JP2001131074A - Medicament containing sialic acid derivative as active component - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new medicaments comprising a sialic acid derivative, especially a low-molecular sialic acid derivative, and more concretely to provide a prophylactic and/or therapeutic agent for viral disease such as influenza virus infection disease. SOLUTION: The medicaments comprise a compound represented by formula I wherein R1 is a 1-6C alkyl group; R2 is a (substituted) phenyl group, a 1-20C alkyl group, a 1-20C alkenyl group or (CH2)k-[NH-(CH2)m-NH]n-Y (wherein Y is phoshatidyl ethanolamine residue, polyglutamic acid residue or the like; k and m are each an integer of 2 to 10); R3 is H or a (substituted) hydroxy group; R4 is H, a halogen, azido group, a (substituted) amino group or a (substituted) hydroxy group; and R5 to R8 are each a (substituted) hydroxy group} or its pharmacologically permissive salt as an active component.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a medicine containing a sialic acid derivative as an active ingredient.

[0002]

2. Description of the Related Art Since viruses, including influenza, multiply depending on the biosynthesis mechanism of the host, it is difficult to develop a drug capable of exhibiting a sufficient antiviral action specifically against the virus, and clinically. There are few known drugs that can exhibit high efficacy. For virus infection,
At present, treatment with vaccines is the most effective means, but if the virus in question is not identified, vaccines cannot be produced and used, and viruses with severe mutations However, there is a problem that the vaccine produced cannot be expected to be effective.

[0003] Influenza viruses include types A, B, and C. It is known that type A virus can pass through the human immune system as a host by drastically changing the structure of a protein as an antigen. Therefore, there is a problem that vaccine treatment cannot sufficiently cope. Influenza virus has two types of spiked glycoproteins,
It has a trimeric hemagglutinin (HA) and a tetrameric sialidase (NA). Influenza virus infects by multivalent specific action of its hemagglutinin (HA) and sialoglycoside receptor of the host cell, and sialidase (NA)
The virus propagated by destroying the virus receptor is released from the host cell. Both of these two types of proteins use a sialo-glycan present on the host cell surface as a ligand. Therefore, it is considered that infection of the influenza virus can be inhibited by interfering with the interaction between these two proteins and the sialo-glycan.

Recently, very effective antiviral agents have been developed based on the analysis of the crystal structure of sialidase, one of two viral membrane proteins of influenza virus (von Itzstein, M. et al., Nature, 363, 188
3). However, since sialidase is not involved in the first stage of infection, there is a problem that this antiviral agent cannot prevent the establishment of infection itself.
Therefore, it is necessary to develop an antiviral agent that can exert a strong inhibitory action on both of two types of membrane proteins (sialidase and hemagglutinin). To date, sialyl lactose-containing polymers have shown a strong inhibitory activity against H1 type virus HA (Japanese Patent Application Laid-Open No. H11-14795), and polymers having sialic acid monosaccharides bound as C- or S-glycosides are H3-type. It is reported to have strong inhibitory activity against viral HA. However,
To date, no compound that is a low-molecular-weight sialic acid derivative capable of exerting a potent inhibitory action on two types of membrane proteins (sialidase and hemagglutinin) has not been reported.

[0005] The present inventor has conducted a study to find a compound having a substituent on the carbon atom at the 3-position for the purpose of providing a compound which can be expected to exhibit excellent antiviral activity against influenza virus and the like. We have succeeded in synthesizing a sialic acid derivative (JP-A-11-343).
295; 20th Carbohydrate Symposium, July 1998). However, it has not been confirmed that these sialic acid derivatives actually have anti-influenza virus activity.

[0006]

An object of the present invention is to provide a novel drug containing a sialic acid derivative, particularly a low molecular weight sialic acid derivative, more specifically, a prophylactic agent for a viral disease such as influenza virus infection and the like. And / or providing a therapeutic agent. Another object of the present invention is to provide a novel drug containing a sialic acid derivative showing resistance to hydrolysis against sialidase, more specifically, a preventive and / or therapeutic agent for a viral disease such as influenza virus infection. It is to be.

[0007]

Means for Solving the Problems The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, sialic acid derivatives which may have a substituent on the carbon atom at the 3-position have a hydrolysis resistance. At the same time as having an inhibitory effect on the membrane-bound protein of influenza virus, thereby completing the present invention. That is, the present invention provides the following formula
(I):

Embedded image [Wherein, R ¹ represents a hydrogen atom or a C _1-6 alkyl group;
² is a phenyl group which may have a substituent, a C _1-20 alkyl group, a C _1-20 alkenyl group,-(CH ₂ ) _k- [NH-
_{(CH 2) m -NH] n} -Y, - (CH 2) k - [NH-
_{(CH 2 CH 2 O) m} -1 -CH 2 CH 2 NH] n -Y (Y represents a phosphatidylethanolamine residue or polyglutamic acid residue, 2 to k and m each independently 10
And n represents 0 or 1, provided that when n represents 1, Y represents a phosphatidylethyl group); R ³ represents a hydrogen atom or a hydroxyl group which may have a substituent; R ⁴ Represents a hydrogen atom, a halogen atom, an azide group,
R ⁵ , R ⁶ , R ⁷ , and R ⁸ each independently represent a hydroxyl group which may have a substituent; an amino group which may have a substituent, or a hydroxyl group which may have a substituent; Or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof as an active ingredient.

