JP2003246792A - Anti-influenza-viral compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound which is formed by bonding a sialic acid derivative or a KDN derivative to a flavone derivative, decreases the amount of the influenza virus, improves the viability of cells infected by the influenza virus, and is useful as a medicine for preventing or curing influenza and also as a food for preventing or curing influenza. <P>SOLUTION: This compound is represented by formula (1) (wherein R<SP>1</SP>and R<SP>7</SP>are each independently H, Na or K; R<SP>2</SP>, R<SP>3</SP>, R<SP>4</SP>, R<SP>5</SP>, R<SP>8</SP>, R<SP>9</SP>, R<SP>10</SP>, and R<SP>11</SP>are each independently H, a sulfuric acid group or an acetyl group; R<SP>6</SP>and R<SP>12</SP>are each independently H or an acetyl, methyl, ethyl, n-propyl, n-butyl, n-octyl, benzyl or allyl group; X and Y are each independently an acetoamino, glycolylamino, hydroxyl or acetyloxy group; and A and B are each independently O or S). <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antiviral compound useful for the prevention / treatment of viral diseases such as influenza.

[0002]

BACKGROUND OF THE INVENTION Influenza is a life-threatening infectious disease for patients with underlying diseases and the elderly. In fact, there is an increase in excess mortality during the flu season. In addition, there are many cases of pneumonia in the elderly and encephalitis in the infant, with death or severe illness (Keizo Matsumoto, Nihon Rinji,
55 (10), 2536-2541 (1997)). Furthermore, in recent years, the emergence of a new subtype of influenza A virus is at stake, and it is predicted that tens of thousands to hundreds of thousands of deaths will occur if an outbreak occurs in Japan. For these reasons, measures against influenza have become an important social issue. It is arguable that vaccination is the most effective measure against influenza (Shinichi Tamura, Tsuyoshi Kurata, Bio Clinica,
11 (9), 665-669 (1996)). But,
Since it takes a certain amount of time to manufacture a vaccine and supply a sufficient amount of it, it is necessary to administer an anti-influenza virus agent to alleviate the symptoms when a new virus outbreak is imminent. .

At present, there is amantadine as an anti-influenza virus drug marketed in Japan, and zanamivir and oseltamivir will be released soon. Amantadine is already approved for insurance against influenza A virus infections. Amantadine acts on the M2 protein of influenza virus. M2 protein is an ion channel existing on the membrane of influenza A virus, and by acidifying the inside of virus particles, it is the cytoplasm of ribonucleoprotein complex (RNP), which is a complex of RNA gene, nucleoprotein, and RNA polymerase. Plays a major role in the release. Amantadine inhibits the function of this M2 protein, suppresses RNP release, and prevents virus growth.

Zanamivir and oseltamivir are effective against both A and B influenza viruses. The action mechanism of these drugs is different from that of amantadine, and exerts its effect by the sialidase (also called neuraminidase) inhibitory action. Sialidase is a glycoprotein present on the surface of viruses A and B, and acts to promote budding by releasing the hemagglutinin-receptor (sialic acid in the receptor) bond when the propagated virus is released from the host cell. . Zanamivir and oseltamivir bind to this active site and suppress its action, thus blocking the release of virus.
The virus that is sequestered in the infected cells binds to other viruses, thus stopping the spread of further infection and eventually ending it.

Zanamivir is also famous as one of the drugs theoretically developed in computer graphics.
It is a drug that emerged because the structure of sialidase and the three-dimensional structure of the active site were clarified by the progress of basic research.
Oseltamivir also acts to inhibit the function of sialidase by binding more strongly to the active site of sialidase that binds to sialic acid. Zanamivir has a low bioavailability when administered orally, so it is necessary to inhale the powder formulation through the nose with an inhaler or the like. In that respect, oseltamivir can be administered orally,
It is a prodrug that is absorbed into the body and then converted into the active form.

[0006] All of these anti-influenza drugs must be administered within 48 hours after the onset. After this, the effect is almost the same as the natural process and almost no effect is observed.
The clinical efficacy was not sufficient. In addition, there is a possibility that resistant viruses will appear in sialidase inhibitors,
There is a need for new anti-influenza drugs.

The inventors of the present invention also confirmed that a sulfated KDN derivative was used in vivo.
Have been found to exhibit anti-influenza virus activity. Furthermore, one of the flavone derivatives, F36 (5,
7, 4'-trihydroxy-8-methoxyflavone) has been reported to have anti-influenza virus activity in vitro and in vivo (T. Nagai, Y. Miyaichi, T. Tomimo).
ri, Y. Suzuki, and H. Yamada, Chem. Pharm. Bull.,
38, 1329-1332 (1990), T. Nagai, Y. Miyaichi, T. Tomi
mori, Y. Suzuki, and H. Yamada, AntiviralRes., 19,
207-217 (1992)), the development of more potent anti-influenza virus compounds has been desired.

[0008]

Therefore, it is an object of the present invention to provide a novel antiviral compound useful for the prevention / treatment of viral diseases, particularly viral diseases such as influenza.

