DPPH radical scavenging reaction of hydroxy- and methoxychalcones

Article abstract of DOI:10.1271/bbb.70.193

The DPPH radical scavenging activity of 2′,4′,6′- trihydroxy- and 2′-hydroxy-4′,6′-dimethoxychalcones carrying a 2,3- and 3,4-dihydroxylated, and 3,4,5-trihydroxylated B-ring was evaluated in alcoholic and non-alcoholic solvents. All test compounds scavenged more than two equivalent of radicals by a possible conversion to the corresponding B-ring quinones and in most cases subsequently underwent cyclization to aurones and flavanones, these being identified in the reaction solutions by an in situ NMR analysis. Interestingly, the reaction between 2′,3,4-trihydroxy-4′, 6′-dimethoxychalcone and the DPPH radical was significantly affected by the solvent used, which might be accounted for by the difference in readiness for cyclization to an aurone.

Full text of DOI:10.1271/bbb.70.193

Biosci. Biotechnol. Biochem., 70 (1), 193–202, 2006
DPPH Radical Scavenging Reaction of Hydroxy- and Methoxychalcones
y
Jun NISHIDA and Jun KAWABATA
Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
Received August 2, 2005; Accepted September 29, 2005
The DPPH radical scavenging activity of 20,40,60-
trihydroxy- and 20-hydroxy-40,60-dimethoxychalcones
carrying a 2,3- and 3,4-dihydroxylated, and 3,4,5-
trihydroxylated B-ring was evaluated in alcoholic and
non-alcoholic solvents. All test compounds scavenged
more than two equivalent of radicals by a possible
conversion to the corresponding B-ring quinones and in
most cases subsequently underwent cyclization to au-
rones and flavanones, these being identified in the
reaction solutions by an in situ NMR analysis. Interest-
ingly, the reaction between 20,3,4-trihydroxy-40,60-dime-
thoxychalcone and the DPPH radical was significantly
affected by the solvent used, which might be accounted
for by the difference in readiness for cyclization to an
aurone.
none with a benzaldehyde, there has been little inves-
tigation on their antiradical mechanism and structure-
activity relationship.
In this study, to examine the effects of the number and
arrangement of hydroxyl groups in the chalcone skeleton
on the antiradical activity, 20,3,4-trihydroxy-40,60-di-
methoxychalcone (1), 20,3,4,40,60-pentahydroxychalcone
(2), 2,20,3-trihydroxy-40,60-dimethoxychalcone (3), 2,20,
3,40,60-pentahydroxychalcone (4), 20,3,4,5-tetrahydroxy-
40,60-dimethoxychalcone (5), and 20,3,4,40,5,60-hexahy-
droxychalcone (6) were prepared and tested for their
DPPH radical scavenging activity in aprotic and protic
solvents.
Materials and Methods
Key words: chalcone; aurone; flavanone; radical scav-
enging reaction; DPPH radical
Chemicals. 3,4-Dihydroxybenzaldehyde, 2,3-dihy-
droxybenzaldehyde, 3,4,5-trihydroxybenzaldehyde, 20,
40,60-trihydroxyacetophenone, phloroglucinol, methoxy-
methyl chloride and chloroacetonitrile were purchased
from Tokyo Kasei Kogyo Co. 20-Hydroxy-40,60-dime-
thoxyacetophenone and 4.0 M HCl solution in dioxane
were purchased from Aldrich Chemical Co. 2,2-Diphen-
yl-1-picrylhydrazyl and other reagents were purchased
from Wako Pure Chemical Industries. All solvents used
were of reagent grade.
The implication of oxidative and free radical medi-
ated reactions in degenerative processes related to aging
and other disease conditions is cause for concern. Free
radicals, reactive oxygen species (ROS), and reactive
nitrogen species (RNS) are implicated in numerous
pathological conditions such as inflammation, metabolic
disorders, cellular aging, reperfusion damage, athero-
sclerosis, and carcinogenesis.1,2)
Polyphenol compounds such as protocatechuic acid,
caffeic acid and a variety of flavonoids are present in
fruits and vegetables and are an integral part of the
human diet. It is already known that dietary polyphenols
show potent antiradical ability. The radical scavenging
abilities of these phenolic compounds depend greatly
on the number and arrangement of phenolic hydroxyl
groups. The antiradical reaction of protocatechuic
acid,3–5) gallic acid,5) caffeic acid,6,7) quercetin8) and
catechin9) have been widely investigated. It has been
that the radical scavenging reactivity of protocatechuic
acid and gallic acid changed in protic and aprotic
solvents.3–5)
Aparatus. Melting point (mp) data were measured on
a hot stage and are uncorrected. Electron ionization mass
spectra (EI-MS) were obtained with Jeol JMS-AX500
and JMS-SX102A instruments. NMR spectra were
recorded by a Bruker AMX500 spectrometer (1H, 500
MHz; 13C, 125 MHz); chemical shifts are expressed
relative to the residual signals of chloroform-d (ꢀH 7.26,
ꢀC 77.0), methanol-d4 (ꢀH 3.30, ꢀC 49.0) or acetone-d6
(ꢀH 2.04, ꢀC 29.8). Optical absorbance was acquired with
a Hitachi U-3210 spectrophotometer. Silica gel column
chromatography was performed with Wakogel C-300
(Wako Pure Chemical Industries). Normal-phase prep-
arative and analytical thin-layer chromatography (TLC)
was performed on silica gel plates, Merck 60 F254
(0.5 and 0.25 mm thickness, respectively), and reverse
phase TLC on RP-18 F254s (Merck, 0.2 mm thickness).
