Structure-activity relationships for α-glucosidase inhibition of baicalein, 5,6,7-trihydroxyflavone: The effect of A-ring substitution

Article abstract of DOI:10.1271/bbb.68.369

In order to estimate the effects of the A-ring hydroxyl group of baicalein (5,6,7-trihydroxyflavone, 1) on rat intestinal α-glucosidase inhibition, flavone, monohydroxyflavones, dihydroxyflavones, and methylated derivatives of 5,6,7-trihydroxyflavone were used for the structure-activity relationship (SAR) study. The importance of the 6-hydroxyl group of baicalein was validated for an exertion of the activity. And also, the tested flavones which lacked a hydroxyl substituent on any of positions 5, 6, or 7, showed no activity. Hence, the 5,6,7-trihydroxyflavone structure was concluded to be crucial for the potent inhibitory activity. In addition, an introduction of electron-withdrawing or electron-donating groups at position 8 of baicalein led to a dramatic decrease for activity, except for 8-fluoro-5,6,7-trihydroxyflavone, which carried a less bulky substituent on position 8. Hence, this result suggested that a sterically bulky substituent on C-8 of baicalein was detrimental for the activity regardless of its electronic nature. Through examining the inhibitory mechanism of baicalein against rat intestinal α-glucosidase, it was suggested to be a mixed type inhibition.

Full text of DOI:10.1271/bbb.68.369

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Structure-activity Relationships for α-Glucosidase
Inhibition of Baicalein, 5,6,7-Trihydroxyflavone: the
Effect of A-Ring Substitution
Hong GAOa, Tetsuo NISHIOKAa, Jun Kawabataa & Takanori KASAIa
a Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of
Agriculture, Hokkaido University
Published online: 22 May 2014.
To cite this article: Hong GAO, Tetsuo NISHIOKA, Jun Kawabata & Takanori KASAI (2004) Structure-activity Relationships
for α-Glucosidase Inhibition of Baicalein, 5,6,7-Trihydroxyflavone: the Effect of A-Ring Substitution, Bioscience,
Biotechnology, and Biochemistry, 68:2, 369-375, DOI: 10.1271/bbb.68.369
To link to this article: http://dx.doi.org/10.1271/bbb.68.369
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Biosci. Biotechnol. Biochem., 68 (2), 369–375, 2004
Structure-activity Relationships for ꢀ-Glucosidase Inhibition of Baicalein,
5,6,7-Trihydroxyflavone: the Effect of A-Ring Substitution
Hong GAO, Tetsuo NISHIOKA, Jun KAWABATA,y and Takanori KASAI
Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-Ku, Sapporo 060-8589, Japan
Received August 28, 2003; Accepted October 17, 2003
In order to estimate the effects of the A-ring hydroxyl
group of baicalein (5,6,7-trihydroxyflavone, 1) on rat
intestinal ꢀ-glucosidase inhibition, flavone, monohy-
droxyflavones, dihydroxyflavones, and methylated de-
rivatives of 5,6,7-trihydroxyflavone were used for the
structure-activity relationship (SAR) study. The impor-
tance of the 6-hydroxyl group of baicalein was validated
for an exertion of the activity. And also, the tested
flavones which lacked a hydroxyl substituent on any of
positions 5, 6, or 7, showed no activity. Hence, the 5,6,7-
trihydroxyflavone structure was concluded to be crucial
for the potent inhibitory activity. In addition, an
introduction of electron-withdrawing or electron-donat-
ing groups at position 8 of baicalein led to a dramatic
decrease for activity, except for 8-fluoro-5,6,7-trihy-
droxyflavone, which carried a less bulky substituent on
position 8. Hence, this result suggested that a sterically
bulky substituent on C-8 of baicalein was detrimental
for the activity regardless of its electronic nature.
Through examining the inhibitory mechanism of baica-
lein against rat intestinal ꢀ-glucosidase, it was suggested
to be a mixed type inhibition.
hydroxyl substituent, did not show any inhibitory
activity, while 1, 6-hydroxyapigenin (40,5,6,7-tetrahy-
droxyflavone) and 6-hydroxyluteolin (30,40,5,6,7-penta-
hydroxyflavone) showed high activity. Hence, this result
suggested that the 6-hydroxyl substituent on 5,6,7-
trihydroxyflavones was crucial for the ꢀ-glucosidase
inhibitory activity.3)
We are interested in the structure-activity relationship
of 1 and the related compounds against rat intestinal ꢀ-
glucosidase. In order to estimate the effects of the
hydroxyl and other groups on A-ring of 1 for inhibition
of the enzyme, we synthesized a series of hydroxy-
flavones and examined their ꢀ-glucosidase inhibitory
activity.
