Hz), 4.80 (1 H, d, J 7.3 Hz),
6.69 (2 H, d, J 8.8 Hz), 7.16–7.22 (4 H, m),
8.16 (1 H, s). FABMS (positive-ion): m/z 433
[M+H]+. FABMS (negative-ion): m/z 431
[M−H]−. HRFABMS, Calcd for C21H19O10:
431.0978; Found: 431.0985. Anal. Calcd for
C21H20O10: C, 58.33; H, 4.66. Found: C, 58.21;
H, 4.70.
4%,8-Diallyloxyisofla6on-7-yl 2,3,4,6-tetra-
O-benzoyl-h-D-mannopyranoside (11).—To a
solution
of
2,3,4,6-tetra-O-benzoyl-a-D-
4%,8-Diallyloxyisofla6on-7-yl 2,3,4,6-tetra-
O-acetyl-i-D-galactopyranoside (7).—2,3,4,6-
Tetra-O-acetyl-a-D-galactopyranosyl bromide
(6) (1.28 g, 3.11 mmol) was treated with 2 as
described in the formation of 3 from 1 and 2
to give 7 (391 mg, 55%) as a gum. [h]2D3 −0.8°
(c 1.05, CHCl3). IR wmax(CHCl3) 1753, 1646,
1607, 1570 cm−1. 1H NMR (CDCl3): l 2.03 (3
H, s), 2.08 (3 H, s), 2.09 (3 H, s), 2.22 (3 H, s),
4.11–4.28 (3 H, m), 4.54–4.59 (3 H, m), 4.70
(1 H, dd, J 5.9, 11.7 Hz), 5.11–5.15 (2 H, m,
containing 1 H, d, J 8.1 Hz, at 5.12 ppm),
5.26–5.49 (5 H, m), 5.61 (1 H, dd, J 8.1, 11.0
Hz), 6.03–6.17 (2 H, m), 6.99 (2 H, d, J 8.8
Hz), 7.21 (1 H, d, J 8.8 Hz), 7.49 (2 H, d, J 8.1
Hz), 7.99–8.01 (2 H, m). FABMS (positive-
ion): m/z 681 [M+H]+. HRFABMS, Calcd
for C35H37O14: 681.2183; Found: 681.2186.
4%,8-Dihydroxyisofla6on-7-yl 2,3,4,6-tetra-
mannopyranosyl bromide (10) (2.01 g, 3.05
mmol) and 2 (352 mg, 1.00 mmol) in pyridine
(20 mL) was added Ag2CO3 (2.18 g, 7.91
mmol). The mixture was stirred for 18 h at rt,
filtered, and concentrated in vacuo to give a
mixture. This mixture was diluted with
EtOAc, washed with 0.1 M HCl, water, satd
aq NaHCO3, and brine, dried over MgSO4,
filtered, concentrated in vacuo, and chro-
matographed on a silica gel column. Elution
with 3:2 cyclohexane–EtOAc gave a mixture
(286 mg) of 11 and unknown byproducts that
could not be separated. The crude product
was employed for the next reaction without
further purification.
4%,8-Dihydroxyisofla6on-7-yl 2,3,4,6-tetra-
O-benzoyl-h-D-mannopyranoside (12).—The
mixture of 11 and byproducts (154 mg) ob-
tained above was treated as described in the
formation of 4 from 3 to give 12 (63.9 mg,
67%) as a white powder; mp 270.7–272.0 °C.
[h]2D4 +32.4° (c 0.80, CHCl3). IR wmax(KBr)
O-acetyl-i-D-galactopyranoside
(8).—Com-
pound 7 (265 mg, 0.390 mmol) was treated as
described in the formation of 4 from 3 to give
8 (145 mg, 62%) as a gum. [h]2D4 −2.9° (c 0.90,
CHCl3). IR wmax(CHCl3) 3691, 3598, 3483,
1
3392, 1730, 1269 cm−1. H NMR (CDCl3): l
4.53 (1 H, dd, J 5.9, 12.5 Hz), 4.66–4.69 (2 H,
m), 5.95 (1 H, d, J 1.5 Hz), 6.09–6.13 (3 H, m,
containing OH), 6.18 (1 H, t, J 9.5 Hz), 6.42
(1 H, s, OH), 6.88 (2 H, d, J 8.8 Hz), 7.25–
8.09 (25 H, m). FABMS (positive-ion): m/z
849 [M+H]+. HRFABMS, Calcd for
C49H37O14, 849.2183; Found: 849.2168.
4%,8-Diacetoxyisofla6on-7-yl 2,3,4,6-tetra-
O-acetyl-h-D-mannopyranoside (13).—Com-
pound 12 (43.5 mg, 0.051 mmol) in 0.2 M
NaOH MeOH solution (2 mL) was stirred for
18 h at rt. The solution was acidified with 1 M
HCl (0.4 mL), and concentrated in vacuo to
give a residue, which was chromatographed
(Cosmosil 140 C18-OPN). Elution with 1:9
MeOH–water gave the product-containing
fractions, which were concentrated in vacuo to
1752, 1647, 1612, 1579 cm−1
.