Although hydroxychalcones are key compounds
precedent to many flavonoids and can be easily
synthesized by condensing the corresponding acetophe-
y
To whom correspondence should be addressed. Tel/Fax: +81-11-706-2496; E-mail: junk@chem.agr.hokudai.ac.jp

194
J. NISHIDA and J. KAWABATA
Preparative HPLC was done with an Inertsil PREP-ODS
column (20:0 ꢀ 250 mm, GL-Science).
was prepared from 2,3-dihydroxybenzaldehyde to give
colorless oil (88%). EI-MS m=z (%): 226 (5, [M]þ);
1H-NMR (CDCl3) ꢀ (ppm): 3.52, 3.58 (each 3H, s,
2 ꢀ OCH3), 5.23, 5.25 (each 2H, s, 2 ꢀ OCH2O), 7.15
(1H, dd, J ¼ 8:1, 7.8 Hz, H-5), 7.41 (1H, dd, J ¼ 8:1,
1.5 Hz, H-6), 7.51 (1H, dd, J ¼ 7:8, 1.5 Hz, H-4).
Colorimetric radical scavenging tests. DPPH radical
scavenging activity was measured as described previ-
ously.4,5) To a solution of the DPPH radical (500 mM,
1 ml) in methanol or acetone was added a test solution
(12.5 mM, 4 ml) in the same solvent in a test tube. The
final molar ratio of the radical and test compound was
10:1. The solution was immediately mixed vigorously
for 10 s by a Vortex mixer and transferred to a cuvette.
The absorbance reading at 517 nm was taken 1, 2, 3, 4,
5, 10, 20 and 30 min after mixing. Acetone and methanol
were respectively chosen as aprotic and protic solvents.
A solution of dl-ꢁ-tocopherol in the same concentration
was measured as a positive control. A reduction in the
absorbance, 0.228, by the positive control is regarded as
corresponding to the consumption of two molecules of
the DPPH radical. All experiments were performed in
triplicate.
3,4,5-Tris(methoxymethoxy)benzaldehyde (10). By
the same procedure as that used for the preparation of
7, 10 was prepared from 3,4,5-trihydroxybenzaldehyde
to give colorless oil (64%). EI-MS m=z (%): 286
(33, [M]þ); 1H-NMR (CDCl3) ꢀ (ppm): 3.52 (6H, s,
2 ꢀ OCH3), 3.62 (3H, s, OCH3), 5.24 (2H, s, OCH2O),
5.26 (4H, s, 2 ꢀ OCH2O), 7.39 (2H, s, H-2 and 5), 9.85
(1H, s, CHO).
20-Hydroxy-40,60-dimethoxy-3,4-bis(methoxymethoxy)-
chalcone (11).10) To a stirred solution of KOH (2.0 g,
45.6 mmol) in water (2 ml) cooled to 0 ꢁC in an ice bath
was added dropwise a solution of 8 (226 mg, 1.0 mmol)
and 20-hydroxy-40,60-dimethoxyacetophenone (7a, 294
mg, 1.5 mmol) in methanol (10 ml) under argon. The
reaction mixture was kept at 0 ꢁC for 3 h, and then at
room temperature for 72 h. The mixture was poured into
ice–water (10 ml), adjusted to pH 3–4 with 1 M HCl, and
then extracted with ethyl acetate. The organic layer was
successively washed with water and saturated brine,
dried over anhydrous Na2SO4 and concentrated under
reduced pressure to yield a yellow solid. Recrystalliza-
tion from hexane–ethyl acetate gave 11 as yellow
powder (392 mg, 97%). Mp 112–113 ꢁC; EI-HR-MS m=z
404.1450 (calcd. for C21H24O8, 404.1471); 1H-NMR
(CDCl3) ꢀ (ppm): 3.52, 3.55, 3.84, 3.92 (each 3H, s,
4 ꢀ OCH3), 5.28 (4H, s, 2 ꢀ OCH2O), 5.97 (1H, d,
J ¼ 2:2 Hz, H-50), 6.11 (1H, d, J ¼ 2:2 Hz, H-30), 7.18
(1H, d, J ¼ 8:6 Hz, H-5), 7.22 (1H, dd, J ¼ 8:6, 1.9 Hz,
H-6), 7.51 (1H, d, J ¼ 1:9 Hz, H-2), 7.73 (1H, d,
J ¼ 15:5 Hz, H-ꢂ), 7.84 (1H, d, J ¼ 15:5 Hz, H-ꢁ).
NMR measurement of the reaction mixture of a test
compound with the DPPH radical. To the DPPH radical
(6.0 equiv, 12.7 mmol) was added a test compound
(2.1 mmol) in an acetone-d6 (400 ml) or methanol-d4–
acetone-d6 solution (3:1, 400 ml). The methanol–acetone
mixture was used as a protic solvent, in which acetone
was added as a co-solvent to enhance the solubility of
the DPPH radical. The mixture was immediately trans-
1
ferred to an NMR tube and mixed vigorously. H-NMR
spectra were recorded 10 min after mixing.
20-Hydroxy-40,60-bis(methoxymethoxy)acetophenone10)
(7). To a mixture of 20,40,60-trihydroxyacetophenone
(168 mg, 1 mmol) and anhydrous K2CO3 (966 mg,
7 mmol) in dry acetone (20 ml) was added dropwise
methoxymethyl chloride (20 mg, 2.5 mmol). The mix-
ture was refluxed for 60 min, cooled to room temper-
ature, filtered, and evaporated under reduced pressure.
The resulting residue was purified by silica gel column
chromatography (hexane–ethyl acetate (7:3)) to afford 7
(135 mg, 60%) as colorless oil. EI-MS m=z (%): 256
20-Hydroxy-3,4,40,60-tetrakis(methoxymethoxy)chal-
cone (12). By the same procedure as the preparation of
11, 12 was prepared from 7 and 8. The residue was
purified by silica gel column chromatography (hexane–
ethyl acetate (7:3)) to give 12 as yellow powders (74%):
Mp 87–88 ꢁC; EI-HR-MS m=z 464.1691 (calcd. for
1
(26, [M]þ); H-NMR (CDCl3) ꢀ (ppm): 2.65 (3H, s),
3.47, 3.51 (each 3H, s, 2 ꢀ OCH3), 5.16, 5.25 (each
2H, s, 2 ꢀ OCH2O), 6.24 (1H, d, J ¼ 2:2 Hz, H-5), 6.26
(1H, d, J ¼ 2:2 Hz, H-3).