Materials and Methods
General experimental procedures. NMR spectra were
recorded with a Bruker AMX500 (1H, 500 MHz; 13C,
125 MHz) instrument. Chemical shifts were calculated
from the residual solvent signals of ꢁH 3.30 ppm in
methanol-d4, ꢁH 7.24 ppm in chloroform-d, and ꢁH 2.49
and ꢁC 39.5 ppm in dimethyl sulfoxide-d6. Field desorp-
tion (FD), FD-high resolution (HR), electron ionization
(EI), and EI-HR mass spectra (MS) were obtained on a
Jeol JMS-SX102A instrument. Melting points were
measured on a hot stage and were uncorrected. The
following experimental conditions were used for chro-
matography: ordinary phase column chromatography;
Silica gel Wakogel C-300 (Wako Pure Chem. Co.,
Osaka, Japan, 40-64 mesh), reverse phase silica gel
column chromatography; Cosmosil 75C18-OPN (Nacalai
Tesque, Inc., Kyoto, Japan). TLC, precoated TLC plates
with Silica gel 60 F254 (Merck, 0.25 mm or 0.5 mm
thickness, normal phase) and Silica gel RP-18 F254s
(Merck, 0.2 mm thickness, reverse phase). Detection
was done by UV lamp (254 nm). Preparative HPLC was
done with an Inertsil PREP-ODS column (20:0ꢀ250
mm, GL-Science). The detailed analytical conditions are
mentioned in each section.
Key words: 5,6,7-trihydroxyflavone; ꢀ-glucosidase in-
hibitor; structure-activity relationship
Mammalian digestive ꢀ-glucosidases, membrane-
bound enzymes at the epithelium of the small intestine,
hydrolyze disaccharides and oligosaccharides to liberate
glucose. Inhibitors of the ꢀ-glucosidases can retard the
decomposition and absorption of dietary carbohydrates
to suppress postprandial hyperglycemia.1) In the course
of our screening study for rat intestinal ꢀ-glucosidase-
inhibiting substances from natural resources, baicalein
(5,6,7-trihydroxyflavone, 1) was isolated from Scutel-
laria baicalensis,2) and also five 6-hydroxyflavonoids
from marjoram (Origanum majorana).3) Baicalein is a
unique flavone bearing a 6-hydroxyl group together with
ordinary 5,7-dihydroxyl substituents. In the assay for rat
intestinal ꢀ-glucosidase inhibitory activity on hydroxy-
flavones, apigenin (40,5,7-trihydroxyflavone) and luteo-
lin (30,40,5,7-tetrahydroxyflavone), which lack a 6-
All reagents were of reagent grade and were pur-
chased from Wako Pure Chem. Co., Osaka, Japan,
y
To whom correspondence should be addressed. Fax: +81-11-706-2496; E-mail: junk@chem.agr.hokudai.ac.jp

370
H. GAO et al.
unless otherwise stated. Rat intestinal acetone powder,
flavone (2), 5-hydroxyflavone (3), 6-hydroxyflavone (4),
and 7-hydroxyflavone (5) were supplied by Sigma
Aldrich Japan Co., Tokyo, Japan. 5,7-Dihydroxyflavone
(7) and 7,8-dihydroxyflavone (10) were purchased from
Tokyo Kasei Kogyo Co., Tokyo, Japan. Acetone and
tetrahydrofuran were dried by storage over 3A molecu-
lar sieves. All solvents were distilled before use. All
nonaqueous reactions were done in dry glassware.
6,7-Dihydroxyflavone (9). A mixture of 1,2,4-trihy-
droxybenzene (5 g, 40 mmol) and ethyl benzoylacetate
(13.5 g, 70 mmol) in diphenyl ether (25 ml) was heated
under reflux for 4 hr. After cooling, the mixture was put
through silica gel column chromatography using hex-
ane-ethyl acetate (7:3) as the eluent to give 9 (3.25 g,
32%) as yellow powders: mp 254–256 ꢁC (lit.6) mp
254 ꢁC); EI-HR-MS m=z 254.0617 (calcd. for C15H10O4,
254.0579).
