Miscrowave-assisted efficient synthesis of polymethoxyacetophenones and natural polymethoxyflavones, and thier inhibitory effects on melanogenesis

Article abstract of DOI:10.3987/COM-07-11054

Microwave, assisted Friedel-Crafts acetylation of polymethoxybenzenes with acetic anhydride in the presence of In(CF₃SO₃)₃ under solvent-free conditions was achieved rapidly to give polymethoxyacetophenones in high yields. Microwave-assisted benzoylation of 2'-hydroxypolymethoxyacetophenones with polymethoxybenzoyl chlorides, followed by the rearrangement of the resulting benzoates and the final formation of natural polymethoxyflavones was achieved rapidly and efficiently. The polymethoxyflavones showed inhibitory effects on melanogenesis in human melanoma cells.

Full text of DOI:10.3987/COM-07-11054

HETEROCYCLES, Vol. 71, No. 7, 2007
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HETEROCYCLES, Vol. 71, No. 7, 2007, pp. 1589 - 1600. © The Japan Institute of Heterocyclic Chemistry
Received, 14th March, 2007, Accepted, 2nd May, 2007, Published online, 8th May, 2007. COM-07-11054
MICROWAVE-ASSISTED
EFFICIENT
SYNTHESIS
OF
POLYMETHOXYACETOPHENONES
AND NATURAL
POLYMETHOXYFLAVONES, AND THEIR INHIBITORY EFFECTS ON
MELANOGENESIS
Masao Tsukayama,a* Eiji Kusunoki,a Mohammad M. Hossain,a Yasuhiko
Kawamura,a and Shinji Hayashib
aDepartment of Chemical Science and Technology, Faculty of Engineering, The
University of Tokushima, Minamijosanjima-cho, Tokushima 770-8506, Japan
bNOF Corporation Oleochemicals Research Laboratory, 1-56 Ohama-cho,
Amagasaki-shi, Hyogo 660-0095, Japan
e-mail:tukayama@mb.tcn.ne.jp
Abstract
polymethoxybenzenes with acetic anhydride in the presence of In(CF3SO3)3
under solvent-free conditions was achieved rapidly to give
–
Microwave-assisted
Friedel-Crafts
acetylation
of
polymethoxyacetophenones in high yields. Microwave-assisted benzoylation of
2’-hydroxypolymethoxyacetophenones with polymethoxybenzoyl chlorides,
followed by the rearrangement of the resulting benzoates and the final formation
of natural polymethoxyflavones was achieved rapidly and efficiently. The
polymethoxyflavones showed inhibitory effects on melanogenesis in human
melanoma cells.
INTRODUCTION
Aromatic ketones are very useful and important as starting materials and/or synthetic intermediates in
synthetic organic chemistry. They have been synthesized by Friedel-Crafts acylation of aromatic
compounds, which needs usually stoichiometric amounts of a Lewis acid, such as AlCl3.1 Recently, the
catalytic Friedel-Crafts acylation of various substituted benzenes have been carried out by use of
hafnium(IV) triflate.2 Aromatic ketones have also been synthesized by solvent-free Friedel-Crafts
acylation of aromatic compounds in the presence of catalytic amounts of perfluoroalkanoic anhydride and
bismath triflate.3 Moreover, microwave(MW)-assisted Friedel-Crafts acylation of aromatic compounds
has been carried out in the presence of catalytic Lewis acids under solvent-free conditions.4-7 However, as
far as we know, the synthesis of highly oxygenated acetophenones has not been conducted yet in the

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presence of a catalytic amount of Lewis acids under microwave irradiation (MWI). The synthetic
problems of polymethoxyacetophenones remain to be resolved. Therefore, we wish to report here on
MW-assisted Friedel-Crafts acylation of polymethoxybenzenes with acetic anhydride in catalytic amounts
of In(CF3SO3)3 under solvent-free conditions.
