Synthesis and evaluation of influenza virus sialidase inhibitory activity of hinokiflavone-sialic acid conjugates

Article abstract of DOI:10.3987/COM-07-11285

The known biflavonoid, hinokiflavone (1) was isolated from the leaves of Metasequoia glyptostroboides Hu et Cheng and displayed influenza A and B virus sialidase inhibitory activity. The unnatural glycoconjugate, hinokiflavone-sialic acid (8) was synthesi

Full text of DOI:10.3987/COM-07-11285

HETEROCYCLES, Vol. 75, No. 4, 2008
879
HETEROCYCLES, Vol. 75, No. 4, 2008, pp. 879 - 885. © The Japan Institute of Heterocyclic Chemistry
Received, 30th November, 2007, Accepted, 8th January, 2008, Published online, 11th January, 2008. COM-07-11285
SYNTHESIS AND EVALUATION OF INFLUENZA VIRUS SIALIDASE
INHIBITORY ACTIVITY OF HINOKIFLAVONE-SIALIC ACID
CONJUGATES
Kazuhiko Miki,a Takayuki Nagai,b,c Takayuki Nakamura,a Mitsuru Tuji,a
Kiyotaka Koyama,a Kaoru Kinoshita,a Kimio Furuhata,d Haruki Yamada,b,c
and Kunio Takahashi* a
aDepartment of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical
University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japan. E-mail address:
b
diamonds@my-pharm.ac.jp Kitasato Institute for Life Sciences and Graduate
School of Infection Control Sciences, Kitasato University, Shirokane 5-9-1,
c
Minato-ku, Tokyo 108-8641, Japan. Oriental Medicine Research Center, The
d
Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyo 108-8642, Japan. School
of Pharmacy, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641,
Japan
Abstract – The known biflavonoid, hinokiflavone (1) was isolated from the
leaves of Metasequoia glyptostroboides Hu et Cheng and displayed influenza A
and B virus sialidase inhibitory activity. The unnatural glycoconjugate,
hinokiflavone-sialic acid (8) was synthesized and exhibited more potent
inhibitory activity.
INTRODUCTION
Flavonoids are widely distributed in the plant kingdom and possess a variety of biological activities.
Biflavonoids are flavonoid-dimers connected by a C-O-C or C-C bond. The biological activities of a
number of biflavonoids were recently investigated and we found that a C-C bond type biflavonoid,
ginkgetin, exhibits inhibitory activity against influenza virus sialidase.1 Here we report a C-O-C type
biflavonoid, hinokiflavone, which was isolated from Metasequoia glyptostroboides Hu et Cheng and
shows inhibitory activity against influenza virus sialidase. As sialic acids are essential components of the
host-cell surface receptors recognized by influenza viruses during the first stage of infection, synthetic
sialic acid-aglycone conjugates might inhibit the virus. Therefore we synthesized a variety of

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HETEROCYCLES, Vol. 75, No. 4, 2008
hinokiflavone-sialic acid conjugates and evaluated their inhibitory activity against influenza virus
sialidase.
RESULTS AND DISCUSSION
The leaves of Metasequoia glyptostroboides Hu et Cheng (289.4 g), collected in Tokyo prefecture, Japan,
were extracted with acetone seven times. The acetone extract (26.2 g) was subjected to silica gel column
chromatography using CHCl3-MeOH-DMSO to obtain known biflavonoid, hinokiflavone (1) (758.4 mg).
Hinokiflavone (1) was identified by comparing its spectroscopic data with reported data.2 The
glycosylation of the arylic hydroxyl group to sialic acid (Neu5Ac) (2) was performed by the Williamson’s
method.3,4 The preparation of the methyl ester of Neu5Ac (3) was carried out by using a dried strong
cation-exchange resin (Dowex-50W-X2) with MeOH. Following acetylation of 3 with Ac2O and pyridine,
the chloro derivative (5) was prepared from 4 by AcCl. Compound 5 was identified by comparing its
spectral data with that reported in the literature.5
OH
HO
OH
HO
OH
HO
OH
HO
a
b
HO
HO
CO2H
O
CO2Me
O
3
AcHN
87%
89%
AcHN
2
OAc
Cl
AcO
AcO
OAc
OAc
AcO
c
AcO
AcO
CO2Me
CO2Me
O
O
5
AcHN
AcHN
100%
AcO
4
Scheme 1. Reagents and conditions: (a) MeOH, Dowex-50W-X2, rt, 48 h; (b) Ac2O, pyridine, rt, 20 h; (c)
AcCl, rt, 48 h.