Assay for rat intestinal ꢀ-glucosidase inhibitory
activity. The ꢀ-glucosidase inhibitory activity was
measured as described previously.4,5) The crude rat
intestinal ꢀ-glucosidase solution prepared from rat
intestinal acetone powder contained 526 mg of protein,
and its relative ꢀ-glucosidase activity against sucrose
was 0.385 unit/mg protein or 0.352 unit/ml enzyme
solution. The reaction mixture consisted of crude
enzyme solution (0.2 ml), 56 mM sucrose in 0.1 M
potassium phosphate buffer (pH 6.3, 0.2 ml), and the
test sample in 50% aqueous dimethyl sulfoxide (DMSO,
0.1 ml). After incubation for 15 min at 37 ꢁC, the
reaction was stopped by adding 0.75 ml of 2 M Tris
HCl buffer (pH 7.0). The reaction mixture was passed
through a short column of basic alumina (ICN Alumina
B, grade I, ICN Biomedical GmbH, Eschwege, Germa-
ny) to remove phenolic compounds which might
interfere with glucose measurement.2) The amount of
liberated glucose was measured by the glucose oxidase
method using a commercial test kit (Glucose B test
Wako, Wako Pure Chem. Co., Osaka, Japan). The
concentration of inhibitors required for inhibiting 50%
of the ꢀ-glucosidase activity under the assay conditions
was defined as the IC50 value. The IC50 value was
measured graphically by a plot of percent inhibition
versus log of the test compound.
5-Hydroxy-6,7-dimethoxyflavone (11) and 5,7-dihy-
droxy-6-methoxyflavone (12). A mixture of 1 (100 mg,
0.37 mmol) and methyl iodide (0.2 ml, 2.5 mmol) in dry
acetone (15 ml) and anhydrous potassium carbonate
(0.35 g, 2.5 mmol) was well stirred under reflux for 2 hr.
The reaction mixture was cooled and filtered. The filtrate
was concentrated and the resulting oil was put through
preparative thin layer chromatography using chloro-
form-methanol-formic acid (50:2:1) as the eluent to
yield 11 (Rf 0.78, 60 mg, 60%) as pale yellow powders:
mp 157–159 ꢁC (lit.7) 146–148.7 ꢁC); EI-HR-MS m=z
298.0827 (calcd. for C17H14O5, 298.0841); and 12 (Rf
0.54, 15 mg, 15%) as yellow powders: mp 181–184 ꢁC
(lit.7) 184–186 ꢁC); EI-HR-MS m=z 284.0695 (calcd. for
C16H12O5, 284.0685).
5,6-Dihydroxy-7-methoxyflavone (13). To a solution
of 1 (0.78 g, 3 mmol) in N,N-dimethylformamide (15 ml)
were added lithium carbonate (0.8 g, 8 mmol) and
methyl iodide (6 mmol). The resulting suspension was
stirred at 55 ꢁC for 17 hr. The reaction mixture was
cooled, poured into water (20 ml) containing concen-
trated hydrochloric acid (10 ml), and extracted with
ethyl acetate (50 mlꢀ3). The extracts were washed with
water and dried over anhydrous sodium sulfate. After
the ethyl acetate was removed, the residue was recrys-
tallized from 70% acetic acid to give 13 (0.61 g, 72%) as
yellow powders: mp 223–224 ꢁC (lit.8) 225–228 ꢁC); EI-
HR-MS m=z 284.0670 (calcd. for C16H12O5, 284.0685).
5,6-Dihydroxyflavone (6) and 5,8-dihydroxyflavone
(8). To a stirred solution of 5-hydroxyflavone (3) (4.76 g,
20 mmol) in water (100 ml) containing sodium hydrox-
ide (4 g, 100 mmol) was added dropwise during 4 hr a
solution of potassium persulfate (6 g, 22 mmol) in water
(200 ml), the temperature being kept at 15–20 ꢁC. After
24 hr, the solution was acidified to pH 4–5 and filtered.
The filtrate was extracted twice with ether. To the
aqueous phase was added concentrated hydrochloric
acid (20 ml), and this was refluxed for 2 hr. The resulting
mixture was cooled and extracted with ethyl acetate.
The organic phase was dried over anhydrous sodium
sulfate. After the ethyl acetate was removed, the
resulting oil was put through silica gel preparative thin
layer chromatography using chloroform-methanol
(100:4) as the eluent to give 6 (Rf 0.46, 254 mg, 5%)
as yellow crystalline solid: mp 193–195 ꢁC (lit.6) mp
189–190 ꢁC); FD-MS m=z (%): 254 (100, [M]þ); and 8
(Rf 0.56, 305 mg, 6%) as yellow powders: mp 230–
232 ꢁC (lit.6) mp 230–231 ꢁC); FD-MS m=z (%): 254
(100, [M]þ).