[0009]

[Means for Solving the Problems] The inventors of the present invention conducted extensive studies to develop anti-influenza virus compounds, and conducted various sialic acid derivatives or KDN (2-keto-3-deoxy) which is one of sialic acids. -D-glycero-D-galacto-2-nonulo
The present invention was completed by discovering that a novel conjugate of a sonic acid) derivative and a flavone derivative exhibits anti-influenza virus activity.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION That is, the present invention includes (1) the following general formula (I)

[Chemical 2] (In the formula, R ¹ and R ⁷ are each independently a hydrogen atom, sodium, potassium, R ² , R ³ , R ⁴ , R ⁵ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are independently hydrogen. Atom, sulfate group, acetyl group, R ⁶ and R ¹² are each independently a hydrogen atom, acetyl group, methyl group, ethyl group, n-propyl group, n-butyl group, n-octyl group, benzyl group, allyl group , X, Y are each independently an acetamino group, a glycolylamino group, a hydroxyl group, an acetyloxy group,
A and B each independently represent an oxygen atom or a sulfur atom), a food or drug containing the compound of (2) (1) as an active ingredient, and a compound of (3) (1) as an active ingredient And a food or medicine for preventing or treating influenza virus.

Since the compound represented by the general formula (I) has an excellent anti-influenza virus action, as will be apparent from the examples described below, foods, foods for specified health uses, and health drinks utilizing this action. , Healthy foods, nutritional foods, and other various types of foods (in the present invention, foods include beverages),
It can also be used as a medicine such as an anti-influenza virus agent. When used as a food, the compound of the present active ingredient may be used as it is, or may be used in combination with other foods or food ingredients in accordance with an ordinary method.
The food according to the present invention using the present active ingredient may be in a solid form (powder, granular form or the like), a paste form, a liquid form or a suspension form, but is commonly used in the production of sweeteners, acidulants, vitamins and other drinks. It is also possible to manufacture into a health drink using the various ingredients mentioned above.

When used as a pharmaceutical, the compound of the present invention can be administered to animals and humans as it is or together with a conventional pharmaceutical carrier by either oral or parenteral administration. Further, the compound of the present invention can be used in various dosage forms such as powders, granules, tablets, dragees, capsules,
It may be an oral administration agent such as ampoule, subcutaneous, intramuscular or intravenous injection, suppository and the like. These formulations include compounds alone or their derivative excipients, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, flavors, coatings. It can be produced by appropriately combining with agents and the like and prescribing. The anti-influenza virus agent of the present invention thus obtained varies depending on the patient's age, body weight, symptom and administration route, but generally, in adults, the compound is 0.6 to 300 mg / day, preferably 5 to The dose is 200 mg / day, and it is usually preferable to administer this in 3 to 4 divided doses per day.

[0013]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0014]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[Example 1] 4 ', 5, 7-Trihydroxy-8-meth
Synthesis of oxyflavone (2) Method by Iinuma et al. (Munekazu Iinuma, Toshiyuki Tanaka, Ki
yoshi Iwashima, Yakugaku Zasshi (Pharmaceutical magazine), 104
(6), 691-694 (1984)) and synthesized by the method shown in FIG.

The melting point (mp) was measured using a Yazawa By-10 melting point measuring device and was not corrected. Mass spectrum (MS) is based on fast atom bombardment (FAB, m-
JEOL JMS-DX300 and JMS-AX505HA
It measured using the apparatus. The infrared absorption (IR) spectrum is
It was measured by the KBr tablet method using a JASCO FT / IR-400 Plus device. Specific rotation ([α] _D ) was measured using JASCO DIP-370 device.
It was measured at ° C. Nuclear Magnetic Resonance (NMR) spectrum is Varian
Using the VXR-300 device, Tetrame
thylsilane (TMS) and Sodium 3- (Trimethylsilyl) -1-p
It was measured using ropanesulfonate (DSS). Silica gel 60 (manufactured by Merck, 70-230 mesh) was used for silica gel column chromatography.