1
C23H28O10, 464.1682); H-NMR (acetone-d6) ꢀ (ppm):
3.45, 3.47, 3.50, 3.56 (each 3H, s, 4 ꢀ OCH3), 5.26,
5.27, 5.28, 5.42 (each 2H, s, 4 ꢀ OCH2O), 6.24 (1H, d,
J ¼ 2:2 Hz, H-50), 6.34 (1H, d, J ¼ 2:2 Hz, H-30), 7.20
(1H, d, J ¼ 8:6 Hz, H-5), 7.34 (1H, dd, J ¼ 8:6, 2.2 Hz,
H-6), 7.56 (1H, d, J ¼ 2:2 Hz, H-2), 7.74 (1H, d,
J ¼ 15:8 Hz, H-ꢂ), 7.96 (1H, d, J ¼ 15:8 Hz, H-ꢁ).
3,4-Bis(methoxymethoxy)benzaldehyde (8). By the
same procedure as the preparation of 7, 8 was prepared
from 3,4-dihydroxybenzaldehyde to give colorless oil
(87%): EI-MS m=z (%): 226 (13, [M]þ); 1H-NMR
(CDCl3) ꢀ (ppm): 3.53 (6H, s, 2 ꢀ OCH3), 5.30, 5.33
(each 2H, s, 2 ꢀ OCH2O), 7.29 (1H, dd, J ¼ 7:9,
1.2 Hz, H-6), 7.52 (1H, d, J ¼ 7:9 Hz, H-5), 7.68 (1H, d,
J ¼ 1:2 Hz, H-2), 9.87 (1H, s, CHO).
20-Hydroxy-40,60-dimethoxy-2,3-bis(methoxymethoxy)-
chalcone (13). By the same procedure as that used for
the preparation of 11, 13 was prepared from 7a and 9 to
give yellow powder (88%). Mp 94–95 ꢁC; EI-HR-MS
m=z 404.1440 (calcd. for C21H24O8, 404.1471); 1H-
2,3-Bis(methoxymethoxy)benzaldehyde (9). By the
same procedure as that used for the preparation of 7, 9

DPPH Radical Scavenging Activity of Chalcones
195
NMR (CDCl3) ꢀ (ppm): 3.51, 3.64, 3.83, 3.90 (each
3H, s, 4 ꢀ OCH3), 5.19, 5.22 (each 2H, s, 2 ꢀ OCH2O),
5.96 (1H, d, J ¼ 2:3 Hz, H-50), 6.11 (1H, d, J ¼ 2:3 Hz,
H-30), 7.08 (1H, dd, J ¼ 8:1, 7.9 Hz, H-5), 7.19 (1H, dd,
J ¼ 8:1, 1.5 Hz, H-6), 7.33 (1H, dd, J ¼ 7:9, 1.5 Hz, H-
4), 7.90 (1H, d, J ¼ 15:8 Hz, H-ꢂ), 8.20 (1H, d, J ¼
15:8 Hz, H-ꢁ).
1 as yellow powder (53 mg, 67%). Mp 174–175 ꢁC; EI-
HR-MS m=z 316.0926 (calcd. for C17H16O6, 316.0947);
1H-NMR (acetone-d6) ꢀ (ppm): 3.86, 4.00 (each 3H, s,
2 ꢀ OCH3), 6.08 (1H, d, J ¼ 2:5 Hz, H-50), 6.11 (1H, d,
J ¼ 2:5 Hz, H-30), 6.89 (1H, d, J ¼ 8:1 Hz, H-5), 7.11
(1H, dd, J ¼ 8:1, 2.0 Hz, H-6), 7.24 (1H, d, J ¼ 2:0 Hz,
H-2), 7.69 (1H, d, J ¼ 15:5 Hz, H-ꢂ), 7.83 (1H, d, J ¼
15:5 Hz, H-ꢁ).
20-Hydroxy-2,3,40,60-tetrakis(methoxymethoxy)chal-
cone (14). By the same procedure as that used for the
preparation of 11, 14 was prepared from 7 and 9 and
purified by silica gel column chromatography (hexane–
ethyl acetate (7:3)) to give yellow powder (440 mg,
95%). Mp 67–68 ꢁC; EI-HR-MS m=z 464.1682 (calcd.
20,3,4,40,60-Pentahydroxychalcone (2). By the same
procedure as that used for the preparation of 1, 2 was
prepared from 12 and purified by silica gel column
chromatography (chloroform–methanol (9:1)) to give
yellow powder (41%). Mp 157–158 ꢁC (lit.11) 249–
252 ꢁC); EI-HR-MS m=z 288.0602 (calcd. for C15H12O6,
1
for C23H28O10, 464.1682); H-NMR (CDCl3) ꢀ (ppm):
1
3.48, 3.51, 3.52, 3.64 (each 3H, s, 4 ꢀ OCH3), 5.19,
5.19, 5.22, 5.29 (each 2H, s, 4 ꢀ OCH2O), 6.26 (1H, d,
J ¼ 2:5 Hz, H-50), 6.32 (1H, d, J ¼ 2:5 Hz, H-30), 7.08
(1H, dd, J ¼ 8:1, 7.9 Hz, H-5), 7.20 (1H, dd, J ¼ 8:1,
1.2 Hz, H-6), 7.32 (1H, dd, J ¼ 7:9, 1.2 Hz, H-4), 7.92
(1H, d, J ¼ 15:8 Hz, H-ꢂ), 8.22 (1H, d, J ¼ 15:8 Hz,
H-ꢁ).