5-Hydroxy-6,7-dimethoxy-8-nitroflavone (14a). So-
dium nitrate (0.85 g, 10 mmol) and lanthanum nitrate
(44 mg, 0.1 mmol) were dissolved in water (10 ml). To
this solution was added 11 (2.98 g, 10 mmol) dissolved
in chloroform (40 ml). The reaction mixture was stirred
for 12 hr at room temperature and then extracted with
chloroform. The organic phase was washed with water
and dried over anhydrous sodium sulfate. After removal
of the chloroform, the resulting oil was put through
silica gel column chromatography using hexane-ethyl
acetate (7:3) as the eluent to yield 14a (1.85 g, 54%) as
pale yellow powders: mp 213–214 ꢁC; EI-HR-MS m=z
343.0664 (calcd. for C17H13O7N, 343.0692); 1H-NMR ꢁ
(chloroform-d) ppm (J in Hz): 3.95 (3H, s, OMe-6), 3.97
(3 H, s, OMe-7), 6.75 (1H, s, H-3), 7.50–7.57 (3H, m, H-
30, 40 and 50), 7.83–7.85 (2H, m, H-20 and 60), 13.28 (1H,
s, OH-5).

ꢀ-Glucosidase Inhibition and A-Ring Substitution on Baicalein
371
5,6,7-Trihydroxy-8-nitroflavone (14). 14a (1 mmol,
343 mg) was dissolved in dichloromethane (30 ml) and
cooled to ꢂ80 ꢁC and 4 ml of 1 M boron tribromide in
dichloromethane (4 mmol) was added dropwise with
stirring. The mixture was stirred for 1 hr at ꢂ80 ꢁC and
for 12 hr at room temperature. The reaction was stopped
by adding ice-water (20 ml). To this mixture was added
1-butanol (50 ml), and the organic phase was separated,
washed with water, and evaporated to dryness. The
residue was taken up in methanol and put through
reverse phase silica gel column chromatography using
water-methanol-formic acid (30:70:0.1) as the eluent to
yield the crude solid of 14. The solid was further purified
by preparative HPLC (mobile phase, water-acetonitrile-
formic acid (60:40:0.1); flow rate, 5.0 ml/min; tR 19
min; detection, UV 254 nm) to give 14 (201 mg, 63%) as
yellow powders: mp 266 ꢁC; FD-HR-MS m=z 315.0361
yellow powders: mp 205–206 ꢁC ; FD-HR-MS m=z
326.0810 (calcd. for C18H14O6, 326.0790); H-NMR ꢁ
(chloroform-d) ppm (J in Hz): 3.95 (3H, s, OMe-6), 4.19
(3H, s, OMe-7), 6.81 (1H, s, H-3), 7.54–7.55 (3H, m, H-
30, 40 and 50), 8.13–8.15 (2H, m, H-20 and 60), 10.43 (1H,
s, CHO-8), 13.69 (1H, s, OH-5).
1
8-Formyl-5,6,7-trihydroxyflavone (15). By the same
method as 14 and 21, 15a (1 mmol, 326 mg) was
demethylated to 15. Crystallization from hexane-ethyl
acetate to give 15 (223 mg, 75%) as yellow powders: mp
274–276 ꢁC; EI-HR-MS m=z 298.0471 (calcd. for
C16H10O6, 298.0477); 1H-NMR ꢁ (methanol-d4) ppm
(J in Hz): 7.19 (1H, s, H-3), 7.58–7.62 (3H, m, H-30, 40,
and 50), 8.21 (2H, d, J¼7:4, H-20 and 60), 10.46 (1H, s,
CHO-8), 13.69 (1H, s, OH-5).
1
(calcd. for C15H9O7N, 315.0379); H-NMR ꢁ (DMSO-
5,6,7-Trihydroxy-8-methylflavone (19). To a solution
of 15 (200 mg, 0.5mmol) and sodium cyanoborohydride
(1.5 mmol, 95 mg) in 30 ml of tetrahydrofuran was
added methyl orange (0.1 mg) as an indictor, giving
the solution a yellow color. The mixture was stirred for
3 hr at room temperature. Hydrochloric acid (1 M) was
occasionally added to the reaction mixture in order to
keep the color of the solution red. The mixture was
diluted with water (50 ml), and extracted with ethyl
acetate (50 mlꢀ3). After removing the ethyl acetate, the
residue was dissolved in a small amount of methanol
and put through preparative HPLC (mobile phase,
water-acetonitrile-formic acid (40:60:0.1); flow rate,
5.0 ml/min; tR 21 min; detection, UV 254 nm) to give
19 (92 mg, 65%) as yellow powders: mp 286 ꢁC (lit.9)
286–288 ꢁC); FD-HR-MS m=z 284.0706 (calcd. for
C16H12O5, 284.0685).
d6) ppm (J in Hz): 7.15 (1H, s, H-3), 7.51–7.62 (3H, m,
H-30, 40 and 50), 7.98 (2H, d, J¼5:1, H-20 and 60), 13.13
(1H, s, OH-5); 13C-NMR ꢁ (DMSO-d6) ppm: 102.6 (C-
10), 105.2 (C-3), 129.5 (C-6), 126.3 (C-20 and 60), 129.3
(C-30 and 50), 146.5 (C-7), 130.2 (C-10), 132.3 (C-40),
121.8 (C-8), 142.8 (C-5), 149.1 (C-9), 162.9 (C-2),
181.6 (C-4).