[0008] Preferably, the medicament of the present invention is a prophylactic and / or therapeutic agent for a viral disease. More preferably,
The medicament of the present invention is a prophylactic and / or therapeutic agent for influenza virus infection. More specifically, the medicament of the present invention has an influenza virus infection and / or growth inhibitory action.

Preferably, in the formula (I), R^TwoIs-(C
H_Two)_k-[NH- (CH_Two)_m-NH] _n-Y or-(C
H_Two)_k-[NH- (CH_TwoCH_TwoO)_m-1-CH_TwoCH_TwoN
H]_n-Y, particularly preferably-(CH_Two)_k-[NH-
(CH_Two)_m-NH]_n-Y (wherein Y is phosphatidyl
Indicates an ethanolamine residue or polyglutamic acid residue
And k and m each independently represent an integer of 2 to 10.
And n represents 0 or 1, provided that when n represents 1,
Y represents a phosphatidylethyl group). Even better
More preferably, in the formula (I), R^TwoIs-(CH_Two)_k−Y (expression
In the formula, Y represents a phosphatidylethanolamine residue;
k represents an integer of 2 to 10). Preferably,
R^ThreeAnd R^FourOne is a hydrogen atom and the other is a halogen
A hydroxyl group which may have an atom or a substituent. Sa
More preferably, R^ThreeIs a hydrogen atom, and R^FourIs halogen
A hydroxyl group which may have an atom or a substituent. Good
Preferably, R¹Is a hydrogen atom.

[0010] In another aspect, the present invention provides a method for producing a medicament (particularly, a prophylactic and / or therapeutic agent for a viral disease such as influenza virus infection).
Use of the compound represented by (I) or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof; and a method for preventing and / or treating a viral disease such as influenza virus infection. A method comprising administering an effective amount of the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof or a hydrate or solvate thereof to a mammal including a human. Provided.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The terms used in the present specification have the following meanings. The alkyl moiety in the alkyl group or the functional group containing the alkyl moiety may be linear, branched, cyclic, or a combination thereof. For example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, a cyclopropylmethyl group, a cyclobutyl group, and the like can be used.

When the phenyl group represented by R ² has a substituent, the number and position of the substituent are not particularly limited. For example, a substituted or unsubstituted C _1-6 alkyl group such as a methyl group, a trifluoromethyl group and a hydroxyethyl group;
A C _1-6 alkoxy group such as a methoxy group and an ethoxy group; a halogen atom (in the present specification, a halogen atom may be any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom); A substituted or unsubstituted C _1-6 alkanoyl group such as a fluoroacetyl group; a substituted or unsubstituted aralkyl group such as a benzyl group, a p-methoxybenzyl group, a p-chlorobenzyl group; a nitro group; a cyano group; Substituted or unsubstituted aryl group such as chlorophenyl group; substituted or unsubstituted aroyl group such as benzoyl group and chlorobenzoyl group; carboxyl group; C _1-6 alkoxycarbonyl group such as ethoxycarbonyl group and tert-butoxycarbonyl group; Substituted or unsubstituted amino group, monomethylamino group, acetylamino group, etc. And the like can be mentioned amino groups, it is not limited thereto. Of these, a nitro group is preferred, and the substitution position is preferably the p-position.

The C _1-20 alkyl group or C _1-20 alkenyl group represented by R ² may be linear, branched, cyclic, or a combination thereof. The number of unsaturated bonds in the alkenyl group is not particularly limited, but is, for example, one to three, and preferably one. -(CH ₂ ) _k- [NH- (C
H _{2) m} -NH] _n -Y or _{- (CH 2) k - [} NH- (C
H ₂ CH ₂ O) _m-1 —CH ₂ CH ₂ NH] _n —Y In the group represented by Y, Y represents a phosphatidylethanolamine residue or a polyglutamic acid residue, where k is 2 and n
Is preferably 0. In this case, Y is a nitrogen atom of phosphatidylethanolamine - (CH _{2) k} - is preferably bound to. It is preferable to use an N-terminal acetylated polyglutamic acid residue as the polyglutamic acid residue, and it is preferable that k is 2 and n is 1. In the present invention, R ² is preferably-(CH ₂ ) <sub>k
- [NH- (CH 2) m</sub> -NH] n -Y or - (CH <sub>2) k
- [NH- (CH 2 CH 2</sub> O) m-1 -CH 2 CH 2 NH] n -
Y (wherein, Y represents a phosphatidylethanolamine residue or a polyglutamic acid residue, k and m each independently represent an integer of 2 to 10, n represents 0 or 1,
However, when n represents 1, Y represents a phosphatidylethyl group), and particularly preferably, R ² is — (CH ₂ ) _k
—Y (wherein, Y represents a phosphatidylethanolamine residue, and k represents an integer of 2 to 10).