[Example 2] Synthesis of Neu5Ac-flavone derivative Methyl N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-2,
3-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl
onate (4) and 4 ', 5,7-trihydroxy-8-methoxyflavonedi-Na
Reaction of salt (3) (Fig. 2) 5-Hydroxy-4 ', 7-di- (methyl N-acetyl-4,7,8,9-tetra-O
-acetyl-3,5-dideoxyl-D-glycero-α-D-galacto-2-nonu
lopyranosylonate) -8-methoxyflavone (5) and 5,7-dihydro
xy-4 '-(methyl N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-
dideoxyl-D-glycero-α-D-galacto-2-nonulopyranosylo
Synthesis of nate) -8-methoxyflavone (6) 2 (80 mg, 0.26 mmol) was dissolved in MeOH (5 ml), and
After 8% NaOMe-MeOH (120 mg) was added, the solvent was evaporated, and the residue was dried under reduced pressure (0.8 mmHg) at room temperature for 3 hours. Na salt of 2
Dissolve (3) in N, N-dimethylformamide (5 ml), and add Methyl N-acetyl-4,7,8,9-tetra-O-acetyl-2-chloro-
2,3-dideoxy-D-glycero-β-D-galacto-2-nonulopyranos
Yylonate (4, 0.75 g, 1.47 mmol) was added under an argon stream and stirred at room temperature for 2 hours, and then the reaction solution was directly subjected to silica gel column chromatography (CHCl ₃ -MeOH, 10: 1) to separate the deoxy derivative. After that, it was separated and purified using re-silica gel column chromatography (AcOEt), and 5 (65 mg,
20%) and 6 (50 mg, 24%) were obtained as powders. 5: [α] _D + 18.0 ° (c = 0.5, CHCl ₃ ), Anal. Calcd for C ₅₆ H ₆₆ NO ₃₀ : C, 53.93; H, 5.33; N,
2.25. Found: C, 55.58; H, 5.32; N, 2.20. FAB-MS m / z: 1248 (M ⁺ +1) IR ν _max (cm ^-1 ): 3380, 1750, 1655, 1610, 1545, 15
00. ¹ H-NMR (CDCl ₃ ) δ: Neu5Ac moiety; 2.24 (dd, 1H, J
= 11.5, 13.0 Hz, 3-Hax), 2.74 (dd, 1H, J = 4.5, 1
3.0 Hz, 3-Heq), 4.06 (dt, 1H, J = 10.5, 10.0Hz, 5-
H), 4.18 (dd, 1H, J = 6.0, 12.5 Hz, 9-H), 4.33 (d
d, 1H, J = 2.5, 12.5 Hz, 9'-H), 4.47 (dd, 1H, J =
1.5, 11.0 Hz, 6-H), 5.03 (ddd, 1H, J = 4.5, 10.5, 1
1.5 Hz, 4-H), 5.34 (dd, 1H, J = 6.5, 1.5 Hz, 7-H),
5,39 (ddd, 1H, J = 6.5, 6.0, 2.0 Hz, 8-H), 5.56
(d, 1H, J = 10.0 Hz, NH), 1.91 (s, 3H, NAc), 2.02
(s, 3H, OAc), 2.04 (s, 3H, OAc), 2.12 (s, 3H, OAc),
2.16 (s, 3H, OAc), 3.66 (s, 3H, CO ₂ Me), Neu5Ac 'm
oiety; 2.28 (dd, 1H, J = 12.0, 13.0 Hz, 3-Hax), 2.
73 (dd, 1H, J = 4.5, 13.0 Hz, 3-Heq), 4.12 (ddd, 1
H, J = 11.15, 10.5, 10.0 Hz, 5-H), 4.10 (dd, 1H, J
= 6.0, 12.5 Hz, 9-H), 4.26 (dd, 1H, J = 2.0, 12.5
Hz, 9'-H), 4.56 (dd, 1H, J = 1.0, 11.0 Hz, 6-H),
4.96 (ddd, 1H, J = 4.5, 10.5, 12.0 Hz, 4-H), 5.36
(dd, 1H, J = 8.5, 1.0 Hz, 7-H), 5,39 (ddd, 1H, J =
8.5, 6.0, 2.0 Hz, 8-H), 5.45 (d, 1H, J = 10.0 Hz,
NH), 1.90 (s, 3H, NAc), 2.01 (s, 3H, OAc), 2.03
(s, 3H, OAc), 2.11 (s, 3H, OAc), 2.14 (s, 3H, OAc),
3.66 (s, 3H, CO ₂ Me), Flavone moiety; 6.66 (1H, br.
s, 3-H), 6.73 (1H, s, 6-H), 3.93 (3H, s, 8-OMe),
7.18 (2H, d, J = 9.0 Hz, 2'-H, 6'-H), 7.86 (2H, d,
J = 9.0 Hz, 3'-H, 5'-H) 6: [α] _D + 8.5 ° (c = 0.45, CHCl ₃ ), Anal.Calcd for C ₃₆ H ₃₉ NO ₁₉ : C, 55.89; H, 5.08; N,
1.81. Found: C, 55.91; H, 4.99; N, 1.75. FAB-MS m / z: 774 (M ⁺ +1) IR ν _max (cm ^-1 ): 3390, 1750, 1655, 1605, 1580, 15
_{^{10. 1 H-NMR (CDCl 3}} ) δ: Neu5Ac moiety; 2.26 (dd, 1H, J
= 11.5, 13.0 Hz, 3-Hax), 2.76 (dd, 1H, J = 4.5, 1
3.0 Hz, 3-Heq), 4.10 (dt, 1H, J = 10.5, 10.0Hz, 5-
H), 4.18 (dd, 1H, J = 6.0, 12.5 Hz, 9-H), 4.40 (d
d, 1H, J = 2.5, 12.5 Hz, 9'-H), 4.48 (dd, 1H, J =
1.0, 10.5 Hz, 6-H), 5.06 (ddd, 1H, J = 4.5, 10.5, 1
1.5 Hz, 4-H), 5.38 (dd, 1H, J = 6.5, 1.5 Hz, 7-H),
5,43 (ddd, 1H, J = 6.5, 6.0, 2.5 Hz, 8-H), 5.43
(d, 1H, J = 10.0 Hz, NH), 1.95 (s, 3H, NAc), 2.03
(s, 3H, OAc), 2.07 (s, 3H, OAc), 2.16 (s, 3H, OAc),
2.19 (s, 3H, OAc), 3.81 (s, 3H, CO ₂ Me), Flavone moi
ety; 6.52 (1H, s, 3-H), 6.72 (1H, s, 6-H), 3.91 (3
H, s, 8-OMe), 7.23 (2H, d, J = 9.0 Hz, 2'-H, 6'-H),
6.91 (2H, d, J = 9.0 Hz, 3'-H, 5'-H), 12.2 (1H, br.
s, 5-OH) Deprotection group reaction 5 (35 mg) was dissolved in MeOH (5 ml), and 28% NaOMe-MeO was added to it.
H (0.1 ml) was added and the mixture was left at room temperature for 2 hours. The reaction solution was evaporated and dried to dryness, water (1 ml) was added and the mixture was left at room temperature for 3 hours. The reaction solution is subjected to gel filtration (Sephadex G-10) as it is, and the filtrate is lyophilized to give 5-hydroxy-4 ', 7-di- (sodium N-acetyl-
3,5-dideoxyl-D-glycero-α-D-galacto-2-nonulopyrano
Sylonate) -8-methoxyflavone (7, 22 mg, 85%) was obtained as a pale yellow powder. 7: [α] _D + 77.3 ° (c = 0.5, H ₂ O) Anal. Calcd for C ₃₈ H ₄₄ N ₂ Na ₂ O ₂₂ : C, 49.25; H, 4.79;
N, 3.02. Found: C, 50.02; H, 4.99; N, 3.11.IR ν _max (cm ^-1 ): 3400, 1655, 1610, 1580, 1500. ¹ H-NMR (CDCl ₃ ) δ: Neu5Ac moiety; 1.97 (dd, 1H, J
= 12.5, 12.0 Hz, 3-Hax), 2.87 (dd, 1H, J = 5.0, 1
2.5 Hz, 3-Heq), 3.59 (dd, 1H, J = 9.5, 1.5 Hz, 7-
H), 3.63 (dd, 1H, J = 5.0, 12.5 Hz, 9-H), 3.81 (dd
d, 1H, J = 5.0, 10.0, 12.0 Hz, 4-H), 3.86 (dd, 1H,
J = 3.0, 12.5 Hz, 9'-H), 3.89 (ddd, 1H, J = 9.5,
5.0, 3.0 Hz, 8-H), 3.96 (q, 1H, J = 10.0 Hz, 5-H),
4.10 (dd, 1H, J = 1.5, 10.0 Hz, 6-H), Neu5Ac 'moi
ety; 1.97 (dd, 1H, J = 12.5, 12.0Hz, 3-Hax), 2.85
(dd, 1H, J = 5.0, 12.5 Hz, 3-Heq), 3.59 (dd, 1H, J
= 9.5, 1.5 Hz, 7-H), 3.63 (dd, 1H, J = 5.0, 12.5
Hz, 9-H), 3.86 (dd, 1H, J = 3.0, 12.5 Hz, 9'-H), 3.
89 (ddd, 1H, J = 9.5, 5.0, 3.0 Hz, 8-H), 3.93 (q,
1H, J = 10.0 Hz, 5-H), 3.77 (ddd, 1H, J = 5.0, 1
0.0, 12.0 Hz, 4-H), 4.04 (dd, 1H, J = 1.5, 10.0 H
z, 6-H), Flavone moiety; 6.77 (1H, br.s, 3-H), 6.
81 (1H, s, 6-H), 3.94 (3H, s, 8-OMe), 7.94 (2H, d,
J = 8.5 Hz, 2'-H, 6'-H), 6.91 (2H, d, J = 8.5 Hz,
Deprotection reaction of 3'-H, 5'-H), 6 (20 mg) was performed in the same manner as in the case of 5 above, 5,7-dihydroxy-4 '-(sodium N-acetyl-3,5 -dideoxyl
-D-glycero-α-D-galacto-2-nonulopyranosylonate) -8-
Methoxyflavone (8, 14 mg, 86%) was obtained as a pale yellow powder. 8: [α] _D + 52.7 ° (c = 0.33, H ₂ O), Anal. Calcd for C ₂₇ H ₂₇ N ₂ Na ₂ O ₁₄ : C, 51.03; H, 4.28;
N, 2.20. Found: C, 50.93; H, 4.33; N, 2.14. IR ν _max (cm ^-1 ): 3400, 1650, 1605, 1575, 1500. ¹ H-NMR (CDCl ₃ ) δ: Neu5Ac moiety; 1.99 (dd, 1H, J
= 12.5, 12.0 Hz, 3-Hax), 2.87 (dd, 1H, J = 5.0, 1
2.5 Hz, 3-Heq), 3.59 (dd, 1H, J = 9.5, 1.5 Hz, 7-
H), 3.65 (dd, 1H, J = 5.0, 12.5 Hz, 9-H), 3.81 (dd
d, 1H, J = 5.0, 10.0, 12.0 Hz, 4-H), 3.88 (dd, 1H,
J = 3.0, 12.5 Hz, 9'-H), 3.89 (ddd, 1H, J = 9.5,
5.0, 3.0 Hz, 8-H), 3.95 (q, 1H, J = 10.0 Hz, 5-H),
4.09 (dd, 1H, J = 1.5, 10.0 Hz, 6-H), Flavone moi
ety; 6.52 (1H, s, 3-H), 6.72 (1H, s, 6-H), 3.91 (3
H, s, 8-OMe), 7.23 (2H, br.s, 2'-H, 6'-H), 6.91 (2
H, br.s, 3'-H, 5'-H),