Flavonoids are present in a wide variety of edible plants, especially in Citrus species. The highly
methoxylated flavones exhibit higher biological activity even though they occur in much lower
concentrations.8
Nobiletin
(3’,4’,5,6,7,8-hexamethoxyflavone)
and
tangeretin
(4’,5,6,7,8-pentamethoxyflavone) in Citrus aurantium were easily synthesized under microwave
irradiation and they have shown inhibitory effects on melanogenesis in human HM3KO melanoma
cells.9,10 Recently, new 5,6,7,8,3’,4’,5’-heptamethoxyflavone (Eupatorium coelestinum) (4),11
5,7,8,3’,4’,5’-hexamethoxyflavone (Murraya paniculata) (5),12 and 5,7,8,3’,4’-pentamethoxyflavone
(Citrus hassaku) (6),13 were isolated and their structures were confirmed by spectroscopic analysis. As a
continuation of our studies on the MW-assisted synthesis of polymethoxyflavones, we have examined the
efficient total synthesis of polyoxygenated flavones 4, 5,
6
and non-natural
5,7,8,2’,6’-pentamethoxyflavone (7) from the obtained polymethoxyacetophenones under solvent-free
conditions or with minimal use of organic solvents by MWI owing to the approach to an
environmentally-friendly methodology. We also wish to report here for the first time on the inhibitory
effects of these flavones on melanogenesis in human melanoma cells.
RESULTS AND DISCUSSION
MW-assisted methylation of phloroglucinol, pyrogallol and resorcinol in a minimal acetone by
(MeO)2SO2-K2CO3 method gave the methyl ethers 1c, 1d and 1e for 4 min in 90, 87 and 91% yields,
respectively. In a similar manner, MW-assisted methylation of 2,5-dihydroxy-1,3-dimethoxybenzene,
which
was
prepared
from
2,6-dimethoxy-1,4-benzoquinone,14
and
2-hydroxy-1,3,4,5-tetramethoxybenzene (2b”), which was synthesized according to Scheme 1, gave
methyl ethers 1b (87% yield) and 1a (88% yield) for 10 min, respectively.
MW-assisted Friedel-Crafts acetylation of polymethoxybenzenes 1a and 1b with acetic anhydride in the
presence of catalytic In(CF3SO3)3 under solvent-free conditions was achieved for 3-15 min to give tetra-
and pentamethoxyacetophenones 2a and 2b in high yields (Table 1). In a similar manner, MW-assisted
Friedel-Crafts acetylation of the other methyl ethers 1c, 1d and 1e was carried out rapidly to give
acetophenones 2c, 2d and 2e in good yields (Table 1). The above results revealed that the MW-assisted
acetylation of polymethoxybenzenes with acetic anhydride in the presence of In(CF3SO3)3 under
solvent-free conditions is an excellent method for the synthesis of polymethoxyacetophenones, because
of rapid reaction, clean experiments, high yields and solvent-free conditions. Pentamethoxyacetophenone

HETEROCYCLES, Vol. 71, No. 7, 2007
1591
Table 1 Microwave-assisted synthesis of polymethoxyacetophenones under solvent-free
conditions
MW
MeO
MeO
Ac2O, In(CF3SO3)3
75-160ºC, 1-15 min
Ac
1a-e
2a-e
Methoxybenzene
In(CF3SO3)3 (equiv)
Time (min)
15
Acetophenone (%)
Temp (ºC)
145-160
OMe
OMe
MeO
MeO
OMe
MeO
MeO
OMe
Ac
0.015
77
OMe
1a
OMe
2a
OMe
OMe
MeO
MeO
OMe
OMe
MeO
MeO
OMe
Ac
0.006
0.009
85-95
3
1
87
80
OMe
OMe
1b
2b
OMe
Ac
110-120
OMe
OMe
1c
OMe
2c
OMe
MeO
MeO
OMe
Ac
MeO
MeO
OMe
OMe
0.012
0.012
75-90
7
6
73
60
1d
1e
2d
2e
OMe
Ac
85-100
OMe
OMe
OMe
OMe
MW (250 W)
MeO
OMe
Ac
MeO
MeO
OMe
OH
+ H2O , NaOH
r t
m-CPBA / TFA
CH2Cl2 , 20-25ºC
5 min
OAc
MeO
2b
MeO
MeO
2b"
MW
2b'
MW
(MeO)2SO2
K2CO3
AlCl3 , MeCN
reflux, 1.5 min
reflux, 10 min
OMe
OMe
OMe
MW
AlCl3 , MeCN
MW
Ac2O
MeO
R
OH
Ac
MeO
MeO
OMe
Ac
MeO
OMe
OMe
reflux, 1.5 min
In(CF3SO3)3
MeO
MeO
2a
145-160ºC, 15 min
MeO
1a
3a
: R=OMe
3b
: R=H
Scheme 1
2a was synthesized according to the pathway of Scheme 1. So far, pentamethoxybenzene (1a) in Table 1
has been synthesized by methylation of 2,3-dihydroxy-1,4,5-trimethoxybenzene, which has been
synthesized in less than 60% yield by Dakin reaction of 2’-hydroxytrimethoxyacetophenone 3b with 6%
H2O2-NaOH.9,15 The oxidative reaction of 2b should be improved to obtain the corresponding phenol 2b”

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HETEROCYCLES, Vol. 71, No. 7, 2007
in a better yield. Baeyer-Villiger reaction of 2b with m-chloroperbenzoic acid (m-CPBA) in the presence
of trifluoroacetic acid (TFA) in CH2Cl2 gave the desired acetate 2b’ for 18 h at 25˚C in 63% yield.