8-Amino-5-hydroxy-6,7-dimethoxyflavone (21a).
A
solution of 14a (172 mg, 0.5 mmol) in tetrahydrofuran
(30 ml) was hydrogenated for 12 hr in the presence of
10% palladium on charcoal (20 mg). The catalyst was
removed by filtration to afford 21a (148 mg, 95%) as
pale yellow powders: mp 134 ꢁC; EI-HR-MS m=z
1
313.0981 (calcd. for C17H15O5N, 313.0950); H-NMR
ꢁ (DMSO-d6) ppm (J in Hz): 3.85 (3H, s, OMe-6), 3.96
(3H, s, OMe-7), 7.05 (1H, s, H-3), 7.56–7.64 (3H, m, H-
30, 40, and 50), 8.27 (2H, d, J¼7:6, H-20 and 60).
8-Bromo-5,6,7-trihydroxyflavone (16). To a suspen-
sion of 1 (0.18 g, 0.67 mmol) in methanol (20 ml), 30%
H2O2 (1 ml) was added dropwise at 0–5 ꢁC and then
47% aqueous hydrobromic acid (0.5 ml) was added
dropwise for 20 min. After this was stirred for 4 hr at 0–
5 ꢁC, the mixture was warmed to 20 ꢁC and further
stirred for 1 hr. The reaction mixture was poured into
water (200 ml). The precipitate was collected by suction
and washed with water. The solid was further purified by
preparative HPLC (mobile phase, water-methanol-for-
mic acid (20:80:0.1); flow rate, 5.0 ml/min; tR 24 min;
detection, UV 254 nm) to give 16 (95 mg, 41%) as
yellow powders: mp 239 ꢁC; FD-HR-MS m=z 347.9655
8-Amino-5,6,7-trihydroxyflavone (21). By the same
method described in 14 from 14a, compound 21 was
obtained from 21a (0.4 mmol, 125 mg). Crystallization
from hexane-ethyl acetate to give 21 (81 mg, 71%) as
yellow powders: mp 204–205 ꢁC; FD-HR-MS m=z
1
285.0652 (calcd. for C15H11O5N, 285.0637); H-NMR
ꢁ (methanol-d4) ppm (J in Hz): 6.68 (1H, s, H-3), 7.55–
7.56 (3H, m, H-30, 40 and 50), 8.06 (2H, d, J¼7:9, H-20
and 60).
8-Formyl-5-hydroxy-6,7-dimethoxyflavone (15a). A
mixture of 11 (2.98 g, 10 mmol), hexamethylenetet-
ramine (1.68 g, 12 mmol), and trifluoroacetic acid
(50 ml) was heated under reflux for 24 hr. The mixture
was poured into 4 M hydrochloric acid (50 ml), stirred
for 1 hr, and extracted with chloroform (200 mlꢀ2). The
combined organic extracts were washed successively
with water, and sat. brine, then dried over anhydrous
sodium sulfate. After removal of the chloroform, the
resulting oil was put through silica gel column chroma-
tography using chloroform as the eluent to give 15a as
1
(calcd. for C15H9O5Br, 347.9633); H-NMR ꢁ (DMSO-
d6) ppm (J in Hz): 7.07 (1H, s, H-3), 7.60–7.61 (3H, m,
H-30, 40 and 50), 8.12 (2H, dd, J¼7:4, 1.1, H-20 and 60),
12.76 (1H, s, OH-5); 13C-NMR ꢁ (DMSO-d6) ppm: 86.9
(C-8), 104.4 (C-10), 104.6 (C-3), 126.3 (C-20 and 60),
129.2 (C-30 and 50), 129.6 (C-6), 130.7 (C-10), 132.1 (C-
40), 146.2 (C-5), 146.6 (C-7), 151.7 (C-9), 162.9 (C-2),
182.1 (C-4).
8-Chloro-5,6,7-trihydroxyflavone (17) and 8-fluoro-

372
H. GAO et al.
1
5,6,7-trihydroxyflavone (18). A solution of 11 (1 mmol,
298 mg) in 5 ml of 1,1,2-trichloroethane was treated with
N-fluoropyridinium triflate (1.2 mmol, 293 mg) under
reflux for 32 hr. The reaction mixture was directly put
through silica gel column chromatography using hex-
ane-ethyl acetate (3:7) as the eluent to obtain a mixture
of 8-chloro-5-hydroxy-6,7-dimethoxyflavone and 8-flu-
oro-5-hydroxy-6,7-dimethoxyflavone (Rf 0.62, 192 mg).