[Example 3] Synthesis of KDN-flavone derivative Methyl 4,5,7,8,9-penta-O-acetyl-2-chloro-2,3-dideo
xy-D-glycero-β-D-galacto-2-nonulopyranosylonate
(9) and 4 ', 5,7-trihydroxy-8-methoxyflavone di-Na salt (3)
Reaction (Fig. 3) 5-Hydroxy-4 ', 7-di- (methyl 4,5,7,8,9-penta-O-acetyl
-3-deoxyl-D-glycero-α-D-galacto-2-nonulopyranosyl
onate) -8-methoxyflavone (10) and 4 ', 5-dihydroxy-7- (m
ethyl 4,5,7,8,9-penta-O-acetyl-3-deoxyl-D-glycero-
α-D-galacto-2-nonulopyranosylonate) -8-methoxyflav
one (11) and 5,7-dihydroxy-4 '-(methyl 4,5,7,8,9-pent
aO-acetyl-3-deoxyl-D-glycero-α-D-galacto-2-nonul
Synthesis of opyranosylonate) -8-methoxyflavone (12) 2 (172 mg, 0.5 mmol) was dissolved in MeOH (5 ml),
After 28% NaOMe-MeOH (240 mg) was added, the solvent was evaporated, and the mixture was dried under reduced pressure (0.8 mmHg) at room temperature for 3 hours. Na salt of 2
Dissolve (3) in N, N-dimethylformamide (15 ml) and add Methyl N-acetyl-4,5,7,8,9-penta-O-acetyl-2-chl.
oro-2,3-dideoxy-D-glycero-β-D-galacto-2-nonulopyr
Anosylonate (9, 0.75 g, 1.5 mmol) was added under an argon stream, and the mixture was stirred at room temperature for 2 hours, and the reaction mixture was directly purified by silica gel column chromatography (AcOEt-hexane, 1: 1). (115 mg, 18%), 11 (30 mg, 7
%), 12 (75 mg, 19%), and deoxy derivatives (methyl
4,5,7,8,9-penta-O-acetyl-2,6-anhydro-3-deoxy-D-gly
cero-D-galacto-non-2-enonate, 301 mg, 41%) was obtained as a powder. 10: [α] _D + 19.7 ° (c = 0.67, CHCl ₃ ), Anal. Calcd for C ₅₆ H ₆₄ O ₃₂ : C, 53.85; H, 5.16. Foun
d: C, 53.58; H, 5.32. FAB-MS m / z: 1249 (M ⁺ +1) IR ν _max (cm ^-1 ): 3380, 1755, 1610, 1610, 1500. ¹ H-NMR (CDCl ₃ ) δ: KDN moiety; 2.26 (dd, 1H, J = 1
1.5, 13.0 Hz, 3-Hax), 2.74 (dd, 1H, J = 4.5, 13.0 H
z, 3-Heq), 4.21 (dd, 1H, J = 5.5, 12.5 Hz, 9-H), 4.
32 (dd, 1H, J = 2.5, 12.5 Hz, 9'-H), 4.94 (t, 1H,
J = 10.0 Hz, 5-H), 4.46 (dd, 1H, J = 1.5, 10.0 Hz,
6-H), 5.04 (ddd, 1H, J = 4.5, 10.0,11.5 Hz, 4-H),
5.37 (dd, 1H, J = 8.0, 1.5 Hz, 7-H), 5,45 (ddd, 1
H, J = 8.0, 5.5, 4.5 Hz, 8-H), 2.00 (s, 3H, OAc),
2.04 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.05 (s, 3H,
OAc), 2.08 (s, 3H, OAc), 2.20 (s, 3H, OAc), 3.67
(s, 3H, CO ₂ Me), KDN 'moiety; 2.26 (dd, 1H, J = 1
2.0, 13.0 Hz, 3-Hax), 2.83 (dd, 1H, J = 4.5, 13.0
Hz, 3-Heq), 4.16 (dd, 1H, J = 3.5, 12.5 Hz, 9-H),
4.32 (dd, 1H, J = 2.5, 12.5 Hz, 9'-H), 4.63 (dd,
1H, J = 1.0, 10.0 Hz, 6-H), 4.90 (t, 1H, J = 10.0 H
z, 5-H), 5.04 (ddd, 1H, J = 4.5, 10.0, 12.0 Hz, 4-
H), 5.39 (dd, 1H, J = 9.0, 1.0 Hz, 7-H), 5,45 (dd
d, 1H, J = 9.0, 3.5, 2.5 Hz, 8-H), 2.03 (s, 3H, O
Ac), 2.04 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.12 (s,
3H, OAc), 2.17 (s, 3H, OAc), 3.80 (s, 3H, CO ₂ Me), F
lavonemoiety; 3.94 (3H, s, 8-OMe), 6.66 (1H, s, 3-
H), 6.77 (1H, s, 6-H), 7.19 (2H, d, J = 9.0 Hz, 2'-
H, 6'-H), 7.87 (2H, d, J = 9.0 Hz, 3'-H, 5'-H), 1
2.37 (1H, s, 5-OH). 11: [α] _D + 19.7 ° (c = 0.36, CHCl ₃ ), Anal. Calcd for C ₃₆ H ₃₈ O ₁₉ : C, 55.82; H, 4.94. Foun
d:. C, 55.95; H , 4.99 FAB-MS m / z: 775 (M + +1) IR ν max (cm -1): 3390, 1755, 1655, 1605, 1510. 1 H-NMR (CDCl 3 ) δ: KDN moiety; 2.26 (dd, 1H, J = 1
1.5, 13.5 Hz, 3-Hax), 2.83 (dd, 1H, J = 4.5, 13.5 H
z, 3-Heq), 4.22 (dd, 1H, J = 5.5, 12.5 Hz, 9-H), 4.
38 (dd, 1H, J = 2.5, 12.5 Hz, 9'-H), 4.46 (dd, 1H,
J = 1.5, 10.5Hz, 6-H), 4.94 (t, 1H, J = 10.0 Hz,
5-H), 5.05 (ddd, 1H, J = 4.5, 10.5, 11.5 Hz, 4-H),
5.39 (dd, 1H, J = 8.0, 1.5 Hz, 7-H), 5,48 (ddd, 1
H, J = 8.0, 5.5, 2.5 Hz, 8-H), 2.02 (s, 3H, OAc),
2.05 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.14 (s, 3H,
OAc), 2.16 (s, 3H, OAc), 3.82 (s, 3H, CO ₂ Me), Flav
one moiety; 3.91 (3H, s, 8-OMe), 6.50 (1H, br.s, 3
-H), 6.73 (1H, s, 6-H), 6.76 (2H, d, J = 9.0 Hz, 2 '