Furthermore, MW (250w)-assisted Baeyer-Villiger reaction of 2b with m-CPBA/TFA in CH2Cl2 gave the
corresponding acetate 2b’ for 5 min at 20-25˚C in 73% yield. From this result, MW-assisted conditions
were found to be more effective for Baeyer-Villiger reaction of acetophenone 2b. Hydrolysis of 2b’ with
aqueous NaOH, followed by the MW-assisted methylation of the resultant phenol 2b” gave the desired
pentamethoxybenzene 1a, which was efficiently converted into the pentamethoxyacetophenone 2a
(Scheme 1 and Table 1). MW-assisted demethylation of the acetophenones 2a and 2b with AlCl3 in
MeCN gave 2’-hydroxytetramethoxyacetophetone 3a and 2’-hydroxytrimethoxyacetophetone 3b for 1.5
min in 84% and 81% yield, respectively (Scheme 1).
MW-assisted benzoylation of acetophenones 3a and 3b with substituted benzoyl chlorides 8-10 in
minimal pyridine was carried out from 1.5 to 5 min under reflux to give the corresponding benzoates
11-17 in 75-89% yields, respectively. MW-assisted Baker-Venkataraman rearrangement of benzoates
11-17 in the presence of KOH in minimal pyridine was achieved for 4-7 min under reflux to afford the
corresponding diketones 12-18 in 86-96% yields, respectively. MW-assisted cyclization reaction of 12-18
O
OMe
OMe
MW
MeO
R
O C
MeO
R
OH
Me
Ar
pyridine
+
Ar COCl
Me
reflux, 1.5-5 min
MeO
O
O
MeO
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
3a
: R=OMe
8: Ar=
11: R=OMe, Ar=
3b
: R=H
13: R=H, Ar=
9
: Ar=
OMe
OMe
OMe
OMe
10
: Ar=
OMe
15: R=H, Ar=
OMe
17
: R=OMe, Ar=
OMe
OMe
OMe
MW
MW
MeO
R
OH
MeO
R
O
pyridine
H2SO4,
KOH,
AcOH
Ar
A
reflux, 1.5-5 min
reflux, 4-7 min
Ar
MeO
O
O
OMe O
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
4: R=OMe, Ar=
5: R=H, Ar=
6: R=H, Ar=
12: R=OMe, Ar=
14: R=H, Ar=
B
OMe
OMe
B
B
OMe
OMe
OMe
OMe
OMe
16: R=H, Ar=
OMe
OMe
OMe
OMe
7
18: R=OMe, Ar=
: R=OMe, Ar=
OMe
Scheme 2

HETEROCYCLES, Vol. 71, No. 7, 2007
1593
with concd H2SO4 in minimal AcOH was conducted from 1.5 to 5 min under reflux to give the
1
corresponding flavones 4-7 in 83-94% yields, respectively (Scheme 2). The H-NMR spectra and other
physical properties of the synthetic flavones 4, 5 and 6 were identical with those of each natural sample of
3’,4’,5’,5,6,7,8-heptamethoxy-, 3’,4’,5’,5,7,8-hexamethoxy-, and 3’,4’,5,7,8-pentamethoxyflavone. From
the results of the MW-assisted total synthesis of flavone 7, three successive reactions were not shown to
be affected by the steric hindrance of 2,6-dimethoxybenzoyl group.
The new synthetic methodology under microwave-enhanced conditions is also environmentally friendly
and results in high yields of the polymethoxyacetophenones and polymethoxyflavones.
