Anti-influenza virus activity of biflavonoids

Article abstract of DOI:10.1016/j.bmcl.2006.10.075

Ginkgetin was found to inhibit the influenza virus sialidase. Ginkgetin-sialic acid conjugates showed a significant survival effect in the influenza-virus-infected mice.

Full text of DOI:10.1016/j.bmcl.2006.10.075

Bioorganic & Medicinal Chemistry Letters 17 (2007) 772–775
Anti-influenza virus activity of biflavonoids
a
b,c
a
a
Kazuhiko Miki, Takayuki Nagai, Kazushige Suzuki, Ryo Tsujimura,
a
a
d
b,c
Kiyotaka Koyama, Kaoru Kinoshita, Kimio Furuhata, Haruki Yamada
a,
*
and Kunio Takahashi
a
Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japan
Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University,
b
Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan
Oriental Medicine Research Center, The Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyo 108-8642, Japan
c
d
School of Pharmacy, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan
Received 24 July 2006; revised 4 October 2006; accepted 25 October 2006
Available online 28 October 2006
Abstract—Ginkgetin was found to inhibit the influenza virus sialidase. Ginkgetin-sialic acid conjugates showed a significant survival
effect in the influenza-virus-infected mice.
Ó 2006 Elsevier Ltd. All rights reserved.
0
5,7,4 -Trihydroxy-8-methoxyflavone (F36) was surveyed
as an influenza virus sialidase inhibitory compound
of glycoconjugate receptors at the first stage of infection.
It may suggest that synthetic sialic acid-aglycone conju-
gates are important to inhibit the proliferation of virus-
es. Therefore, ginkgetin-sialic acid conjugates were
synthesized herewith.
1
among many flavonoids. It also showed an anti-influen-
za virus activity in Madin–Darby canine kidney
(
egg. In the influenza-virus-infected mice, F36 revealed
MDCK) cells and in the allantoic sac of embryonated
1
–3
3
–5
a distinct survival effect. Compound F36 was charac-
terized as an 8-methoxy-flavone derivative, which does
not frequently occur in nature. On the other hand,
biflavonoids including amentoflavone-type compounds
were reported to show the inhibitory activity against
N-Acetylneuraminic acid 1 is widely distributed inter-
nally in animals playing crucial roles in various biologi-
cal events such as infection, cell adhesion, immune
response, and neural function. The glycosylation of the
6
7
8
8
respiratory syncytial, herpes , and measles viruses.
OH
OH
OH
OH
Accordingly, some naturally occurring biflavonoids
were screened for the inhibitory activities against influ-
enza virus sialidase.
H
COOH
OH
H
COOMe
OH
O
a
c
O
b
OH
AcHN
OH
AcHN
OH
OH
As the result, it was demonstrated that ginkgetin isolat-
ed from Ginkgo biloba L. and Cephalotaxus harringtonia
K. Koch showed a potent inhibitory activity against
influenza virus sialidase.
1
2
OAc
H
OAc
H
OAc
OAc
COOMe
CL
O
O
On the other hand, influenza A and B viruses are known
to bind themselves on the host cells via sialic acid residue
OAc
COOMe
OAc
AcHN
OAc
AcHN
OAc
OAc
3
4
Keywords: Influenza; Ginkgetin; Sialic acid; Ginkgo biloba; Biflavo-
noid; Sialidase.
Scheme 1. Reagents and conditions: (a) Dowex-50 W-X2, dry MeOH,
rt, under Ar, 24 h (92%); (b) Ac O, DMAP, dry pyridine, rt, under Ar,
*
Corresponding author. Tel.: +81 424 95 8912; fax: +81 424 95
912; e-mail: diamonds@my-pharm.ac.jp
2
8
24 h (83%); (c) AcCl, HCl gas, 0 °C to rt, 24 h (98%).
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.075