-H, 6'-H), 7.72 (2H, d, J = 9.0 Hz, 3'-H, 5'-H), 12.
40 (1H, s, 5-OH) 12: [α] _D + 20.5 ° (c = 0.3.7, CHCl ₃ ), Anal. Calcd
for C ₃₆ H ₃₈ O ₁₉ : C, 55.82; H, 4.94. Found: C, 55.9
. 7; H, 4.56 FAB- MS m / z: 775 (M + +1) IR ν max (cm -1): 3390, 1755, 1655, 1610, 1510. 1 H-NMR (CDCl 3) δ: KDN moiety; 2.23 (dd, 1H, J = 1
1.5, 13.0 Hz, 3-Hax), 2.81 (dd, 1H, J = 4.5, 13.0
Hz, 3-Heq), 4.22 (dd, 1H, J = 5.0, 12.5 Hz, 9-H),
4.32 (dd, 1H, J = 2.5, 12.5 Hz, 9'-H), 4.48 (dd, 1
H, J = 2.0, 10.0Hz, 6-H), 4.90 (t, 1H, J = 10.0 H
z, 5-H), 5.05 (ddd, 1H, J = 5.0, 10.0,11.5 Hz, 4-
H), 5.37 (dd, 1H, J = 8.0, 2.0 Hz, 7-H), 5,44 (dd
d, 1H, J = 8.0, 5.0, 2.5 Hz, 8-H), 2.00 (s, 3H, OA
c), 2.04 (s, 3H, OAc), 2.12 (s, 3H, OAc), 2.17 (s,
3H, OAc), 2.35 (s, 3H, OAc), 3.81 (s, 3H, CO ₂ Me),
Flavone moiety; 3.91 (3H, s, 8-OMe), 6.67 (1H, s,
3-H), 6.88 (1H, s, 6-H), 7.28 (2H, d, J = 9.0 Hz,
2'-H, 6'-H), 7.85 (2H, d, J = 9.0 Hz, 3'-H, 5'-H),
12.31 (1H, s, 5-OH) deprotection reaction 10 (50 mg) was dissolved in MeOH (5 ml), and 28% NaOMe-Me was added to it.
OH (0.1 ml) was added and the mixture was left at room temperature for 2 hours. The reaction solution was evaporated and dried to dryness, water (1 ml) was added and the mixture was left at room temperature for 3 hours. The reaction solution is subjected to gel filtration (Sephadex G-10) as it is, and the solution is freeze-dried to give 5-Hydroxy-4 ', 7-di- (sodium 3,5-dideox).
yl-D-glycero-α-D-galacto-2-nonulopyranosylonate)-
8-Methoxyflavone (13, 29 mg, 86%) was obtained as a pale yellow powder. 13: [α] _D + 52.6 ° (c = 0.35, H ₂ O) Anal. Calcd for C ₃₄ H ₃₈ Na ₂ O ₂₂ : C, 48.35; H, 4.53. F
ound: C, 49.11; H, 4.86. IR ν _max (cm ^-1 ): 3400, 1655, 1605, 1500. ¹ H-NMR (CDCl ₃ ) δ: KDN moiety; 1.94 (dd, 1H, J = 1
2.5, 12.0 Hz, 3-Hax), 2.81 (dd, 1H, J = 5.0, 12.5 H
z, 3-Heq), 3.63 (t, 1H, J = 9.5 Hz, 5-H), 3.99 (d
d, 1H, J = 1.5, 10.0 Hz, 6-H). KDN moiety; 1.97 (d
d, 1H, J = 12.5,12.0 Hz, 3-Hax), 2.78 (dd, 1H, J =
5.0, 12.5 Hz, 3-Heq), 3.64 (t, 1H, J = 9.5 Hz, 5-
H), 4.04 (dd, 1H, J = 1.5, 10.0 Hz, 6-H), Flavon
e moiety; 6.70 (1H, br.s, 3-H), 6.80 (1H, s, 6-H),
3.93 (3H, s, 8-OMe), 7.24 (2H, d, J = 8.5 Hz, 2'-
H, 6'-H), 7.90 (2H, d, J = 8.5 Hz, 3'-H, 5'-H). The deprotection reaction of 11 (20 mg) was carried out in the same manner as in 10 above. Done 4 ', 7-dihydroxy-5- (sodium 3-deoxyl-D-glycero-
α-D-galacto-2-nonulopyranosylonate) -8-methoxyflav
One (14, 14 mg, 91%) was obtained as a pale yellow powder. 14: [α] _D + 57.2 ° (c = 0.28, H ₂ O), Anal. Calcd for C ₂₅ H ₂₄ Na ₂ O ₁₄ : C, 50.51; H, 4.07. F
ound:. C, 49.96; H , 4.68 IR ν max (cm -1): 3400, 1605, 1575, 1500. 1 H-NMR (CDCl 3) δ: KDN moiety; 1.99 (dd, 1H, J = 1
2.5, 12.0 Hz, 3-Hax), 2.87 (dd, 1H, J = 4.5, 12.5 H
z, 3-Heq), 3.67 (t, 1H, J = 9.5 Hz, 5-H), 4.09 (b
rd, 1H, J = 9.5 Hz, 6-H), Flavone moiety; 6.19 (1
H, s, 3-H), 6.61 (1H, s, 6-H), 3.92 (3H, s, 8-OMe),
7.23 (2H, d, 2'-H, 6'-H), 6.91 (2H, br.s, 3'-H,
The deprotection group reaction of 5'-H). 12 (15 mg) was performed in the same manner as in the case of the above 10, 5,7-dihydroxy-4 '-(sodium 3,5-dideoxyl-D-glycol.
ero-α-D-galacto-2-nonulopyranosylonate) -8-methoxy
Flavone (15, 10 mg, 86%) was obtained as a pale yellow powder. 15: [α] _D + 38.37 ° (c = 0.23, H ₂ O), Anal. Calcd for C ₂₅ H ₂₄ Na ₂ O ₁₄ : C, 50.51; H, 4.07. F
ound: C, 49.88; H, 4.57. IR ν _max (cm ^-1 ): 3400, 1650, 1605, 1575, 1500. ¹ H-NMR (CDCl ₃ ) δ: KDN moiety; 2.00 (br.t, 1H, J =
12.5 Hz, 3-Hax), 2.82 (dd, 1H, J = 5.0, 12.5 Hz, 3
-Heq), 3.66 (t, 1H, J = 9.5 Hz, 5-H), 4.00 (dd, 1H,
J = 1.5, 9.5 Hz, 6-H), Flavone moiety; 6.35 (1H,
s, 3-H), 6.68 (1H, s, 6-H), 3.90 (3H, s, 8-OMe), 6.
40 (2H, br.s, 2'-H, 6'-H), 7.35 (2H, br.s, 3'-H,
5'-H).