The cytotoxicity of the synthetic polymethoxyflavones (4, 5, nobiletin and tangeretin) on human HM3KO
melanoma cells10 was investigated by MTT assay, and the results are shown in Figure 1. The inhibitory
effects of the synthetic polymethoxyflavones (4, 5 and 6) as well as nobiletin and tangeretin on
melanogenesis in HM3KO melanoma cells LINE, HM3KO,10 were carried out at different concentrations,
and the results are shown in Figure 2. From Figure 1, it is obvious that the highly methoxylated B-ring (4,
5 and nobiletin; see Scheme 2) has a propensity to increase the cytotoxicity of polymethoxyflavones (4, 5
and nobiletin) at concentrations of 10-20 µM. In contrast, polymethoxyflavones having the completely
methoxylated A-ring (4, nobiletin and tangeretin; see Scheme 2) show the upward tendency of inhibitory
effect on melanogenesis. Figure 2 shows that the polymethoxflavones (4, 5, nobiletin and tangeretin)
suppress the production of melanin upto 40-60% at concentrations of 5-10 µM. These results clearly
indicate that both the cytotoxicity and inhibitory effects of polymethoxflavones on melanogenesis are
affected by the number of methoxy groups on a flavone skeletone. Therefore, hepta- and
hexamethoxyflavones (4 and 5) as well as nobiletin and tangeretin would be utilizable as an agent for the
treatment of skin pigmentation such as spots and freckles induced by UV light.10
120
100
80
PMF (4)
PMF (5)
60
nobiletin
tangeretin
40
20
0
1
10
100
Concentration of Polymethoxyfalvones (μM)
Fig 1 Cytotoxicity Assay of Polymethoxyflavones on Human Melanoma Cells

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HETEROCYCLES, Vol. 71, No. 7, 2007
120
100
80
60
40
20
0
5μM
10μM
Control
PMF(4)
PMF(5)
PMF(6)
nobiletin
tangeretin
Concentration of Polymethoxyfalvones (μM)
Fig 2 Inhibitory Effects of Polymethoxyflavones on Melanogenesis
EXPERIMENTAL
All the melting points were taken on a Yanaco MP-J3 micro melting-point apparatus and were
1
uncorrected. The H-NMR spectra were recorded with a JEOL EX-400 spectrometer (400 MHz), using
tetramethylsilane as internal standard (δ, ppm). The IR spectra were recorded on an FT/IR-460 Plus
(JASCO) spectrophotometer using KBr pellets, and the UV spectra were recorded on a Hitachi U-2000
spectrophotometer. Elemental analyses were performed with a J-Science Lab. CHN corder JM-10. A
microwave oven (650 W and 2.45 GHz, modified properly by fitting a condenser and a thermo-sensor
through the holes made in the roof; Shikoku Instrumentation Co., Ltd, Japan) was used as a reaction
apparatus. Column chromatography and thin-layer chromatography (TLC) were carried out on Kieselgel
60 (70-230 mesh) and Kieselgel 60 F-254 (Merck).
Polymethoxybenzenes (1c-e) from Phloroglucinol, Pyrogallol and Resorcinol.
A mixture of polyphenols (each 500 mg, 3.96-4.45 mmol), (MeO)2SO2 (1.9-2.2 mL) and K2CO3 (25-27
mmol) in a minimal acetone (8-9 mL) was refluxed with stirring for 4 min (30 s × 8 times, 30 s
interval/irradiation) under MWI. The reaction mixture was extracted with AcOEt, and the extract was
washed with 6% HCl, water and dried (Na2SO4). The resulting compounds were chromatographed over a
silica gel column to give polymethoxybenzenes (1c, 1d and 1e) in high yields: 1,3,5-trimethoxybenzene
(1c, mp 51-53 ˚C, 91% yield as colorless needles); 1,2,3-trimethoxybenzene (1d, mp 43-45 ˚C, 87% yield
as colorless plates); 1,3-dimethoxybenzene (1e, 91% yield as a pale yellow oil) (Table 1).
Polymethoxyacetophenones (2c-e).
A mixture of polymethoxybenzenes 1c-e (each 500 mg, 2.97-3.62 mmol), Ac2O (2 equiv) and
In(CF3SO3)3 (0.006-0.012 equiv) was stirred for 1-7 min (30 s × 2-14 times, 30 s interval/irradiation) at
75-120 ˚C under MWI. The resulting compounds were chromatographed over a silica gel column to give

HETEROCYCLES, Vol. 71, No. 7, 2007
1595
the acetophenones (2c-e); acetophenone 2c: mp 101-103 ˚C, acetophenone 2d: a pale yellow oil and
acetophenone 2e: a pale yellow oil (Table 1).
1,2,3,5-Tetramethoxybenzene (1b).
A mixture of 2,5-dihydroxy-1,3-dimethoxybenzene9 (5.00 g, 29.4 mmol), (MeO)2SO2 (11.2 mL, 118
mmol) and K2CO3 (12.2 g, 88.1 mmol) in acetone (25 mL) was refluxed with stirring for 4 min (30 s × 8
times, 30 s interval/irradiation) under MWI. The resulting compounds were chromatographed over a silica
gel column to give the tetramethoxybenzene 1b14 (5.17 g, 87%) as a pale yellow oil (Table 1).