To synthesize hinokiflavone-sialic acid (6), a Williamson’s reaction using sodium hydride was carried out
to combine the chloro derivative of Neu5Ac (5) with the C-7’’ position of 1. Compound 6 was assigned
the molecular formula of C50H45NO22 by its positive HRFABMS spectral data m/z 1012.2524 [M+H]+,
(calcd 1012.2512, C50H45NO22, [M+H]+). The UV spectrum (MeOH) of 6 showed absorption maxima at
λ
max nm (log ε): 209 (4.80), 269 (4.56) and 336 (4.68) nm, indicating the presence of aromatic rings. The
IR spectrum exhibited the presence of hydroxyl or amino groups (3430 cm-1) and carbonyl (1750, 1660,
1610 cm-1) groups. The 1H-NMR spectrum showed six singlet peaks at δ 1.71, 1.87, 1.91, 2.03, 2.16 and
3.68 (each 3H, s) each due to a methyl group. The last methyl group was assigned to the methyl ester. The
spectrum exhibited signals in the aromatic region (δ 6.20-8.15) due to the hinokiflavone skeleton, and
between δ 1.91-5.40 due to the sialic acid part. The carboxyl methyl proton at δ 3.68 showed NOESY

HETEROCYCLES, Vol. 75, No. 4, 2008
881
correlations with the hinokiflavone part at δ 7.10 (8’’ position) and δ 8.04 (2’ and 6’ position). This
NOESY correlation data suggested that Neu5Ac was linked to 1 at the C-7’’ position. In order to
methylate the hydroxyl groups at 7 and 4’’’ positions, 6 was treated with CH2N2 in CHCl3 and DMF.
Finally the O-acetyl and methyl ester groups of 7 were hydrolyzed with NaOMe, MeOH and H2O to yield
(8).
3'''
4'''
OH
OH
2'''
1'''
AcO
OAc
CO2Me
O
O
1''
8''
2''
9''
O
HO 7''
O
AcO
5'''
AcHN
AcO
6'''
a
6''
b
3'
6'
3''
10''
4'
O
O
2'
1'
5''
4''
1
32%
61%
8
OH O
6
OH O
2
9
7
HO
O
HO
O
5'
6
3
10
1
4
5
OH O
OH O
OMe
OMe
AcO
OAc
CO2Me
HO
OH
CO2H
O
O
O
O
AcO
O
HO
O
AcHN
AcO
AcHN
HO
c
O
O
OH O
OH O
8
21%
MeO
O
MeO
O
7
OH O
OH O
Scheme 2. Reagents and conditions: (a) 5, NaH, DMF, rt, 18 h; (b) CH2N2, CHCl3, DMF, rt, 3 h; (c)
NaOMe, MeOH, H2O, rt, 23 h.
The glycosylation of Neu5Ac by Williamson’s method reacts by SN2 reaction mechanism and only gives
α-glycoside.3,4 It was slightly synthesized a glycoconjugate which was linked to 1 at the C-7 position. It
seems that C-7 glycosidic linkage is very unstable to be hydrolyzed easily. In contrast, compound 6 might
be stable with its complex structure.
Table 1. Inhibition of influenza A and B virus sialidases by hinokiflavone-sialic acid conjugates
Sialidase inhibitory activity [IC50 (µg/mL)]
Compound
A/New Caledonia/20/99
(H1N1)
A/Guizhou/54/89
(H3N2)
B/Ibaraki/2/85
F36
1
55.3
41.8
>100
>100
19.1
55.0
47.2
>100
>100
13.5
34.0
24.8
>100
66.7
8.2
6
7
8

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Compounds 6 and 7 both showed weak inhibitory activity against sialidase of influenza A (H1N1, H3N2)
and B viruses, whereas compounds 1 and 8 exhibited significant inhibitory activity. In particular,
compound 8 exhibited higher inhibitory activity than F36 (5, 7, 4’-trihydroxy-8-methoxyflavone), which
was isolated from the roots of Scutellaria baicalensis6 and used as a positive control (Table 1).