288.0634); H-NMR (acetone-d6) ꢀ (ppm): 5.95 (2H, s,
H-30 and 50), 6.88 (1H, d, J ¼ 8:1 Hz, H-5), 7.07 (1H,
dd, J ¼ 8:1, 1.8 Hz, H-6), 7.19 (1H, d, J ¼ 1:8 Hz, H-2),
7.69 (1H, d, J ¼ 15:5 Hz, H-ꢂ), 8.05 (1H, d, J ¼ 15:5
Hz, H-ꢁ). The structure was confirmed by satisfactory
matching of the analytical data compared the reference
data, although melting point was significantly lower than
the reference figure.
20-Hydroxy-40,60-dimethoxy-3,4,5-tris(methoxymethoxy)-
chalcone (15). By the same procedure as that used for
the preparation of 11, 15 was prepared from 7a and 10
to give yellow powder (64%). Mp 110–111 ꢁC; EI-HR-
MS m=z 464.1635 (calcd. for C23H28O10, 464.1682);
1H-NMR (acetone-d6) ꢀ (ppm): 3.51 (6H, s, 2 ꢀ OCH3),
3.57, 3.87, 4.00 (each 3H, s, 3 ꢀ OCH3), 5.15 (2H, s,
OCH2O), 5.30 (4H, s, 2 ꢀ OCH2O), 6.09 (1H, d, J ¼
2:2 Hz, H-50), 6.12 (1H, d, J ¼ 2:2 Hz, H-30), 7.24 (2H,
s, H-2 and 6), 7.66 (1H, d, J ¼ 15:5 Hz, H-ꢂ), 7.94 (1H,
d, J ¼ 15:5 Hz, H-ꢁ).
2,20,3-Trihydroxy-40,60-dimethoxychalcone (3). By
the same procedure as that used for the preparation of
1, 3 was prepared from 13 to give yellow powder (60%).
Mp 175–176 ꢁC; EI-HR-MS m=z 316.0918 (calcd. for
1
C17H16O6, 316.0947); H-NMR (acetone-d6) ꢀ (ppm):
3.83, 3.94 (each 3H, s, 2 ꢀ OCH3), 6.04 (1H, d, J ¼ 2:4
Hz, H-50), 6.07 (1H, d, J ¼ 2:4 Hz, H-30), 6.68 (1H, dd,
J ¼ 8:1, 7.2 Hz, H-5), 6.87 (1H, d, J ¼ 7:2 Hz, H-6),
7.11 (1H, d, J ¼ 8:1 Hz, H-4), 8.12 (2H, m, H-ꢁ and ꢂ).
2,20,3,40,60-Pentahydroxychalcone (4). By the same
procedure as that used for the preparation of 1, 4 was
prepared from 14 and purified by silica gel column
chromatography (chloroform–methanol (9:1)) to give
yellow powder (12%). Mp 166–167 ꢁC; EI-HR-MS m=z
288.0627 (calcd. for C15H12O6, 288.0634); 1H-NMR
(acetone-d6) ꢀ (ppm): 5.96 (2H, s, H-30 and 50), 6.72
(1H, t, J ¼ 7:9 Hz, H-5), 6.89 (1H, dd, J ¼ 7:9, 1.3,
H-6), 7.15 (1H, dd, J ¼ 7:9, 1.3 Hz, H-4), 8.21 (1H, d,
J ¼ 15:6 Hz, H-ꢂ), 8.28 (1H, d, J ¼ 15:6 Hz, H-ꢁ).
20-Hydroxy-3,4,40,5,60-pentakis(methoxymethoxy)chal-
cone (16). By the same procedure as that used for the
preparation of 11, 16 was prepared from 7 and 10 and
purified by silica gel column chromatography (hexane–
ethyl acetate (7:3)) to give yellow powder (65%).
Mp 89–90 ꢁC; EI-HR-MS m=z 524.1908 (calcd. for
1
C25H32O12, 524.1894); H-NMR (CDCl3) ꢀ (ppm): 3.48
(3H, s, OCH3), 3.51 (6H, s, 2 OCH3), 3.54, 3.62 (each
3H, s, OCH3), 5.19, 5.19 (each 2H, s, 2 ꢀ OCH2O),
5.23 (4H, s, 2 ꢀ OCH2O), 5.30 (2H, s, OCH2O), 6.28
(1H, d, J ¼ 2:5 Hz, H-50), 6.31 (1H, d, J ¼ 2:5 Hz,
H-30), 7.16 (2H, s, H-2 and 6), 7.69 (2H, d, J ¼ 15:5 Hz,
H-ꢂ), 7.86 (1H, d, J ¼ 15:5 Hz, H-ꢁ).
20,3,4,5,-Tetrahydroxy-40,60-dimethoxychalcone (5).
By the same procedure as that used for the preparation
of 1, 5 was prepared from 15 to give yellow powder
(25%). Mp 163–164 ꢁC; EI-HR-MS m=z 332.0919
(calcd. for C17H16O7, 332.0896); 1H-NMR (acetone-
d6) ꢀ (ppm): 3.86, 4.00 (each 3H, s, 2 ꢀ OCH3), 6.07
(1H, d, J ¼ 2:4 Hz, H-50), 6.11 (1H, d, J ¼ 2:4 Hz, H-
30), 6.81 (2H, s, H-2 and 6), 7.61 (1H, d, J ¼ 15:5 Hz, H-
ꢂ), 7.80 (1H, d, J ¼ 15:5 Hz, H-ꢁ).