2’,3’,4’,6’-Tetramethoxyacetophenone (2b).
A mixture of the tetramethoxybenzene 1b (5.00 g, 25.2 mmol), Ac2O (4.77 mL, 50.1 mmol) and
In(CF3SO3)3 (97 mg, 0.17 mmol) was irradiated with stirring for 3 min (15 s × 12 times, 30 s
interval/irradiation) under MWI. The reaction mixture was extracted with AcOEt, and the extract washed
with water, and dried (Na2SO4). The resulting compound was chromatographed over a silica gel column
(AcOEt:hexane=1:2 as a solvent) to give the acetophenone 2b14 (2.25 g, 87%) as colorless needles, mp
49.5-51 ˚C.
2-Acetoxy-1,3,4,5-tetramethoxybenzene (2b’).
CF3CO2H (0.11 ml, 1.5 mmol) was added into a solution of m-CPBA (460 mg, 1.99 mmol) in CH2Cl2 (3
mL) with stirring in an ice-water bath, and then the mixture was stirred for 30 min. The whole mixture,
which tetramethoxyacetophenone 2b (300 mg, 1.25 mmol) was added to the above mixture, was
irradiated with stirring at 20-25 ˚C for 5 min (10 s × 30 times, 30 s interval cooling by ice/irradiation)
under MWI (250W). The resulting compound was filtered and the filtrate was extracted with CH2Cl2, and
the extract washed with aqueous NaHCO3 solution and water, and dried (Mg2SO4). The resulting
compound was chromatographed over a silica gel column to give the desired acetoxybenzene 2b’ (232
mg, 73%) as colorless needles, mp 60-62 ˚C. 1H-NMR (CDCl3) δ 2.33 (3H, s, OCOCH3), 3.80, 3.83, 3.87
and 3.88 (each 3H, s, OCH3), 6.33 (1H, s, Ar-H).
2,3,4,6-Tetramethoxyphenol (2b”).
The acetate 2b’ (500 mg, 1.95 mmol) was hydrolyzed with 10% aqueous NaOH for 30 min at 25 ˚C to
give the phenol 2b” (368 mg, 87%) as pale yellow needles, mp 84-85˚C. 1H-NMR (CDCl3) δ 3.83, 3.84,
3.87 and 3.89 (each 3H, s, OCH3), 5.25 (1H, s, OH), 6.33 (1H, s, Ar-H).
1,2,3,4,5-Pentamethoxybenzene (1a).
A mixture of 2b” (500 mg, 2.33 mmol), (MeO)2SO2 (0.45 mL, 4.7 mmol) and K2CO3 (691 mg, 5.0 mmol)
in acetone (5 mL) was irradiated with stirring for 10 min (30 s × 20 times, 30 s interval/irradiation) under
MWI. The resulting compound was chromatographed over a silica gel column to give the
1
pentamethoxybenzene 1a14 (471 mg, 88%) as pale yellow needles, mp 55.5-57 ˚C. H-NMR (CDCl3) δ
3.83 (6H, s, OCH3 × 2), 3.85 (6H, s, OCH3 × 2), 3.95 (3H, s, OCH3), 6.30 (1H, s, Ar-H).

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HETEROCYCLES, Vol. 71, No. 7, 2007
2’,3’,4’,5’,6’-Pentamethoxyacetophenone (2a).
A mixture of 1a (500 mg, 2.19 mmol), Ac2O (10 mL) and In(CF3SO3)3 (8 mg, 0.015 mmol) was
irradiatied with stirring for 15 min (30 s × 30 times, 30 s interval/irradiation) under MWI. The resulting
compound was chromatographed over a silica gel column to give the acetophenone 2a14 (456 mg, 77%)
as a pale yellow oil.
2’-Hydroxy-3’,4’,5’,6’-tetramethoxyacetophenone (3a).
A solution of 2a (1.06 g, 3.93 mmol) and AlCl3 (1.05 g, 7.86 mmol) in MeCN (8 mL) was irradiated with
stirring for 1.5 min (10 s × 9 times, 15 s interval/irradiation) under MWI. To the reaction mixture was
added 6% HCl (10 mL) and the whole mixture was stirred for 30 min. The resulting compound was
chromatographed over a silica gel column (AcOEt:hexane=2:3 as a solvent) to give the
1
2’-hydroxyacetophenone 3a14 (850 mg, 85%) as a pale yellow oil. H-NMR (CDCl3) δ 2.67 (3H, s,
COCH3), 3.81, 3.86, 3.95 and 4.08 (each 3H, s, OCH3), 13.16 (1H, s, OH).