EXPERIMENTAL
General Experimental Procedures. Melting points were determined on a Yanaco MP. Optical rotation
was measured with a HORIBA SEPA-300 polarimeter. The IR spectra were recorded with a JASCO IR
Report-100 spectrophotometer, and the UV spectra were recorded with a Shimadzu UV-240
spectrophotometer. The MS spectra were measured with a JEOL JMS-700 spectrometer. The 1H- and 13C-
NMR spectra were measured with a JEOL JNM-LA500 MHz and JNM-AL400 MHz spectrometers
(DMSO with TMS). Column chromatography was performed using KANTO CHEMICAL silica gel 60N
(63-210 µm), GE Healthcare Sephadex LH-20 and Mitsubishi Chemical DIAION HP-20. HPLC was
performed using a Senshu Scientific SSC-346 PUMP and measured with a SSC-5410 UV/VIS Detector
using a Senshu Pak PEGASIL-ODS column. The procedure for the influenza A and B virus sialidase
inhibition assay was the same as described previously.1
Plant material. The leaves of Metasequoia glyptostroboides Hu et Cheng were collected in Meiji
Pharmaceutical University, Tokyo prefecture, Japan, in December 2005.
Extraction and isolation. The dried leaves of Metasequoia glyptostroboides Hu et Cheng (289.4 g) were
extracted with acetone (4 L × 7 times). The concentrated acetone extract (26.2 g) was subjected to silica
gel column chromatography (column A;
7
×
200 mm) eluted with CHCl3-MeOH
(100:1→50:1→30:1→10:1), MeOH and DMSO to afford 11 fractions. Fraction A11 (1.02 g) was
precipitated with MeOH and filtered, isolating hinokiflavone (1) (758.4 mg) as a solid. Hinokiflavone (1).
Yellow powder; mp 253~258 °C; IR ν max (KBr) cm-1: 3200, 1660, 1610, 1500, 1460, 1365, 1290, 1250,
1175, 1100, 1030, 840; UV (MeOH) λmax nm (log ε): 213 (4.57), 271 (4.36), 339 (4.42); EIMS m/z (rel.
int. ): 538 ([M]+, 100), 270 (12), 257 (19), 254 (11); HREIMS m/z 538.0895 (calcd for C30H18O10, [M]+
538.0900); 1H-NMR (DMSO-d6) δ 6.20 (d, 1H, J = 2.0 Hz), 6.48 (d, 1H, J = 2.0 Hz), 6.72 (s, 1H), 6.84 (s,
1H) , 6.84 (s, 1H), 6.94 (d, 2H, J = 8.7 Hz), 7.96 (d, 2H, J = 8.7 Hz), 7.04 (d, 2H, J = 9.1 Hz), 8.01 (d, 2H,
13
J = 9.1 Hz), 12.88 (s, 1H), 13.20 (s, 1H); C-NMR (DMSO-d6) δ 94.0, 94.6, 98.9, 102.5, 103.8, 103.9,
104.1, 115.3, 115.3, 116.0, 116.0, 121.1, 124.2, 124.7, 128.3, 128.3, 128.6, 128.6, 153.1, 153.7, 157.2,
157.3, 160.6, 161.3, 161.4, 163.1, 164.1, 164.2, 181.7, 182.0.
General procedure for the preparation of 3 from 2. A solution of 2 (0.016 mol) and dried strong
cation-exchange resin (Dowex-50W-X2) (5.0 g) in MeOH (200 mL) was stirred at rt under Ar gas. After
the reaction mixture had been stirred for 48 h, the MeOH solution was concentrated to give 3 (89 %).

HETEROCYCLES, Vol. 75, No. 4, 2008
883
White powder; mp 170~173 °C; [α]D -105.0 (c 0.5, MeOH); IR ν max (KBr) cm-1: 3350, 2940, 1740, 1640,
1550, 1430, 1370, 1010, 890; FABMS (positive) m/z : 324 [M+H]+, 309; HRFABMS (positive) m/z
324.1292 (calcd for C12H22NO9, [M+H]+ 324.1295); 1H-NMR (DMSO-d6) δ 1.89 (s, 3H), 2.03 (dd, 1H, J
13
= 12.6, 4.7 Hz), 3.70 (s, 3H), 3.3-6.5 (m, 13H), 8.18 (d, 1H, J = 8.0 Hz); C-NMR (DMSO-d6) δ 22.4,
48.5, 52.2, 53.0, 63.5, 65.3, 69.0, 69.5, 70.4, 94.8, 170.1, 171.9.