20,3,4-Trihydroxy-40,60-dimethoxychalcone10) (1). To
a stirred solution of 11 (101 mg, 0.25 mmol) in methanol
(5 ml) was added dropwise 3 M HCl (2 ml). The mixture
was refluxd for 45 min, diluted with water (5 ml), and
extracted with ethyl acetate. The organic layer was
successively washed with water and brine, dried over
anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography (chloroform–methanol (100:3)) to give
20,3,4,40,5,60-Hexahydroxychalcone (6). By the same
procedure as that used for the preparation of 1, 6 was
prepared from 16 and purified by silica gel column

196
J. NISHIDA and J. KAWABATA
R5
B
R5
R2
R4'O
OR6'
O
R4
R3
R4
R3
R4'O
OR6'
O
A
a
+
OHC
OH
R2
OH
11 R2 = R5 = H, R3 = R4 = OCH2OMe, R4' = R6' = Me
12 R2 = R5 = H, R3 = R4 = OCH2OMe, R4' = R6' = CH2OMe
13 R4 = R5 = H, R2 = R3 = OCH2OMe, R4' = R6' = Me
14 R4 = R5 = H, R2 = R3 = OCH2OMe, R4' = R6' = CH2OMe
15 R2 = H, R3 = R4 = R5 = OCH2OMe, R4' = R6' = Me
16 R2 = H, R3 = R4 = R5 = OCH2OMe, R4' = R6' = CH2OMe
7
R4' = R6' = CH2OMe 8a R2 = R5 = H, R3 = R4 = OCH2OMe
7a R4' = R6' = Me
9a R4 = R5 = H, R2 = R3 = OCH2OMe
10a R2 = H, R3 = R4 = R5 = OCH2OMe
R5
R4'O
OR6'
O
R4
R3
b
A
B
OH
R2
1 R2 = R5 = H, R3 = R4 = OH, R4' = R6' = Me
2 R2 = R5 = R4' = R6' = H, R3 = R4 = OH
3 R4 = R5 = H, R2 = R3 = OH, R4' = R6' = Me
4 R4 = R5 = R4' = R6' = H, R2 = R3 = OH
5 R1 = H, R3 = R4 = R5 = OH, R4' = R6' = Me
6 R1 = R4' = R6' = H, R3 = R4 = R5 = OH
Scheme 1. Synthesis of 1–6.
Reagents and conditions: (a) KOH, methanol, stirred for 72 h, rt.; (b) 3 M HCl, methanol, refluxed for 45 min.
chromatography (chloroform–methanol (9:1)) to give
yellow powders (11%). Mp 179–180 ꢁC; EI-HR-MS m=z
304.0566 (calcd. for C15H12O7, 304.0583); 1H-NMR
(acetone-d6) ꢀ (ppm): 5.95 (2H, s, H-30 and 50), 6.77
(2H, s, H-2 and 6), 7.61 (1H, d, J ¼ 15:6 Hz, H-ꢂ), 8.03
(1H, d, J ¼ 15:6 Hz, H-ꢁ).
water and brine, dried over Na2SO4 and evaporated to
give pure 18 (1.3 g, 71%). EI-MS m=z (%): 166 (100,
[M]þ).
Aureusidin12,13) (19). To a solution of 18 (100 mg,
0.6 mmol) and 3,4-dihydroxybenzaldehyde (83 mg, 0.6
mmol) in acetic acid (5 ml) was added conc. HCl
(0.2 ml). After stirring for 5 hr at room temperature, the
solution was poured into water and extracted with ethyl
acetate. The ethyl acetate solution was dried with
anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was subjected to preparative
HPLC (ODS, methanol–water–formic acid (60:40:0.1);
5.0 ml/min flow rate; UV 254 nm detection) to give 19
(tR 14 min) as yellow powder (12 mg, 7%). Mp 246–
248 ꢁC; EI-HR-MS m=z 286.0507 (calcd. for C15H10O6,
2-Chloro-20,40,60-trihydroxyacetophenone12) (17). To
a mixture of phloroglucinol (5.0 g, 39.7 mmol) and
chloroacetonitrile (2.5 ml, 39.7 mmol) in dioxane
(20 ml) was added anhydrous ZnCl2 (0.54 g, 3.96 mmol).
The solution was cooled to 0 ꢁC, and a 4.0 M HCl
solution in dioxane (60 ml) was added to it. The mixture
was left overnight, and further 4.0 M HCl in dioxane
(20 ml) was added to it. The precipitated imine was
collected by filtration and washed three times with ether.
The imine was dissolved in 100 ml of 1 M HCl and
heated at 100 ꢁC for 1 h. The solid was collected by
filtration, washed three times with water and dried under
vacuum to yield a pure acetophenone (17) as a pale
white solid which was used without further purification
(2.2 g, 28%). EI-MS m=z (%): 202 (26, [M]þ).
1
286.0477); H-NMR (methanol-d4) ꢀ (ppm): 6.02 (1H,
brs, H-7), 6.20 (1H, brs, H-5), 6.56 (1H, s, H-ꢁ), 6.82
(1H, d, J ¼ 8:1 Hz, H-50), 7.18 (1H, brd, 8.1 Hz, H-60),
7.47 (1H, brs, H-20).
4,6-Dimethoxybenzofuran-3(2H)-one13) (20). To a
solution of 18 (332 mg, 2.0 mmol) in DMF (10 ml) was
added K2CO3 (0.55 g, 4.0 mmol) and MeI (0.37 ml,
6.0 mmol). The solution was heated at 80 ꢁC for 1 h,
poured into water and extracted with ethyl acetate. The
ethyl acetate solution was dried with anhydrous Na2SO4,
filtered and evaporated under reduced pressure to yield
brown viscous oil. This oil was subjected to silica gel
column chromatography (hexane–ethyl acetate (1:1)) to
4,6-Dihydroxybenzofuran-3(2H)-one12) (18). To a
solution of 17 (2.2 g, 11.1 mmol) in methanol (50 ml)
was added NaOMe (1.6 g, 29.3 mmol), and the mixture
was refluxed for 2 h. After cooling, the solution was
acidified with 1 M HCl and evaporated under reduced
pressure. The resulting residue was extracted with ethyl
acetate, and the extract was successively washed with

DPPH Radical Scavenging Activity of Chalcones
197
give 20 as yellow powder (290 mg, 75%). EI-MS m=z
(%):194 (100, [M]þ).