1H NMR
(CDCl3): l 2.05 (2 H, s), 2.09 (3 H, s), 2.15 (3
H, s), 2.22 (3 H, s), 4.10–4.28 (3 H, m), 5.06
(1 H, d, J 7.3 Hz), 5.17 (1 H, dd, J 2.9, 10.3
Hz), 5.49–5.53 (2 H, m), 6.12 (1 H, s, OH),
6.45 (1 H, s, OH), 6.87 (2 H, d, J 8.8 Hz), 7.09
(1 H, d, J 8.8 Hz), 7.38 (2 H, d, J 8.8 Hz), 7.80
(1 H, d, J 8.8 Hz), 8.02 (1 H, s). FABMS
(positive-ion): m/z 601 [M+H]+. HR-
FABMS, Calcd for C29H29O14: 601.1557;
Found: 601.1564.
4%,8-Dihydroxyisofla6on-7-yl i-D-galactopy-
ranoside (9).—Compound 8 (55.2 mg, 0.092
mmol) was treated as described in the forma-
tion of 5 from 4 to give 9 (48.2 mg, 100%) as
a yellow powder; mp 237.0–239.5 °C. [h]D23

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Y. Watanabe et al. / Carbohydrate Research 335 (2001) 283–289
give a residue (18.3 mg). As the residue was
not pure, it was purified again after the next
acetylation of hydroxyl groups.
4%,8-Dihydroxyisofla6on-7-yl 2,3,4,6-tetra-
O-benzoyl-i-D-allopyranoside (17).—Com-
pound 16 (163 mg, 0.176 mmol) was treated as
described in the formation of 4 from 3 to give
17 (120 mg, 80%) as a white powder; mp
269.0–271.5 °C. [h]2D4 +19.2° (c 0.78, CHCl3).
A solution of the residue (16.5 mg, 0.038
mmol) in Ac2O (0.5 mL) and pyridine (1 mL)
was stirred for 1 h at rt. The mixture was
concentrated in vacuo, diluted with EtOAc,
washed with water and brine, dried over
MgSO4, and chromatographed on a silica gel
column. Elution with 2:3 cyclohexane–EtOAc
1
IR wmax(KBr) 3420 (broad), 1731 cm−1. H
NMR (CDCl3): l 4.51 (1 H, dd, J 5.9, 11.7
Hz), 4.75–4.83 (2 H, m), 5.62 (1 H, dd, J 2.9,
9.5 Hz), 5.70 (1 H, d, J 8.1 Hz), 5.72 (1 H, s,
OH), 5.75 (1 H, dd, J 2.9, 8.1 Hz), 6.37 (1 H,
t, J 2.9 Hz), 6.67 (1 H, s, OH), 6.87 (2 H, d,
J 9.5 Hz), 7.18 (1 H, d, J 9.5 Hz), 7.33–7.72
(15 H, m), 7.88–8.10 (9 H, m). FABMS (pos-
itive-ion): m/z 849 [M+H]+. HRFABMS,
Calcd for C49H37O14, 849.2183; Found:
849.2181.
gave 13 (19.8 mg, 60%) as a gum. [h]D23
+
73.1° (c 0.95, CHCl3). IR wmax(CHCl3) 1753,
1
1652, 1625, 1508 cm−1. H NMR (CDCl3): l
2.05 (6 H, s), 2.08 (3 H, s), 2.22 (3 H, s), 2.33
(3 H, s), 2.54 (3 H, s), 4.05–4.07 (2 H, m),
4.29 (1 H, dd, J 5.9, 12.5 Hz), 5.38–5.47 (3 H,
m), 5.66 (1 H, s), 7.17 (2 H, d, J 8.1 Hz), 7.33
(1 H, d, J 8.8 Hz), 7.56 (2 H, d, J 8.1 Hz), 7.97
(1 H, s), 8.16 (1 H, d, J 9.5 Hz). FABMS
(positive-ion): m/z 707 [M+Na]+, 685 [M+
H]+. HRFABMS, Calcd for C33H32NaO16,
707.1588; Found: 707.1559.
4%,8-Diacetoxyisofla6on-7-yl 2,3,4,6-tetra-
O-acetyl-i-D-allopyranoside
(18).—Com-
pound 17 (97.5 mg, 0.115 mmol) was treated
as described in the formation of 13 from 12 to
give 18 (55.8 mg, 72%) as a gum. [h]2D3 −13.0°
(c 0.40, CHCl3). IR wmax(KBr) 1754, 1654,
1624 cm−1. 1H NMR (CDCl3): l 2.04 (3 H, s),
2.09 (3 H, s), 2.10 (3 H, s), 2.19 (3 H, s), 2.33
(3 H, s), 2.40 (3 H, s), 4.27–4.35 (3 H, m),
5.04 (1 H, dd, J 2.9, 10.3 Hz), 5.34 (1 H, dd,
J 2.9, 8.1 Hz), 5.49 (1 H, d, J 8.1 Hz), 5.73 (1
H, t, J 2.9 Hz), 7.18 (2 H, d, J 8.8 Hz), 7.22 (1
H, d, J 8.8 Hz), 7.56 (2 H, d, J 8.8 Hz), 7.96
(1 H, s), 8.17 (1 H, d, J 8.8 Hz). FABMS
(positive-ion): m/z 723 [M+K]+ (on addition
of aq NaI solution); 685 [M+H]+. HR-
FABMS, Calcd for C33H32KO16, 723.1327;
Found: 723.1323. Anal. Calcd for C33H32O16
(684.6): C, 57.90; H, 4.71. Found: C, 57.67; H,
4.83.