By the same method as 14, the crude mixture was
demethylated by boron tribromide to give a mixture of
two main products. The resultant solid was separated by
preparative HPLC (mobile phase, water-acetonitrile-
formic acid (40:60:0.1); flow rate, 5.0 ml/min; detec-
tion, UV 254 nm) to give 21 mg of 17 (tR 15.6 min) as
yellow powders: mp 264–265 ꢁC; EI-HR-MS m=z
(calcd. for C15H10O6, 286.0477); H-NMR ꢁ (DMSO-
d6) ppm (J in Hz): 6.89 (1H, s, H-3), 7.56–7.61 (3H, m,
H-30, 40 and 50), 8.13–8.15 (2H, m, H-20 and 6), 12.16
(1H, s, OH-5); 13C-NMR ꢁ (DMSO-d6) ppm: 102.9 (C-
10), 104.1 (C-3), 125.5 (C-8), 126.4 (C-20 and 60), 129.0
(C-30 and 50), 129.6 (C-6), 131.1 (C-10), 131.8 (C-40),
139.0 (C-7), 139.7 (C-5), 143.8 (C-9), 162.6 (C-2),
182.5 (C-4).
5,6,7-Trihydroxy-8-piperidinomethylflavone (22). To
a stirred solution of 1 (0.54 g, 2 mmol) in ethanol (20 ml)
was added a solution of methylene-bis-piperidine
(0.73 g, 4 mmol) in ethanol (10 ml). The reaction
mixture was refluxed for 1 hr. After cooling overnight
at 4 ꢁC, the solid formed was separated and washed
twice with cold ethanol. Recrystallization from ethanol
gave 22 as yellow powders: mp 220–221 ꢁC; FD-HR-
MS m=z 367.1400 (calcd. for C21H21O5N, 367.1420);
1H-NMR ꢁ (methanol-d4) ppm (J in Hz): 4.60 (2H, s,
CH2-8), 4.85 (10H, m, piperidine), 6.83 (1 H, s, H-3),
7.59–7.61 (3H, m, H-30, 40, and 50), 8.03 (2H, dd, J¼5:2,
1.7, H-20 and 60).
1
304.0126 (calcd. for C15H9O5Cl, 304.0139); H-NMR
ꢁ (DMSO-d6) ppm (J in Hz): 7.04 (1H, s, H-3), 7.58–
7.59 (3H, m, H-30, 40 and 50), 8.08 (2H, d, J¼6:9, H-20
and 60), 9.53 (1H, s, OH-7), 10.91 (1H, s, OH-6), 12.69
(1H, s, OH-5); 13C-NMR ꢁ (DMSO-d6) ppm: 98.0 (C-8),
104.2 (C-10), 104.7 (C-3), 126.2 (C-20 and 60), 129.2 (C-
30 and 50), 129.7 (C-6), 130.7 (C-10), 132.1 (C-40), 145.6
(C-5), 145.7 (C-7), 150.5 (C-9), 162.8 (C-2), 182.1 (C-
4); and 86 mg of 18 (tR 20.1 min) as yellow powders: mp
251 ꢁC; EI-HR-MS m=z 288.0446 (calcd. for C15H9O5F,
Results and Discussion
1
288.0434); H-NMR ꢁ (DMSO-d6) ppm (J in Hz): 6.97
To embark upon the synthesis of 5,6-dihydroxyfla-
vone (6) and 5,8-dihydroxyflavone (8), 5-hydroxyfla-
vone (3) was chosen as the starting material, which was
prepared by the method of Bois et al.11) Compounds 6
and 8 were obtained from 3 by Elb’s persulfate
oxidation.12) Treatment of 1,3,4-trihydroxybenzene with
ethyl benzoylacetate in boiling diphenyl ether yielded
6,7-dihydroxyflavone (9) in 32% yield.13) Methylation of
1 with methyl iodide and potassium carbonate gave 5-
hydroxy-6,7-dimethoxyflavone (11) and 5,7-dihydroxy-
6-methoxyflavone (12),14) while 5,6-dihydroxy-7-me-
thoxyflavone (13) was obtained selectively from 1 with
methyl iodide and lithium carbonate.15) UV shift tests
for the position of 5 and 7-OH on hydroxyflavones, and
comparison of mass and NMR spectra with reference
data confirmed the structures of 3, 6, 8, 9, and 11–13.