6
5
4
3
2
1
0
A
4,6-Di-O-methylaureusidin (21). By the same proce-
dure as that used for the preparation of 19, 21 was
prepared from 20 and 3,4-dihydroxybenzaldehyde as
yellow powder (94%). Mp 170–171 ꢁC; EI-HR-MS m=z
314.0800 (calcd. for C17H14O6, 314.0790); 1H-NMR
(methanol-d4) ꢀ (ppm): 3.92, 3.93 (each 3H, s, 2 ꢀ
OCH3), 6.28 (1H, d, J ¼ 1:7 Hz, H-7), 6.53 (1H, d, J ¼
1:7 Hz, H-5), 6.62 (1H, s, H-ꢁ), 6.82 (1H, d, J ¼ 8:4 Hz,
H-50), 7.22 (1H, dd, J ¼ 8:4, 2.0 Hz, H-60), 7.47 (1H, d,
J ¼ 2:0 Hz, H-20).
Eriodictyol14) (22). To a solution of 2 (432 mg, 1.5
mmol) in methanol (50 ml) was added KF (100 mg,
1.7 mmol). The solution was refluxed for 24 h. The
reaction mixture was diluted with water (50 ml) and
extracted with ethyl acetate. The resulting ethyl acetate
extract was dried with anhydrous Na2SO4 and evapo-
rated under reduced pressure. The resulting residue was
subjected to HPLC (ODS, methanol–water–formic acid
(70:30:0.1); 5.0 ml/min flow rate; UV 254 nm detec-
tion), to give 22 (tR 12 min) as pink powder (173 mg,
40%). Mp 240–242 ꢁC (lit.15) 265–266 ꢁC); EI-HR-MS
288.0673 (calcd. for C15H12O6, 288.0634); 1H-NMR
(methanol-d4) ꢀ (ppm): 2.69 (1H, dd, J ¼ 17:0, 3.0 Hz,
H-3), 3.06 (1H, dd, J ¼ 17:0, 12.5 Hz, H-3), 5.27 (1H,
dd, J ¼ 12:5, 3.0 Hz, H-2), 5.87 (1H, d, J ¼ 2:3 Hz,
H-8), 5.89 (1H, d, J ¼ 2:3 Hz, H-6), 6.78–6.91 (3H, m,
H-20,50,60).
0
10
20
30
Time (min)
6
5
4
3
2
1
0
B
Results and Discussion
0
10
20
30
Chalcones 1–6 were prepared by condensation of
the corresponding acetophenones and benzaldehydes
(Scheme 1). Time-course characteristics for the radical
scavenging activity of 1–6 in acetone and methanol are
shown in Fig. 1. At 30 min, the relative radical scav-
enging equivalence of each compound, when that of ꢁ-
tocopherol in ethanol as a standard is designated as 2,
was as follows: 1, 2.1; 2, 3.8; 3, 1.6; 4, 3.4; 5, 4.4; 6, 4.6
in acetone; and 1, 5.8; 2, 4.0; 3, 4.8; 4, 3.9; 5, 5.6; 6, 5.1
in methanol.
Time (min)
Fig. 1. DPPH Radical Scavenging Activity of 1 ( ), 2 ( ), 3 ( ),
4 ( ), 5 ( ) and 6 ( ) in Acetone (A) and in Methanol (B).
at 7.50 (d, J ¼ 15:5 Hz) and ꢀ 8.09 (d, J ¼ 15:5 Hz),
and two meta-coupled doublet peaks of ꢀ 6.12 (d, J ¼
2:0 Hz) and 6.14 (d, J ¼ 2:0 Hz) were observed. These
signals are assignable to the corresponding quinone, 1q.
In addition, there appeared minor signals of ꢀ 6.45 (d,
J ¼ 10:1), 6.39, 6.41 and 6.92 (each s), and ꢀ 7.78 (dd,
J ¼ 10:1, 1.7 Hz). It is suggested that this latter set of
signals was due to a ring-closed aurone derivative.
Hence, the corresponding aurone, 4,6-di-O-methylaur-
eusidin (21) was synthesized according to the procedure
shown in Scheme 2, and oxidized with the DPPH radical
for comparison. In the 1H-NMR spectrum of the reaction
mixture of 21 and the DPPH radical in acetone-d6
(Fig. 2B), the same set of signals appeared as that in the
mixture of 1 and the DPPH radical.
Compound 1 has a 20-hydroxy-40,60-dimethoxylated
A-ring and 3,4-dihydroxylated B-ring, whereas 2 has a
20,40,60-trihydroxylated A-ring and the same B-ring as 1.
Interestingly, 1 showed a dramatic increase in DPPH
radical consumption when changing the solvent from
aprotic acetone to protic methanol, while 2 showed little
difference change in radical scavenging activity in eiter
solvent. The reaction mixture of 1 (or 2) with DPPH
radical in acetone-d6 (or methanol-d4) was directly
1
1
analyzed by H-NMR. In the H-NMR spectrum of the
reaction mixture of 1 with the DPPH radical in acetone-
d6 (Fig. 2A), characteristic signals of a 4-substituted o-
quinone ring at ꢀ 6.50 (d, J ¼ 10:3 Hz), ꢀ 6.68 (brs) and
7.69 (dd, J ¼ 10:3, 1.7 Hz), trans-double bond protons
1
The H-NMR analysis indicated the following reac-
tion of 1 in acetone: Compound 1 scavenges two
equivalents of the DPPH radical and is converted into

198
J. NISHIDA and J. KAWABATA
Fig. 2. 1H NMR Spectra of the Reaction Mixtures of 1 (A) and 21 (B) with the DPPH Radical in Acetone-d6.