K. Miki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 772–775
773
arylic hydroxyl group to sialic acid is performed by Wil-
9
lower inhibitory activities against A/PR/8/34 (H1N1)
and B/Ibaraki/2/85 sialidases comparing with F36. How-
ever, 6S and 7R showed the increase in the activity
against A/Guizhou/54/89 (H3N2) (Table 1).
,10
liamson’s method.
neuraminoside is outlined in Schemes 1 and 2. In the
The synthetic route for ginkgetin-
treatment of 1 with strong cation-exchange resin (Dow-
1
1
ex-50W-X2), MeOH afforded 2. Followed by acetyla-
,10
9
tion of 2 with Ac O, DMAP, and pyridine,
2
The atropic isomers of ginkgetin mononeuraminoside
(8R, 8S) and bisneuraminoside (9R, 9S), respectively,
inhibited both A/PR/8/34 (H1N1) and A/Guizhou/54/
89 (H3N2) sialidases more potently than F36.
compound 4 was prepared from 3 by AcCl and HCl
9
gas. The glycosides of 6–7 were prepared from 4 and
9
,10,12
ginkgetin 5 with NaH, DMF.
When N-acetylneu-
00
raminic acid was combined with C-7 position, it caused
a stereochemical obstruction to yield atropic isomers (R
and S formation). These atropisomeric compounds, 6R
and 6S, 7R and 7S, were separated by HPLC, respec-
tively. Compounds 8R, 8S, 9R, and 9S were prepared
from 6R, 6S, 7R, and 7S by hydrolysis of the acetyl
Especially, 8R showed the most potent inhibitory activ-
ity whose IC50 value against A/PR/8/34 (H1N1) sialidase
was 5.50 lg/mL, and that against A/Guizhou/54/89
(H3N2) sialidase was 0.82 lg/mL. On the other hand,
8R, 8S, 9R, and 9S showed lower activities than F36
against B/Ibaraki/2/85 sialidase (Fig. 1).
group with NaOMe, MeOH, and H O, respectively.
2
The influenza A and B virus sialidase inhibitory activi-
Next, proliferation of influenza virus and the cytotoxic-
ities were tested for compounds 6R–9S by using MDCK
cells infected with A/PR/8/34 (H1N1) (Fig. 2). The assay
was carried out by the described method. It means that
the prevention of virus proliferation reflects the decrease
of the sialidase activity, and the damage of MDCK cells
treated by compounds causes to decrease formasan
1
3
ties of ginkgetin, ginkgetin-sialic acid conjugates 6R–
S, are shown in Table 1, and F36 was used as a positive
standard. The IC50 values of ginkgetin against A/PR/8/
9
1
4
3
5
4 (H1N1) and A/Guizhou/54/89 (H3N2) sialidases were
5.00 and 9.78 lg/mL, respectively. 6R, 6S, 7R, and 7S,
which are ginkgetin acetylated neuraminosides, showed
OMe
OMe
HO
O
HO
O
O
O
a
OR2
OH
OMe
O
OMe
1
HO
R O
O
1
2
6
6
7
7
R R = sial-Ac, R = H
1
2
S R = sial-Ac, R = H
1
2
R R = sial-Ac, R = sial-Ac
OH
O
OH
O
1
2
S R = sial-Ac, R = sial-Ac
5
b
OAc
OAc
H
COOMe
O
O
= sial-Ac
OAc
AcHN
OMe
OAc
HO
O
O
OH
OH
OR2
H
COOH
O
OMe
O
O
= sial
1
1
2
R O
OH
AcHN
8R R = sial, R = H
8
9
1
2
S R = sial, R = H
OH
1
2
R R = sial, R = sial
1
2
OH
O
9S R = sial, R = sial
Scheme 2. Reagents and conditions: (a) 4, NaH, dry DMF, rt, under Ar, 24 h (5% for 6R, 4% for 6S, 12% for 7R, 8% for 7S); (b) NaOMe, dry
MeOH, H
O, rt, 24 h (6R ! 8R, 96%; 6S ! 8S, 76%; 7R ! 9R, 80%; 7S ! 9S, 69%).
2

774
K. Miki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 772–775
Table 1. Inhibition of influenza A and B virus sialidases by ginkgetin-
sialic acid conjugates
OH
Compound
Sialidase inhibitory activity [IC50 (lg/mL)]
HO
O
A/PR/8/34
(H1N1)
A/Guizhou/54/89
(H3N2)
B/Ibaraki/2/85
OH
OMe
O
HO
O
O
F36
Ginkgetin
9.78
55.00
79.20
8.95
9.78
6.58
>100.00
>100.00
>100.00
>100.00
>100.00
9.11
OH
OH
O
HO
6R
6S
7R
7S
8R
8S
9R
9S
>10.00
1.10
1.35
>100.00
>100.00
>100.00
5.50
OH
>10.00
0.82
OH
O
F36
Amentoflavone
6.35
6.61
0.99
6.57
8.64
9.05
6.99
0.94
9.34
OMe
HO
O
formation. Ginkgetin, which was found as a sialidase
inhibitor in the former experiment, showed a cytotoxic-
ity, whereas its neuraminoside derivatives have no cyto-
toxic effects to MDCK cells. Among these compounds
O
OH
OMe
HO
O
6
R–9S, O-acetylation of sialic acid moiety did not affect
the inhibitory activity of virus proliferation. Monone-
uraminoside, 8R and 8S, significantly showed the inhib-
itory activity of virus proliferation in comparison to
bisneuraminosides, 9R and 9S, at least 3- and 8-fold,
respectively. These results revealed that neuraminosyla-
tion of ginkgetin lowered the cytotoxicity and enhanced
inhibitory activity against influenza virus sialidase of
aglycone.
OH
O
Ginkgetin
Figure 1.
as a positive control gave the similar result in compari-
son to the control. On the other hand, compounds 8R,
8S, 9R, and 9S prolonged the survival days. Especially,
compound 8R exhibited the highest survival rates, 75%
at day 10 and 62.5% at day 21 after the A/PR/8/34
The efficacies of intranasally administered compounds,
8
R–9S and F36, were tested in the influenza virus
A/PR/8/34 (H1N1)-infected mice on the basis of the
1
5
survival days (Table 2).
(H1N1) virus infection (p = 0.0385, Logrank test, Kap-
The control mice began to die from day 8 after the virus
infection, and the survival rate was 22% at day 21. F36
lan–Meier method), while 8S showed almost the same
results, 78% at day 10 and 56% at day 21 (p = 0.0714),
F36
Ginkgetin
Cytotoxicity (+)
6
R
6
S
7
R
7
S
8
R
8
S
9
R
9
S
0
10
20
Inhibition of proliferation of influenza virus A/PR/8/34 (%)
Figure 2. Anti-influenza virus activity of ginkgetin-sialic acid conjugates on MDCK cells (concentration of samples was 12.5 lg/mL).
30
40
50
60
70
80
90
100