Compound 11 was treated by sodium nitrate in a two-
phase system (water-chloroform) in the presence of a
catalytic amount of lanthanum nitrate to give 5-hydroxy-
6,7-dimethoxy-8-nitroflavone (14a) in 54% yield.16) 14a
was demethylated by boron tribromide to yield 5,6,7-
trihydroxy-8-nitroflavone (14).17) Hydrogenation of 14a
afforded 21a, which was then treated with boron
tribromide to give 8-amino-5,6,7-trihydroxyflavone
(21).18) Compound 11 was formylated with Duff
reaction conditions, using hexamethylenetetramine and
TFA to yield 8-formyl-5-hydroxy-6,7-dimethoxyflavone
(15a), which was demethylated to obtain 8-formyl-5,6,7-
trihydroxyflavone (15).19) Compound 15 was reduced
with sodium cyanoborohydride to give 5,6,7-trihydroxy-
8-methylflavone (19) in 65% yield.20) Bromination of 1
with HBr-H2O2 gave 8-bromo-5,6,7-trihdyroxyflavone
(16).21) Treatment of 11 with N-fluoropyridinium triflate
(1H, s, H-3), 7.56–7.62 (3H, m, H-30, 40 and 50), 8.02–
8.03 (2H, m, H-20 and 60), 9.38 (1H, s, OH-7), 10.85
(1H, s, OH-6), 12.26 (1H, s, OH-5); 13C-NMR ꢁ
(DMSO-d6) ppm: 102.4 (C-10), 104.8 (C-3), 126.3 (C-
20 and 60), 129.2 (C-30 and 50), 130.2 (C-6), 132.1 (C-40),
134.1 (C-7), 132.3 (C-8), 142.6 (C-9), 142.3 (C-5),
162.8 (C-2), 181.9 (C-4).
5,8-Dihydroxy-6,7-dimethoxyflavone (20a). By the
same method as 6 and 8, compound 20a was obtained
from 11 by the oxidation with potassium persulfate.
Silica gel column chromatography using hexane-ethyl
acetate (10:1) as the eluent gave 20a (378 mg, 12%) as a
colorless solid: mp 230–231 ꢁC (lit.10) 232 ꢁC); EI-HR-
MS m=z 314.0762 (calcd. for C17H14O6, 314.0790).
5,6,7,8-Tetrahydroxyflavone (20). A mixture of 20a
(157 mg, 0.5 mmol), 47% aqueous hydrobromic acid
(0.7 ml, 6.2 mmol), and acetic acid (15 ml) was heated
under reflux for 23 hr. After cooling to room temper-
ature, the reaction mixture was extracted with 1-butanol
(50 mlꢀ2). The organic phase was washed with water
and dried over anhydrous sodium sulfate. After the 1-
butanol was removed, the residue was dissolved in a
small amount of methanol and put through reverse phase
silica gel column chromatography using water-metha-
nol-formic acid (50:50:0.1) as the eluent to yield crude
20. The solid was further purified with preparative
HPLC (mobile phase, water-methanol-formic acid
(70:30:0.1); flow rate, 4.0 ml/min; tR 17.6 min; detec-
tion, UV 254 nm) to give 20 (117 mg, 82%) as yellow
powders: mp 232–233 ꢁC; EI-HR-MS m=z 286.0502

ꢀ-Glucosidase Inhibition and A-Ring Substitution on Baicalein
373
in boiling 1,1,2-trichloroethane gave a mixture of 8-
chloro-5-hydroxy-6,7-dimethoxyflavone and 8-fluoro-5-
hydroxy-6,7-dimethoxyflavone, which was demethylat-
ed to give 8-chloro-5,6,7-trihdyroxyflavone (17) and 8-
fluoro-5,6,7-trihydroxyflavone (18), respectively.22)
Compound 11 was converted to 5,8-dihydroxy-6,7-
dimethoxyflavone (20a) by alkaline persulfate oxida-
tion, and was then demethylated to give 5,6,7,8-
tetrahydroxyflavone (20).23) Condensation of 1 with
methylene-bis-piperidine in refluxing ethanol gave
5,6,7-trihydroxy-8-piperidinomethylflavone (22) in good
yield.24) The structures of 14–22 were confirmed by their
mass and NMR spectra.
In order to estimate the effect of the A-ring hydroxyl
group of 1 on ꢀ-glucosidase inhibitory activity, 1 and
related compounds (2–13) were tested for inhibition of
rat intestinal ꢀ-glucosidase with sucrose as the substrate
(Table 1). As previously reported, 1 was a potent
inhibitor of the ꢀ-glucosidase with IC50¼45 ꢂM. On the
other hand, 7, which lacks a 6-hydroxyl group, did not
show any activity. Hence, it was concluded that the 6-
hydroxyl substituent of 1 was necessary for the activity.
And also, removal of any hydroxyl group at positions
5,6,7 led to a dramatic loss of the inhibitory potency (2–
10). This result indicated that the 5,6,7-trihydroxyfla-
vone structure was crucial for high activity. Further
evidence supporting this rationale is that replacement of
the hydroxyl group with a methoxyl group at position 6
or 7 (11–13) appeared to be detrimental for the activity.