General procedure for the preparation of 4 from 3. To a solution of 3 (0.030 mol) in pyridine (200
mL) was added Ac2O (2.66 mol), which was then stirred at rt under Ar gas for 20 h. The solution was
concentrated in vacuo, and diluted with water and CHCl3. The organic layer was separated, dried over
anhydrous sodium sulfate and evaporated. The crude product was purified by column chromatography on
silica gel (CHCl3-isopropanol) to afford 4 (87 %). White powder; mp 80~81 °C; [α]D -24.8 (c 1.0,
CHCl3); IR ν max (KBr) cm-1: 3300, 2900, 1750, 1670, 1550, 1450, 1380, 1230, 1050; FABMS (positive)
m/z : 534 [M+H]+, 474, 414; HRFABMS (positive) m/z 534.1818 (calcd for C22H32NO14, [M+H]+
1
534.1823); H-NMR (DMSO-d6) δ 1.70 (s, 3H, α-NHCOCH3), 1.70 (s, 3H, β-NHCOCH3), 1.94-2.09 (s,
15H, α-OCOCH3), 1.94-2.13 (s, 15H, β-OCOCH3), 1.90 (m, 1H), 2.42 (dd, 1H, J = 13.5, 5.0 Hz,
β-H-3eq), 2.66 (dd, 1H, J = 12.9, 5.0 Hz, α-H-3eq), 3.68 (s, 3H, β-COOCH3), 3.70 (s, 3H, α-COOCH3),
3.97 (m, 1H), 4.11 (d, 1H, J = 10.5 Hz), 4.26 (m, 1H), 4.95 (m, 1H), 5.05 (m, 1H), 5.20 (m, 1H), 7.81 (d,
1H, J = 9.3 Hz, β-NH), 7.87 (d, 1H, J = 9.3 Hz, α-NH); 13C-NMR (DMSO-d6) δ (α) 20.4, 20.4, 20.5, 20.5,
20.6, 22.5, 35.9, 47.5, 52.7, 61.5, 66.8, 68.6, 68.7, 72.5, 96.3, 167.6, 168.3, 168.9, 169.0, 169.1, 169.6,
169.9, δ (β) 20.4, 20.4, 20.5, 20.5, 20.6, 22.5, 35.6, 47.4, 52.8, 61.3, 66.8, 68.5, 69.1, 71.5, 96.7, 166.0,
168.0, 168.9, 169.0, 169.2, 169.7, 169.9.
General procedure for the preparation of 5 from 4. Compound 4 (1.88 mmol) was dissolved in AcCl
(0.42 mol) and stirred slowly for 48 h at rt. The solution was concentrated in vacuo, and diluted with
water and CHCl3. The organic layer was separated and dried over anhydrous sodium sulfate. Evaporation
of the CHCl3 layer yielded 5 (100 %). White powder; mp 112~114 °C; [α]D -28.2 (c 0.7, CHCl3); IR ν max
(KBr) cm-1: 3300, 2860, 1750, 1660, 1540, 1440, 1220, 1035; FABMS (positive) m/z : 510 [M+H]+, 492,
1
474, 414; HRFABMS (positive) m/z 510.1369 (calcd for C20H29NO12Cl, [M+H]+ 510.1378); H-NMR
(DMSO-d6) δ 1.72 (s, 3H), 1.96 (s, 3H), 1.99 (s, 3H), 2.01 (s, 6H), 2.32 (dd, 1H, J = 13.9, 11.1 Hz), 2.69
(dd, 1H, J = 13.9, 4.4 Hz), 3.79 (s, 3H), 4.01 (m, 2H), 4.24 (d, 1H, J = 12.3 Hz), 4.39 (d, 1H, J = 10.7 Hz),
5.06 (m, 1H), 5.17 (m, 1H), 5.32 (d, 1H, J = 11.9 Hz), 7.95 (d, 1H, J = 9.5 Hz); 13C-NMR (DMSO-d6) δ
20.4, 20.5, 20.5, 20.6, 22.6, 35.6, 45.3, 52.3, 61.6, 66.9, 68.8, 69.2, 75.9, 96.7, 169.0, 169.0, 169.1, 169.8,
169.8, 169.9.