Example 4 Activity Measurement of Water-Soluble Sample MDCK (Madin-Darby cani) cultured in 96-well culture plate
0.001 MOI (multiplicity of infectio) of influenza virus A / PR / 8/34 in the presence of trypsin
n; multiplicity of infection), and samples (final concentration 100
μg / mL) or Ribavirin solution (final concentration 12 or 24 μg / mL)
Was added. This was cultured at 37 ° C. for 3 days under 5% CO ₂ . As an index of anti-influenza virus activity, the virus amount in the culture supernatant was measured by the sialidase activity of the virus, and the cell survival rate was measured by the MTT method. Regarding cytotoxicity, as in the case of the anti-influenza virus activity measurement, the sample or Ribavirin lysed in the same manner as described above was added to influenza virus-uninfected MDCK cells cultured on a 96-Well plate. These are 5% CO ₂
Under the conditions, the cells were cultured at 37 ° C. for 3 days, and the cell viability was measured by the MTT method. The results are shown in FIGS. 5 and 6. Among the measured compounds, compounds 8, 7, 15, and 14 significantly reduced the amount of virus in the culture supernatant (Fig. 5). But,
Of these, only Compound 7 showed a clear improvement in cell viability (FIG. 6). Compound 8 did not show a significant improvement in cell viability and Compounds 15 and 14 were cytotoxic. Since compound 7 was found to have a reduced amount of virus in the culture supernatant, an effect of improving the survival rate of virus-infected cells, and no cytotoxicity, it was confirmed that the compound 7 was used in the system using cultured cells. It became clear that it exhibits influenza virus activity. After adding compound 7 to MDCK cells and culturing for 3 days, the cells were observed under a microscope and as a result, many granules were observed in the cells. It was considered that Compound 7 was probably taken up into the cell. From this, it is expected that, when compound 7 is intranasally administered to mice in an in vivo anti-influenza virus activity experiment, it is taken up by nasal mucosal cells and retained in the nasal cavity to exhibit anti-influenza virus activity.