4%,8-Dihydroxyisofla6on-7-yl h-D-mannopy-
ranoside (14).—Compound 13 (15.5 mg, 0.023
mmol) was treated as described in the forma-
tion of 5 from 4 to give 14 (10.8 mg, 100%) as
a yellow powder; mp 174.5–177.0 °C. [h]D23
+85.8° (c 0.80, MeOH). IR wmax(KBr) 3365
1
(broad), 1626, 1610, 1515 cm−1. H NMR
(CD3OD): l 3.73–3.80 (4 H, m), 4.06 (1 H,
m), 4.19 (1 H, m), 5.63 (1 H, s), 6.85 (2 H, d,
J 8.8 Hz), 7.37–7.42 (3 H, m), 7.59 (1 H, d, J
8.8 Hz), 8.23 (1 H, s). FABMS (positive-ion):
m/z 433 [M+H]+. HRFABMS, Calcd for
C21H21O10, 433.1135; Found: 433.1096.
4%,8-Diallyloxyisofla6on-7-yl 2,3,4,6-tetra-
O-benzoyl-i-D-allopyranoside (16).—2,3,4,6-
Tetra-O-benzoyl-a-D-allopyranosyl bromide
(15) (2.05 g, 3.11 mmol) was treated with 2 as
described in the formation of 3 from 1 and 2
to give 16 (207 mg, 22%) as a gum. [h]D23
+20.1° (c 0.40, CHCl3). IR wmax(CHCl3) 1734,
4%,8-Dihydroxyisofla6on-7-yl i-D-allopyran-
oside (19).—Compound 18 (28.6 mg, 0.042
mmol) was treated as described in the forma-
tion of 5 from 4 to give 19 (23.6 mg, 100%) as
a yellow powder; mp 249.0–251.5 °C. [h]D24
−64.1° (c 0.21, MeOH). IR wmax(KBr) 3348
1
1646, 1603 cm−1. H NMR (CDCl3): l 4.45–
4.52 (3 H, m), 4.57–4.58 (2 H, m), 4.74–4.79
(2 H, m), 5.01–5.45 (4 H, m), 5.62 (1 H, dd, J
2.9, 10.3 Hz), 5.81 (1 H, dd, J 2.9, 8.1 Hz),
6.07 (1 H, m), 6.35 (1 H, t, J 2.9 Hz), 6.99 (2
H, d, J 8.8 Hz), 7.31–7.38 (5 H, m), 7.46–7.67
(10 H, m), 7.88–8.06 (10 H, m). FABMS
(positive-ion): m/z 929 [M+H]+. HR-
FABMS, Calcd for C55H45O14, 929.2809;
Found: 929.2807.
(broad), 1596, 1559 cm−1
.
1H NMR
(CD3OD): l 3.62 (1 H, dd, J 2.9, 9.5 Hz), 3.68
(1 H, dd, J 2.9, 8.1 Hz), 3.73 (1 H, dd, J 5.1,
11.7 Hz), 3.85–3.92 (2 H, m), 4.19 (1 H, t, J
2.9 Hz), 5.16 (1 H, d, J 8.1 Hz), 6.69 (2 H, d,
J 8.1 Hz), 7.17–7.23 (4 H, m), 8.16 (1 H, s).
FABMS (positive-ion): m/z 433 [M+H]+.
HRFABMS, Calcd for C21H21O10, 433.1135;

Y. Watanabe et al. / Carbohydrate Research 335 (2001) 283–289
289
changes at 415 nm being directly proportional
to the amount of liberated 4-nitrophenol were
measured by a microplate reader.
Found: 433.1143. Anal. Calcd for C21H20O10:
C, 58.33; H, 4.66. Found: C, 58.12; H, 4.78.
Method for rat li6er microsome h- -glucosi-
D
dase measurement.—For the measurement of
a-glucosidase inhibition, rat liver microsome
fraction solubilized with 25% Triton X-100
was used as an enzyme. The reaction was
carried out at 37 °C for 20 min in a 96-well
microtiter plate in a total volume of 150 mL
consisting of 70 mL of phosphate buffer (100
mM potassium phosphate with 20 mM
EDTA, pH 6.8), 10 mL of varying concentra-
tion of inhibitors, 50 mL of 10 mM 4-nitro-
phenyl a-D-glucopyranoside, and 20 mL of
diluted enzyme solution (approximately 3×
10−4 units), added finally. Then, 20 mL of 4
M sodium glycinate buffer (pH 10.4) was
added to stop the reactions, and colorimetric
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