Having identified the importance of the 5,6,7-tri-
hydroxyl group of 1, we decided to explore incorpo-
ration of an additional hydroxyl group onto position 8 of
1 to afford 20. Unfortunately, this also led to a reduction
in potency. So, the question remains, why 20 decreases
the activity despite having the 5,6,7-trihdyroxyflavone
structure. Two possibilities are electronic and steric
effects.
With the initial structure-activity relationship (SAR)
results, we turned our attention to an electronic effect of
substituents at position 8 of 1 on the activity. We used
the Hammett substituent constant25) to evaluate the
electronic influence of 1 and 14–21 (Table 2). An
introduction of an electron-withdrawing group (14–17)
at position 8 of 1 resulted in disappearance of ꢀ-
glucosidase inhibitory activity, though 18 was tolerated
with a minor loss of potency. However, 19–21, which
have an electron-donating group at position of 8, were
also inactive, although 20 (IC50¼960 ꢂM) and 21
(IC50¼1000 ꢂM) were very weak inhibitors. Except for
18, an introduction of an electron-withdrawing or
electron-donating group at position 8 of 1 resulted in a
detrimental effect on the potent activity. Hence, it was
considered that an electronic effect could not account for
this reduction in potency. These 8-position substitutions
(14–17, 19–21) of 1, which resulted in a disadvantage
for ꢀ-glucosidase inhibitory activity, was then due
possibly to a steric interference to the interaction
between the 5,6,7-trihydroxyl group of 1 and the
enzyme. Compound 22, which has a large piperidino-
methyl group at position 8, was also inactive, while 18
(IC50¼86 ꢂM) which has a less bulky fluorine at position
8 showed moderate activity compared to other 8-
substitued 5,6,7-trihydroxyflavones (14–17, 19–21),
suggesting that excess steric bulkiness around position
8 of 1 is detrimental for the potent inhibitory activity.
In order to examine the types of inhibition of rat
intestinal ꢀ-glucosidase by 1, ꢀ-glucosidase solution
Table 1. Rat Intestinal ꢀ-Glucosidase Inhibition of Hydroxylated
Flavone Derivatives
Table 2. The Hammett Substituent Constant of 8-Substituted Groups
of 5,6,7-Trihdyroxyflavones, and Their Inhibitory Activity against Rat
Intestinal ꢀ-Glucosidase
R4
R3
R2
O
R
R1
R2
O
HO
HO
O
IC50
(ꢂM)
Compound
R1
R3
R4
H
OH
O
1
OH
OH
OH
45
IC50
(ꢂM)
a
2a
3a
4a
5a
6
H
OH
H
H
H
H
H
H
H
NIb
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Compound
R
ꢁp
OH
H
H
H
1
14
15
16
17
18
19
20
21
22
H
NO2
CHO
Br
0
45
NIb
NI
H
OH
H
H
0.81
NDc
OH
OH
OH
H
OH
H
H
7
OH
H
H
0.26
0.24
NI
NI
8
H
OH
H
Cl
F
9
OH
H
OH
OH
OMe
OH
OMe
0.15
86
NI
10
11a
12
13
H
OH
H
CH3
OH
NH2
ꢂ0:14
ꢂ0:38
ꢂ0:57
ND
OH
OH
OH
OMe
OMe
OH
960
1000
NI
H
d
b
H
C5H10NCH2
a
b
a
Hammett substituent constant.25)
less than 30% inhibition at the
d
The sample was dissolved in 100% DMSO in this assay. less than 30%
inhibition at the concentration of 1000 ꢂM.
c
concentration of 1000 ꢂM. no data available. piperidin-1-ylmethyl.

374
H. GAO et al.
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Fig. 1. Lineweaver-Burk Plots of the Inhibition of Rat Intestinal ꢀ-
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Mixed inhibition, Ki¼0:11 mM, Km¼12:3 mM.
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method for the synthesis of baicalein (5,6,7-trihdyroxy-
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In summary, 1 is a potent rat intestinal ꢀ-glucosidase
inhibitor with mixed inhibitory mechanism. The SAR
studies indicated that 5,6,7-trihdyroxyflavone structure
was crucial for the activity, and validated the importance
of the 6-hydroxyl substitution previously described. And
also, 8-substituted derivatives of 1 tended to decrease
the activity regardless of electronic nature of the
substituents. Hence, it was suggested that possible
excess steric bulkiness around position 8 was detrimen-
tal for the potent inhibitory activity.
Acknowledgments
We are grateful to Mr. Kenji Watanabe and Dr. Eri
Fukushi, GC-MS & NMR Laboratory, Faculty of
Agriculture, Hokkaido University, for mass spectral
measurements.
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