Example 5 Activity Measurement of Water-Insoluble Sample The anti-influenza virus activity was measured as follows. That is, a sample or a methanol solution of F36 (1 mg /
(25 mL) was soaked in a paper disk (8 mm in diameter) and air-dried to remove the solvent. Influenza virus A / PR / 8/34 was added to MDCK cells cultured in a 48-well culture plate.
Was infected, the sample was soaked, and an air-dried paper disk was put thereinto. In addition, a negative control was prepared by injecting an air-dried disc soaked only with the solvent methanol. This is 37 ° C and 5% CO ₂ condition 3
After culturing for a day, the viability of the cells was measured by the MTT method using the virus amount of the culture supernatant as the sialidase activity of the virus. Further, a cell disk was soaked with the sample without being infected with the virus, and an air-dried paper disk was put thereinto to measure cytotoxicity. In the conjugates of the water-insoluble sialic acid derivative or KDN derivative and the flavone derivative (F36) of 5 samples, the cell viability was improved in 6, 5, and 11 as compared with the control (FIG. 7). Of these, 6 and 11 showed some cytotoxicity, whereas 5 did not show cytotoxicity (Fig. 7) and showed a remarkable improving effect on cell viability (Fig. 7). On the other hand, among the 5 water-insoluble samples, only 5 significantly reduced the amount of virus in the culture supernatant as compared with the control, and almost completely suppressed the virus growth (FIG. 8). The in vitro anti-influenza virus activity of compound 5 was stronger than that of the flavone F36 used as a positive control. Of the newly synthesized sialic acid derivative or KDN derivative and F36 conjugate, 5 water-insoluble samples were analyzed by in v
The itro anti-influenza virus activity was measured. As a result, compound 5 was found to have a marked decrease in the amount of virus in the culture supernatant, an effect of improving the survival rate of virus-infected cells, and no cytotoxicity. Therefore, cultured cells were used. It was revealed that the system exhibits anti-influenza virus activity.