K. Miki et al. / Bioorg. Med. Chem. Lett. 17 (2007) 772–775
775
Table 2. Effects of ginkgetin-sialic acid conjugates on survival rates of
a
influenza virus-infected mice
6. Tomimori, T.; Miyaichi, Y.; Kizu, H. Yakugaku Zasshi
1982, 102, 388 (Journal written in Japanese).
b
7. Ma, S.-C.; But, P. P.-H.; Ooi, V. E.-C.; He, Y.-H.; Lee, S.
H.-S.; Lee, S.-F.; Lin, R.-C. Biol. Pharm. Bull. 2001, 24,
Compound
No. of survivors/total no. of mice
1
0 days after infection 21 days after infection
3
11.
Control (saline) 3/9
2/9
2/9
5/8
5/9
4/9
4/9
8. Lin, Y.-M.; Flavin, M. T.; Schure, R.; Chen, F.-C.;
Sidwell, R.; Barnard, D. L.; Huffman, J. H.; Kern, E. R.
Planta Med. 1999, 65, 120.
9. Myers, R. W.; Lee, R. T.; Lee, Y. C.; Thomas, G. H.;
Reynolds, L. W.; Uchida, Y. Anal. Biochem. 1980, 101,
166.
F36
5/9
6/8
7/9
5/9
8/9
8
8
9
9
R
S
R
S
1
1
1
1
0. Furuhata, K. Trends Glycosci. Glycotechnol. 2004, 16,
a
Mice were dosed with compound and then infected with influenza
virus A/PR/8/34 (H1N1).
1
43.
1. Kuhn, R.; Lutz, P.; MacDonald, D. L. Chem. Ber. 1966,
9, 611.
b
A single dose of ginkgetin-sialic acid conjugates and F36 were
administered intranasally at 0.5 mg/kg.
9
2. Furuhata, K.; Komiyama, K.; Ogura, H.; Hata, T. Chem.
Pharm. Bull. 1991, 39, 255.
3. Influenza virus sialidase inhibition activity (A/PR/8/34,
H1N1; A/Guizhou/54/89, H3N2; B/Ibaraki/2/85) was
compared with those of 8R. Bisneuraminoside 9R and
S exhibited slightly weak results in comparison to
mononeuraminosides, 8R and 8S, respectively.
9
determined in
a fluorometric assay. The reaction
mixture containing 0.1 mM 4-MU-NeuAc (50 ll),
sample solution (10 ll), influenza vaccine (10 ll), and
buffer was incubated at 37 °C for 10 min in 96-well
microtiter plate.
In summary, we presented new influenza virus sialidase
inhibitors, which were prepared by the conjugation of
biflavonoid ginkgetin from the plant source and sialic
acid from the animal origin. We also revealed that the
ginkgetin-sialic acid conjugates remarkably presented
potent anti-influenza virus activities in vivo.
1
4. In vitro anti-influenza virus activity was assayed by using
MDCK cells. Influenza virus A/PR/8/34 was inoculated
into confluent monolayers of MDCK cells in 96-well
culture plate, and then sample solutions were put into the
well. The plate was cultured at 37 °C for 3 days under a
5
2
% CO atmosphere. Inhibition of proliferation of influ-
enza virus was determined in a fluorometric assay by
measuring sialidase activity and the viable cells were
determined by MTT assay.
References and notes
1
2
3
4
5
. Nagai, T.; Miyaichi, Y.; Tomimori, T.; Suzuki, Y.;
Yamada, H. Chem. Pharm. Bull. 1990, 38, 1329.
. Nagai, T.; Moriguchi, R.; Suzuki, Y.; Tomimori, T.;
Yamada, H. Antiviral Res. 1995, 26, 11.
. Nagai, T.; Suzuki, Y.; Tomimori, T.; Yamada, H. Biol.
Pharm. Bull. 1995, 18, 295.
. Nagai, T.; Miyaichi, Y.; Tomimori, T.; Suzuki, Y.;
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. Nagai, T.; Nishibe, Y.; Makino, Y.; Tomimori, T.;
Yamada, H. Biol. Pharm. Bull. 1997, 20, 1082.
15. In vivo anti-influenza virus activity was assayed in
the influenza virus A/PR/8/34-infected mice. BALB/c
mice (female, 6-week-old) were anesthetized by an
intraperitoneal injection of amobarbital sodium. Test
samples were dissolved in saline and 10 ll of the
solution was administered to the mouse intranasally.
Then 20 ll mouse-adapted influenza virus A/PR/8/34
suspension in 0.1% BSA in PBS was infected by
intranasal inoculation. The survival rates were
observed for 21 days.