Scheme 2. Synthesis of 19, 21 and 22.
Reagents and conditions: (a) i. ZnCl2, dioxane, HCl–dioxane solution; ii. 1 N HCl, 100 ꢁC, 1 h; (b) MeONa, methanol, refluxed for 2 h;
(c) 3,4-dihydroxybenzaldehyde, HOAc, conc. HCl, stirred for 5 h, rt.; (d) MeI, K2CO3, DMF, 80 ꢁC, 1 h; (e) KF, methanol, refluxed for 24 h.

DPPH Radical Scavenging Activity of Chalcones
199
- 2 H+
- 2
MeO
OMe
O
OH
OH
MeO
MeO
MeO
OMe
O
O
O
in acetone
O
O
H
H
1q
1
OH
O
O
OMe O
1q'
O
O
H
OH
OMe O
in methanol
- 2 H+
- 2
MeO
O
O
MeO
OH
OH
O
O
Complex
mixture
OMe O
OMe O
21
21q
Fig. 3. Plausible Oxidation Mechanism of 20,3,4-Trihydroxy-40,60-dimethoxychalcone (1) by the DPPH Radical.
the corresponding quinone, 1q. The A-ring of 1q then
slowly rotates to yield a 1q0 conformer, by which
intramolecular addition of a phenolic OH on the A-ring
to an ꢁ-carbon of the bridged enone and successive
deprotonation gives aurone 21. Resulting 21 can
scavenge two more DPPH radicals and is converted
into 21q (Fig. 3). Although 21q was detected in the 1H-
NMR spectrum of the reaction mixture, the DPPH
radical scavenging equivalence of 1 at 30 min was 2.1 in
acetone, and hence, contribution of the formation of 21q
to the antiradical efficiency of 1 might be limited.
In contrast, the 1H-NMR spectrum of the reaction
mixture of between 1 and the DPPH radical in methanol
gave a considerably complex pattern composed of many
small peaks (data not shown). By taking into account
nearly six DPPH radical consumption of 1 in methanol,
the intermediate quinone (1q) produced was quickly
cyclized to yield aurone 21 and then its quinone 21q,
which might be further oxidized to a complex mixture.
The higher reaction rate of 1 in methanol compared to
that in acetone was probably due to solvation of the A-
ring hydroxyl and carbonyl groups, which would reduce
the intramolecular hydrogen bond between them and
favor the rotation of the A-ring to a suitable position for
cyclization to aurone 21.
cyclized by the DPPH radical to give ring closure
products. Therefore, the corresponding aurone (aureusi-
din, 19) and flavanone (eriodictyol, 22) were synthesized
according to the procedure shown in Scheme 2, respec-
tively, for comparison. The 1H-NMR spectra of the
reaction mixtures of 19 and 22 with the DPPH radical
in acetone-d6 are shown in Fig. 4B and 4C, respectively.
In Fig. 4B, signals characteristic of aurone quinone 19q
were observed, which are supported by comparing with
the signals in Fig. 2B. The higher field shift of H-ꢁ,
ꢀ 6.42, in 19q compared with ꢀ 6.68 in 21q can be
accounted for by the presence of an intramolecular
hydrogen bond in 19q, which would reduce the electron-
withdrawing nature of the ketone conjugated with an
exocyclic double bond. In Fig. 4C, the characteristic
doublet and doublet of doublet signals of H-50 and 60 in
corresponding 22q at ꢀ 6.48 (d, J ¼ 10:0 Hz) and 7.42
(dd, J ¼ 10:0, 2.0 Hz), respectively, and a broad singlet
peak at ꢀ 6.53 assignable to H-20 are shown, together
with A-ring protons of ꢀ 6.00 and 6.07. All of the signals
found in Fig. 4B and 4C also exist in Fig. 4A, which
suggests that 2 was readily oxidized by DPPH radical
and spontaneously converted to both 19q and 22q.
These results indicate the reaction of 2 in acetone:
Compound 2 scavenges two equivalents of the DPPH
radical and is converted to 2q. Resulting 2q cyclizes
more readily than 1q, since free 60-OH exists in 2q in
place of 60-OMe in 1q. When an intramolecular
Michael-type addition occurs on C-ꢂ, regeneration of
Figure 4A shows the 1H-NMR spectrum of the
reaction mixture of 2 with the DPPH radical in
acetone-d6. The Lack of signals of the trans-double
bond protons indicats that 2 was readily oxidized and

200
J. NISHIDA and J. KAWABATA
Fig. 4. 1H NMR Spectra of the Reaction Mixtures of 2 (A), 19 (B) and 22 (C) with the DPPH Radical in Acetone-d6.
the C-4 ketone would directly give 22q. However, if
cyclization occurs on C-ꢁ, re-aromatization of the B-
ring by deprotonation at C-ꢂ would yield catechol 19,
which could spontaneously undergo oxidation by con-
suming two equivalents of the DPPH radical and convert
to its quinone, 19q. The peak area ratio of the
cyclization products in Fig. 4A indicats that the intra-
molecular attack occurred preferentially on C-ꢁ to give
aurone quinone 19q, rather than 22q. This characteristic
is in accordance with the radical scavenging activity of
2, reaching four equivalents. Unexpectedly, the radical
scavenging reaction of 2 in methanol was basically the
respectively. In each case, the activity was lower than
that of 1 and 2. This result suggests that 3 and 4 were
reluctantly oxidized and converted into 3q and 4q
compared to the rapid oxidation of 1 and 2. The slower
conversion rate to their quinones might have resulted
in reduction of the total radical scavenging activity,
although partial cyclization occurred from 3q and 4q.