[0021]

According to the present invention, a sialic acid derivative or
The compound combining the KDN derivative and the flavone derivative can improve the survival rate of cells infected with influenza virus, is a compound derived from a natural product compound, and has high safety, and is useful as an agent for preventing or treating influenza. In addition, it can be used as a food for preventing or treating influenza.

[Brief description of drawings]

FIG. 1 is a diagram showing a method for synthesizing a flavone derivative.

FIG. 2 is a diagram showing a method for synthesizing a conjugate of sialic acid and a flavone derivative.

FIG. 3 is a diagram showing a method for synthesizing a conjugate of KDN and a flavone derivative.

FIG. 4 is a diagram showing structures of compounds whose antiviral activity was measured.

FIG. 5 shows the effect of water-soluble compounds on the survival rate of MDCK cells infected with influenza virus. The black bar shows the survival rate with virus addition, and the shaded bar shows the survival rate without virus addition.

FIG. 6 is a graph showing the influence of water-soluble compounds on influenza virus growth in MDCK cells.

FIG. 7 shows the effect of water-insoluble compounds on the viability of MDCK cells infected with influenza virus. The black bar shows the survival rate with virus addition, and the shaded bar shows the survival rate without virus addition.

FIG. 8 shows the effect of water-insoluble compounds on influenza virus growth in MDCK cells.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Katsumi Ajisaka             540 Narita, Odawara City, Kanagawa Prefecture, Meiji Dairy Industry             Nutritional Science Institute Co., Ltd. F term (reference) 4B018 LB10 LE01 LE05 MD42 ME09                       MF10                 4C057 BB05 CC03 DD01 DD02 JJ55                       KK08                 4C086 AA01 AA02 AA03 EA04 EA11                       MA01 MA04 NA14 ZB33

Claims (3)

[Claims] 1. The following general formula (I): (In the formula, R ¹ and R ⁷ are each independently a hydrogen atom, sodium, potassium, R ² , R ³ , R ⁴ , R ⁵ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are independently hydrogen. Atom, sulfate group, acetyl group, R ⁶ and R ¹² are each independently a hydrogen atom, acetyl group, methyl group, ethyl group, n-propyl group, n-butyl group, n-octyl group, benzyl group, allyl group , X, Y are each independently an acetamino group, a glycolylamino group, a hydroxyl group, an acetyloxy group,
A and B each independently represent an oxygen atom or a sulfur atom). 2. A food or medicine containing the compound according to claim 1 as an active ingredient. 3. A food or medicine for preventing or treating influenza virus, which comprises the compound according to claim 1 as an active ingredient.