2’-Hydroxy-3’,4’,6’-trimethoxyacetophenone (3b).
A solution of 2b (6.0 g, 25 mmol) and AlCl3 (6.60 g, 49.9 mmol) in MeCN (30 ml) was irradiated with
stirring for 1.5 min (10 sec × 9 times, 15 sec interval/irradiation) under MWI. The resulting compound
was chromatographed over a silica gel column to give the 2’-hydroxyacetophenone 3b16 (4.49 g, 81%) as
pale yellow needles, mp 111-113 ˚C. 1H-NMR (CDCl3) δ 2.62 (3H, s, COCH3), 3.82, 3.90 and 3.94 (each
3H, s, OCH3), 5.97 (1H, s, Ar-H), 13.80 (1H, s, OH).
2’-(3,4,5-Trimethoxybenzoyloxy)-3’,4’,5’,6’-tetramethoxyacetophenone (11).
A mixture of 3a (410 mg, 1.60 mmol), fresh 3,4,5-trimethoxybenzoyl chloride (8) (424 mg, 1.94 mmol)
and pyridine (3 mL) was refluxed with stirring for 1.5 min (30 s × 3 times, 30 s interval/irradiation) under
MWI. The resulting mixture was worked up in the same manner as in the case of 3a to give the ester 11
(541 mg, 75%) as colorless needles, mp 73.5-75.5 ˚C. 1H-NMR (CDCl3) δ 2.49 (3H, s, COCH3), 3.83 (3H,
s, OCH3), 3.90 (3H, s, OCH3), 3.93 (6H, s, OCH3 × 2), 3.94(3H, s, OCH3), 3.95 (3H, s, OCH3), 3.99 (3H,
s, OCH3), 7.41 (2H, s, Ar-H × 2).
1-(2-Hydroxy-3,4,5,6-tetramethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,3-propanedione (12).
A mixture of 11 (480 mg, 1.06 mmol), powdered KOH (300 mg, 5.33 mmol) and pyridine (5 mL) was
refluxed with stirring for 5 min (30 s × 10 times, 30 s interval/irradiation) under MWI. The reaction
mixture was extracted with AcOEt, after which the extract was washed with water and dried (Na2SO4).
The resulting compound was chromatographed over a silica gel column to give the diketone 12 (460 mg,
92%) as pale yellow needles, mp 79-81 ˚C.
5,6,7,8,3’,4’,5’-Heptamethoxyflavone (4).
A solution of the diketone 12 (300 mg, 0.666 mmol) and concd H2SO4 (0.1 mL) in AcOH (4 mL) was
irradiated with stirring for 3 min (15 s × 12 times, 30 s interval/irradiation) under MWI. The reaction

HETEROCYCLES, Vol. 71, No. 7, 2007
1597
mixture was poured into ice-water and extracted with AcOEt, after which the extract was washed with 3%
aqueous NaOH, then water, and dried (Na2SO4). The resulting compound was recrystallized from
AcOEt/hexane to give the desired flavone 4 (251 mg, 87%) as pale yellow needles, mp 101.5-102.5 ˚C.
IR (KBr) ν 3427, 2957, 2839, 1644, 1590 cm-1; UV λmax (log ε) (MeOH) 223 (4.33), 273 (4.19), 321
(4.27); 1H-NMR δ 3.93 (3H, s, OCH3), 3.95 (9H, s, OCH3 × 3), 3.96 (3H, s, OCH3), 4.02 (3H, s, OCH3),
4.11 (3H, s, OCH3), 6.64 (1H, s, C3-H), 7.17 (2H, s, Ar-H × 2); Anal. Calcd for C22H24O9: C, 61.10; H,
5.59. Found: C, 60.95; H, 5.55.
2’-(3,4,5-Trimethoxybenzoyloxy)-3’,4’,6’-trimethoxyacetophenone (13).
A mixture of 3a (1.00 g, 4.42 mmol), benzoyl chloride 8 (1.17 g, 5.08 mmol) and pyridine (3 mL) was
irradiated with stirring for 2 min (30 s × 4 times, 30 s interval/irradiation) under MWI. The resulting
compound was worked up in the same manner as in the case of 11 to give the ester 13 (1.52 g, 82%) as
pale yellow needles, mp 122-124 ˚C. 1H-NMR (CDCl3) δ 2.48 (3H, s, COCH3), 3.77 (3H, s, OCH3), 3.89
(3H, s, OCH3), 3.93 (9H, s, OCH3 × 3), 3.95 (3H, s, OCH3), 6.45 (1H, s, Ar-H), 7.43 (2H, s, Ar-H × 2).