General procedure for the preparation of 6 from 1. A solution of 1 (0.37 mmol) and NaH (0.83 mmol)
in DMF (15 mL) was stirred at rt under Ar gas. After stirring the reaction mixture for 1 h, compound 5

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HETEROCYCLES, Vol. 75, No. 4, 2008
(1.18 mmol) was added and the mixture stirred for 17 h at rt under Ar gas. The solution was diluted with
saturated aqueous NaCl and CHCl3. The organic layer was separated and dried over anhydrous sodium
sulfate. After the CHCl3 layer was evaporated, the residue was purified by Sephadex LH-20 column
chromatography eluted with MeOH to give 6 (32 %). Yellow powder; mp 194~199 °C; [α]D24 +3.9 (c 0.8,
MeOH); IR ν max (KBr) cm-1: 3430, 1750, 1660, 1610, 1450, 1360, 1290, 1240, 1170, 1030, 840; UV
(MeOH) λmax nm (log ε): 209 (4.80), 269 (4.56), 336 (4.68); FABMS (positive) m/z : 1012 [M+H]+, 539,
1
414; HRFABMS (positive) m/z 1012.2524 (calcd for C50H46NO22, [M+H]+ 1012.2512); H-NMR
(DMSO-d6) δ 1.71 (s, 3H), 1.87 (s, 3H), 1.91 (s, 3H), 1.91 (m, 1H), 2.03 (s, 3H), 2.16 (s, 3H), 2.40 (dd,
1H, J = 12.9, 4.6 Hz), 3.68 (s, 3H), 3.94 (q, 1H, J = 10.2 Hz), 4.10 (dd, 1H, J = 12.2, 6.3 Hz), 4.22 (dd,
1H, J = 12.2, 2.9 Hz), 4.45 (dd, 1H, J = 10.7, 1.5 Hz), 4.72 (ddd, 1H, J = 10.9, 10.9, 4.6 Hz), 5.17 (dd, 1H,
J = 8.8, 1.5 Hz), 5.40 (m, 1H), 6.20 (d, 1H, J = 2.2 Hz), 6.49 (d, 1H, J = 2.2 Hz), 6.89 (s, 1H), 6.93 (d, 2H,
J = 8.8 Hz), 6.99 (s, 1H), 7.07 (d, 2H, J = 8.8 Hz), 7.10 (s, 1H), 7.80 (d, 1H, J = 9.8 Hz), 8.04 (d, 2H, J =
8.8 Hz), 8.15 (d, 2H, J = 8.8 Hz), 10.55 (br. s, 1H), 10.76 (br. s, 1H), 12.87 (s, 1H), 13.23 (s, 1H);
13C-NMR (DMSO-d6) δ 20.4, 20.4, 20.5, 20.5, 22.5, 37.7, 47.6, 53.4, 62.0, 66.8, 67.7, 67.9, 73.2, 93.9,
97.3, 98.8, 100.0, 102.8, 103.7, 104.0, 107.2, 115.5, 115.5, 115.8, 115.8, 120.7, 124.5, 126.7, 128.1, 128.1,
128.9, 128.9, 151.8, 152.7, 152.9, 157.2, 160.3, 161.3, 161.4, 164.1, 162.8, 164.9, 167.0, 169.0, 169.1,
169.4, 169.4, 169.7, 181.6, 182.1.