In contrast, pyrogallol analogues 5 and 6 showed
similar DPPH radical consumption of 4.4 and 4.6 in
acetone, and of 5.6 and 5.1 in methanol, respectively. It
is known that the pyrogallol-type phenolic acid, gallic
acid, consumed more than four DPPH radicals in both
aprotic and protic solvents.5) In addition, the oxidized
galloquinone has a tendency to dimerize, since an
additional hydroxyl group has an electron-releasing
nature; thus, the quinone could act as a nucleophile to
add another molecule of quinone. This easy intermo-
lecular-coupling nature complicates the whole oxidation
reaction of pyrogallols and may be a cause of their
enhanced radical scavenging efficiency. However, the
detailed reaction scheme is still unclear, since NMR
profiles of the reaction mixtures of 5 and 6 with the
DPPH radical were complicated and difficult to fully
analyze.
1
same as that in acetone. An analysis of H-NMR data
from the reaction mixture of 2 with the DPPH radical in
methanol suggests that 2 was oxidized by the DPPH
radical and converted to 22q and 19q (data not shown)
as shown with the reaction in acetone. It is still unclear
why 19q did not undergo further oxidation which might
have been triggered by nucleophilic addition of a
methanol molecule to the B-ring quinone2) unlike 21q.
The presence of an intramolecular hydrogen bond in 19q
could reduce the electron-withdrawing property of the
ketone as shown by the chemical shift difference in H-ꢁ
of 19q (ꢀ 6.42) and 21q (ꢀ 6.68) just described. Thus, the
electron density of the quinone ring would be higher in
19q than that in 21q, which would be unfavorable for
undergoing nucleophilic attack (Fig. 5).
In summary, the radical scavenging activity of
polyhydroxychalcones was dependent on their hydroxyl-
ation pattern. Concerning the B-ring, pyrogallol-type
3,4,5-trihydroxylated chalcones showed higher activity
than the catechol-type 3,4-dihydroxylated analogues,
whereas the activity of the 2,3-dihydroxylated isomers
was low. This trend is comparable to those found in
Compounds 3 and 4 are the 2,3-dihydroxylated
analogues of 1 and 2, respectively. In acetone, the
radical scavenging equivalence of 3 is 1.6, and of 4 is
3.4, while in methanol, the figures are 4.8 and 3.9,

DPPH Radical Scavenging Activity of Chalcones
201
O
HO
O
O
O
O
H
22q
b
- 2 H+
- 2
HO
OH
a
O
O
HO
OH
O
OH
OH
b
in acetone
O
O
O
H
H
in methanol
2
2q
19
a
- 2 H+
- 2
HO
OH
OH
HO
O
O
O
O
O
O
O
O
H
H
19q
Fig. 5. Plausible Oxidation Mechanism for 20,3,4,40,60-Pentahydroxychalcone (2) by the DPPH Radical.
Biochem., 68, 1221–1227 (2004).
other flavonoids and catechines. Interestingly, methyl
etherification of the A-ring hydroxyls in 3,4-hydroxy-
lated chalcones greatly affected their antiradical effi-
ciency. This phenomenon might be accounted for by a
difference in the readiness for cyclization of the
chalcone quinones to aurones and/or flavanones and
by intramolecular hydrogen bonding between an A-ring
hydroxyl and a C-ring carbonyl in the resulting cyclized
quinone intermediates.
5) Kawabata, J., Okamoto, Y., Kodama, A., Makimoto, T.,
and Kasai, T., Oxidative dimmers produced from proto-
catechuic and gallic esters in the DPPH radical scaveng-
ing reaction. J. Agric. Food Chem., 50, 5468–5471
(2002).
6) Tazaki, H., Taguchi, D., Hayashida, T., and Nabeta, K.,
Stable isotope-labeling studies on the oxidative coupling
of caffeic acid via o-quinone. Biosci. Biotechnol. Bio-
chem., 65, 2613–2621 (2001).
7) Tazaki, H., Kawabata, J., and Fujita, T., Novel oxidative
dimer from caffeic acid. Biosci. Biotechnol. Biochem.,
67, 1185–1187 (2003).
Acknowledgment
8) Krishnamachari, V., Levine, H. L., and Pare, W. P.,
Flavonoid oxidation by the radical generator AIBN:
a unified mechanism for quercetin radical scavenging.
J. Agric. Food Chem., 50, 4357–4363 (2002).
We are grateful to Mr. Kenji Watanabe and Dr. Eri
Fukushi, of the GC–MS and NMR Laboratory of our
school, for measuring mass spectra.
9) Sang, S., Cheng, X., Stark, E. R., Rosen, T. R., Yang,
S. C., and Ho, C.-T., Chemical studies on antioxidant
mechanism of tea catechins: analysis of radical reaction
products of catechin and epicatechin with 2,2-diphenyl-
1-picrylhydrazyl. Bioorg. Med. Chem., 10, 2233–2237
(2002).
10) Wang, Y., Tan, W., Li, Z. W., and Li, Y., A facile
synthetic approach to prenylated flavanones: first total
syntheses of (ꢂ)-bonannione A and (ꢂ)-sophoraflava-
none A. J. Nat. Prod., 64, 196–199 (2001).
11) Sogawa, S., Nihiro, Y., Ueda, H., Izumi, A., Miki, T.,
Matsumoto, H., and Satoh, T., 3,4-Dihydroxychalcones
as potent 5-lipoxygenase and cyclooxygenase inhibitors.
J. Med. Chem., 36, 3904–3909 (1993).
12) Beney, C., Mariotto, M.-A., and Boumendjel, A., An
efficient synthesis of 4,6 dimethoxyaurones. Hetero-
cycles, 55, 967–972 (2001).
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