1-(2-Hydroxy-3,4,6-trimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-1,3-propanedione (14).
A mixture of 13 (500 mg, 1.19 mmol) and KOH (334 mg, 5.95 mmol) in pyridine (5 mL) was irradiated
with stirring for 5 min (30 s × 10 times, 30 s interval/irradiation) under MWI. The resulting compound
was worked up in the same method as in the case of 12 to give the diketone 14 (440 mg, 88%) as pale
yellow needles, mp 164-166 ˚C.
5,7,8,3’,4’,5’-Hexamethoxyflavone (5).
A solution of the diketone 14 (300 mg, 0.714 mmol) and conc H2SO4 (0.1 mL) in AcOH (4 mL) was
irradiated with stirring for 1.5 min (15 s × 6 times, 30 s interval/irradiation) under MWI. The resulting
compound was worked up in the same method as in the case of 4 to give the flavone 5 (272 mg, 94%) as
colorless needles, mp 197-199 ˚C IR (KBr) ν 3427, 2942, 2843, 1679, 1610 cm-1; UV λmax (log ε)
(MeOH) 271 (4.19), 319 (4.15), 341 (4.13); 1H-NMR (CDCl3) δ 3.93 (3H, s, OCH3), 3.95 (6H, s, OCH3 ×
2), 3.97 (3H, s, OCH3), 4.01 (3H, s, OCH3), 4.02 (3H, s, OCH3), 6.45 (1H, s, Ar-H), 6.65 (1H, s, C3-H),
7.19 (2H, s, Ar-H × 2); Anal. Calcd for C21H22O8: C, 62.68; H, 5.51. Found: C, 62.56; H, 5.52.
2’-(3,4-Dimethoxybenzoyloxy)-3’,4’,6’-trimethoxyacetophenone (15).
A solution of 3b (1.00 g, 4.42 mmol), benzoyl chloride 9 (1.02 g, 5.08 mmol) in pyridine (3 mL) was
irradiated with stirring for 2 min (30 s × 4 times, 30 s interval/irradiation) under MWI. The resulting
compound was worked up in the same manner as in the case of 11 to give the ester 15 (1.53 g, 89%) as
1
colorless needles, mp 201-203 ˚C. H-NMR (CDCl3) δ 2.47 (3H, s, COCH3), 3.76(3H, s, OCH3), 3.88
(3H, s, OCH3), 3.93 (3H, s, OCH3), 3.95 (3H, s, OCH3), 3.96 (3H, s, OCH3), 6.45 (1H, s, Ar-H), 6.93 (1H,
d, J=8.6 Hz, Ar-H), 7.64 (1H, d, J=2.2 Hz, Ar-H), 7.84 (1H, dd, J=2.2 and 8.6 Hz, Ar-H).

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1-(2-Hydroxy-3,4,6-trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1,3-propanedione (16).
A mixture of 15 (1.00 g, 2.56 mmol) and KOH (720 mg, 12.8 mmol) in pyridine (10 mL) was irradiated
with stirring for 4 min (30 s × 8 times, 30 s interval/irradiation) under MWI. The resulting compound was
worked up in the same method as in the case of 12 to give the diketone 16 (860 mg, 86%) as pale yellow
needles.
5,7,8,3’,4’-Pentamethoxyflavone (6).
A solution of the diketone 16 (835 mg, 2.14 mmol) and conc H2SO4 (0.6 mL) in AcOH (8 mL) was
irradiated with stirring for 2 min (30 s × 4 times, 30 s interval/irradiation) under MWI. The resulting
compound was worked up in the same method as in the case of 4 to give the flavone 6 (660 mg, 83%,
crystallized from MeOH) as pale yellow needles, mp 195-197 ˚C. IR (KBr) ν 3310, 2885, 2848, 1662,
1609 cm-1; UV λmax (log ε) (MeOH) 249 (4.34), 269 (4.39), 337 (4.27); 1H-NMR (CDCl3) δ 3.96 (6H, s,
OCH3 × 2), 3.98 (3H, s, OCH3), 3.99 (3H, s, OCH3), 4.02 (3H, s, OCH3), 6.44 (1H, s, Ar-H), 6.62 (1H, s,
C3-H), 7.00 (1H, d, J=8.6 Hz, C5’-H), 7.42 (1H, d, J=2.0 Hz, C2’-H), 7.59 (1H, dd, J=2.0 and 8.6 Hz,
C6’-H); ; Anal. Calcd for C20H20O7: C, 64.51; H, 5.41. Found: C, 64.48; H, 5.46.