General procedure for the preparation of 7 from 6. A solution of 6 (0.028 mmol) and CH2N2 (2 mL)
in CHCl3 (5.0 mL) and DMF (1.0 mL) was stirred at rt for 3 h. After the solution was evaporated, the
residue was purified by column chromatography on silica gel (CHCl3-MeOH) to afford 7 (61 %). Yellow
powder; mp 160~162 °C; [α]D28 +5.4 (c 0.4, MeOH); IR ν max (KBr) cm-1: 3420, 1750, 1660, 1620, 1500,
1290, 1230, 1120, 1040, 840; UV (MeOH) λmax nm (log ε): 209 (4.85), 270 (4.61), 329 (4.74); FABMS
(positive) m/z : 1040 [M+H]+, 567; HRFABMS (positive) m/z 1040.2822 (calcd for C52H50NO22, [M+H]+
1
1040.2825); H-NMR (DMSO-d6) δ 1.71 (s, 3H), 1.87 (s, 3H), 1.91 (s, 3H), 1.94 (m, 1H), 2.03 (s, 3H),
2.19 (s, 3H), 2.40 (dd, 1H, J = 12.5, 4.6 Hz), 3.69 (s, 3H), 3.87 (s, 3H), 3.87 (s, 3H), 3.94 (q, 1H, J = 10.2
Hz), 4.10 (dd, 1H, J = 12.4, 6.3 Hz), 4.21 (dd, 1H, J = 12.3, 3.0 Hz), 4.46 (dd, 1H, J = 10.6, 1.3 Hz), 4.72
(ddd, 1H, J = 11.0, 11.0, 4.6 Hz), 5.17 (dd, 1H, J = 8.9, 1.3 Hz), 5.41 (m, 1H), 6.38 (d, 1H, J = 2.2 Hz),
6.77 (d, 1H, J = 2.2 Hz), 6.95 (s, 1H), 7.08 (s, 1H), 7.09 (d, 2H, J = 9.0 Hz), 7.11 (s, 1H), 7.12 (d, 2H, J =
9.0 Hz), 7.80 (d, 1H, J = 9.8 Hz), 8.07 (d, 2H, J = 9.0 Hz), 8.27 (d, 2H, J = 9.0 Hz), 12.87 (s, 1H), 13.17
(s, 1H); 13C-NMR (DMSO-d6) δ 20.4, 20.5, 20.5, 20.8, 22.6, 37.7, 47.6, 53.4, 55.5, 56.0, 61.9, 66.7, 67.6,
67.9, 73.1, 92.6, 97.1, 97.9, 99.9, 103.4, 104.1, 104.6, 107.1, 114.3, 114.3, 115.4, 115.4, 122.3, 124.2,
126.6, 128.1, 128.1, 128.5, 128.5, 151.7, 152.6, 152.8, 156.9, 160.2, 160.8, 162.3, 163.0, 164.2, 164.9,
166.8, 168.8, 168.9, 169.2, 169.2, 169.6, 181.6, 182.0.

HETEROCYCLES, Vol. 75, No. 4, 2008
885
General procedure for the preparation of 8 from 7. A solution of 7 (0.017 mmol) and NaOMe (28 %
in MeOH) (4 mL) in MeOH (4 mL) was stirred at rt for 6 h under Ar gas. Then H2O (8 mL) was added
and stirred for 17 h at rt. The reaction mixture was treated with 2M HCl and the solution was evaporated.
The residue was purified by column chromatography on DIAION HP-20 (H2O→MeOH) and the solvent
was evaporated. The MeOH layer was purified by ODS-HPLC (80 % MeOH) to afford 8 (21 %). Yellow
29
powder; mp (dec.) 218 °C; [α]D +24.1 (c 0.06, MeOH); IR ν max (KBr) cm-1: 3450, 1650, 1620, 1500,
1450, 1380, 1250, 1180, 1120, 1030, 840; UV (MeOH) λmax nm (log ε): 212 (4.73), 271 (4.48), 333
(4.59); FABMS (negative) m/z : 856 [M-H]-, 565; HRFABMS (negative) m/z 856.2087 (calcd for
1
C43H38NO18, [M-H]- 856.2089); H-NMR (DMSO-d6) δ 1.33 (dd, 1H, J = 11.6, 11.6 Hz), 1.89 (s, 3H),
2.72 (dd, 1H, J = 11.6, 4.8 Hz), 3.34-3.84 (m, 7H), 3.87 (s, 3H), 3.87 (s, 3H), 6.38 (d, 1H, J = 2.2 Hz),
6.82 (d, 1H, J = 2.2 Hz), 6.94 (s, 1H), 6.97 (s, 1H), 7.11 (d, 2H, J = 8.7 Hz), 7.14 (d, 2H, J = 8.7 Hz), 7.66
(s, 1H), 8.04 (d, 2H, J = 8.7 Hz), 8.11 (d, 2H, J = 8.7 Hz), 8.20 (br.s), 12.89 (s, 1H), 12.89 (s, 1H).
ACKNOWLEDGEMENTS
This study was partly supported by Sasakawa Scientific Research Grant from Japan Science Society
(2007-4) and a Grant of the 21st Century COE Program, Ministry of Education, Culture, Sports, Science
and Technology in Japan respectively.
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