2’-(2,6-Dimethoxybenzoyloxy)-3’,4’,5’,6’-tetramethoxyacetophenone (17).
A solution of 3a (500 mg, 1.95 mmol) and benzoyl chloride 10 (467 mg, 2.33 mmol) in pyridine (2 mL)
was irradiated with stirring for 3.5 min (30 s × 7 times, 30 s interval/irradiation) under MWI. The
resulting compound was worked up in the same manner as in the case of 11 to give the ester 17 (620 mg,
1
76%) as a pale yellow oil. H-NMR (CDCl3) δ 2.52 (3H, s, COCH3), 3.88 (9H, s, OCH3 × 3), 3.90, 3.94
and 3.98 (each 3H, s, OCH3), 6.59 (2H, d, J=8.5 Hz, C3-H and C5-H ), 7.33 (each 1H, d, J=8.5 Hz, C4-H
overlap).
1-(2-Hydroxy-3,4,5,6-tetramethoxyphenyl)-3-(2,6-dimethoxyphenyl)-1,3-propanedione (18).
A mixture of 17 (520 mg, 1.26 mmol) and KOH (353 mg, 6.29 mmol) in pyridine (5 mL) was irradiated
with stirring for 7 min (30 s × 12 times, 30 s interval/irradiation) under MWI. The resulting compound
was worked up in the same method as in the case of 12 to give the diketone 18 (494 mg, 95%) as a pale
yellow oil.
5,6,7,8,2’,6’-Hexamethoxyflavone (7).
A solution of the diketone 18 (470 mg, 1.12 mmol) and conc H2SO4 (0.2 mL) in AcOH (5 mL) was
irradiated with stirring for 5 min (30 s × 10 times, 30 s interval/irradiation) under MWI. The resulting
compound was worked up in the same method as in the case of 4 to give the flavone 7 (410 mg, 91%),
which was chromatographed over a silica gel column (AcOEt:hexane=1:1 as a solvent), as colorless
needles, mp 99-100.5 ˚C. IR (KBr) ν 3427, 2938, 2826, 1609, 1592 cm-1; UV λmax (log ε) (MeOH) 231
(4.19), 257 (4.25); 1H-NMR (CDCl3) δ 3.80 (6H, s, OCH3 × 2), 3.92 (3H, s, OCH3), 3.95 (3H, s, OCH3),
3.96 (3H, s, OCH3), 4.08 (3H, s, OCH3), 6.28 (1H, s, C3-H), 6.63 (2H, d, J=8.6 Hz, C3’-H and C5’-H

HETEROCYCLES, Vol. 71, No. 7, 2007
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overlap), 7.40 (1H, d, J=8.6 Hz, C4’-H overlap); Anal. Calcd for C21H22O8: C, 62.68; H, 5.51. Found: C,
62.66; H, 5.52.
Cell culture. Human HM3KO melanoma cells were maintained in culture medium consisting of
Dulbecco’s modified Eagle medium supplement with 10% fetal bovine serum, 100,000 U/L
benzylpenicillin potassium and 100 mg/L kanamycin sulfate.
Cytotoxicity assay. HM3KO melanoma cells (10,000cells/well) were seeded on 96-well plates in 100
μL of culture medium and maintained for 8 h. Then culture medium containing test compound was
applied for 3 days. The culture medium in the well was exchanged with 100 μL of culture medium
containing 10 μL of 0.5% MTT solution and incubation was continued for 2 h. Then the culture medium
containing the MTT solution in the well was exchanged with 100 μL of stop solution (0.04 M HCl in
2-propanol) and the absorbance was determined at 570 nm with a reference wavelength of 630 nm using a
microplate reader. The cell growth rate was calculated from the absorbance compared with the control.
Melanin assay. HM3KO melanoma cells were seeded in 85 mm dishes at a density of 4 × 105 cells per
dish and cultured. After incubation for 9 h, the cells were treated with various concentrations of test
reagents. Three days later, the medium was changed and the test reagents at the same concentration were
added to the medium. The cells were cultured for an additional 2 days, after which they were harvested by
trypsinization, washed with phosphate-buffered saline (PBS). The cells were washed with 5%
trichloroacetic acid aqueous solution, ethanol/ether (3/1) and ether in turn, and dissolved in 1 mL of 2 M
NaOH. Inhibitory activity of melanogenesis was calculated by measuring the absorbance at 410 nm of the
solution.
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