When R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , or R ⁸ is a hydroxyl group having a substituent, the substituent on the hydroxyl group is not particularly limited. And a substituent used for eliminating a hydroxyl group can be used. Examples of such a substituent include a substituted or unsubstituted C _1-6 alkanoyl group such as an acetyl group and a trifluoroacetyl group; and a substituted or unsubstituted group such as a benzyl group, a p-methoxybenzyl group, and a p-chlorobenzyl group. Aralkyl group; alkylsilyl group such as trimethylsilyl group; substituted or unsubstituted C _1-6 alkyl group such as methyl group, trifluoromethyl group, hydroxyethyl group; substituted or unsubstituted aryl group such as phenyl group and chlorophenyl group A substituted or unsubstituted aroyl group such as a benzoyl group and a chlorobenzoyl group; a C _1-6 alkoxycarbonyl group such as an ethoxycarbonyl group and a tert-butoxycarbonyl group; a p-toluenesulfonyl group, a methanesulfonyl group and a trifluoromethanesulfonyl And other substituted sulfonyl groups Possible, without being limited thereto.

When R ⁴ represents an amino group having a substituent, one or two substituents on the amino group may be the same or different, and the type thereof is not particularly limited. It is preferable to use a commonly used substituent or the like as the protecting group. Examples of such a substituent include a substituted or unsubstituted C _1-6 alkanoyl group such as an acetyl group and a trifluoroacetyl group; a benzyl group,
Substituted or unsubstituted aralkyl groups such as methoxybenzyl group and p-chlorobenzyl group; substituted or unsubstituted C _1-6 alkyl groups such as methyl group, trifluoromethyl group and hydroxyethyl group; phenyl group and chlorophenyl group A substituted or unsubstituted aryl group; a substituted or unsubstituted aroyl group such as a benzoyl group and a chlorobenzoyl group; and a C _1-6 alkoxycarbonyl group such as an ethoxycarbonyl group and a tert-butoxycarbonyl group. However, the present invention is not limited to these.

The compound represented by the formula (I) may form a salt depending on the type of the substituent. In the medicament of the present invention, a pharmaceutically acceptable salt of the compound represented by the formula (I) may be used, for example, mineral salts such as hydrochloride and sulfate; or p-toluenesulfonic acid Organic salts such as salts; metal salts such as sodium, potassium and calcium salts; ammonium salts; organic ammonium salts such as methylammonium salts; and amino acid salts such as glycine salts, but are not limited thereto. Never. In addition to the compound in free form or a salt thereof, any hydrate or solvate thereof may be used.

The compound represented by the formula (I) has an asymmetric carbon and may exist as an optically active substance. However, stereoisomers such as optically active substances and diastereoisomers, and stereoisomers Any mixture, racemate, and the like can be used in the present invention. Note that, regarding the steric notations for R ⁶ and R ⁷ in the formula (I), they indicate their relative configurations, and are interpreted as indicating the absolute configuration of the compound represented by the formula (I). Not be.

Among the compounds of the present invention, compounds in which R ² is a phenyl group which may have a substituent include, for example, known bromohydrin compounds (Okamoto, K., et al., Bull.
l. Chem. Soc. Jpn., 60, pp. 631-636, 1987) according to the following scheme. In the scheme, Ac represents an acetyl group, Me represents a methyl group,
Tf indicates a trifluoromethanesulfonyl group, Py indicates pyridine, DMF indicates dimethylformamide, THF indicates tetrahydrofuran, and TASF indicates tris (dimethylamino) -sulfonium difluorotrimethylsilicate.

[0019]

Embedded image

In the examples of JP-A-11-343295, the production methods of the representative compounds described in the above-mentioned schemes are described specifically and in detail. Any compound included in the general formula (I) can be easily produced by appropriately selecting reaction raw materials, reaction reagents, reaction conditions, and the like according to the method, and adding appropriate modifications or alterations as necessary. . JP-A-11-
All the contents described in JP-A-343295 are incorporated herein by reference. However, the method for producing the compound used in the present invention is not particularly limited, and any compound produced by any method can be used in the present invention.

Compounds in which R ² is a C _1-20 alkyl group or a C _1-20 alkenyl group are described in a paper by Okamoto et al.
K., et al., Bull. Chem. Soc. Jpn., 60, pp.637-643,
1987), an alkyl group or an alkenyl group may be introduced into the epoxy compound, and deprotection may be performed if necessary. A compound having an alkenyl group (for example, an allyl group) introduced therein is subjected to ozonolysis, and the obtained ketone compound (or aldehyde compound) is reacted with phosphatidylethanolamine to obtain-(CH ₂ ) _k -Y (where Y is Is a phosphatidylethanolamine residue). Further, H ₂ N— (CH ₂ ) _m —N
H ₂ or _{H 2 N- (CH 2 CH 2} O) m-1 -CH 2 CH 2 NH 2
By reacting a diamine compound represented by as a linker and further reacting a reactive acetylated polyglutamic acid derivative with the terminal amino group of the reaction product, a compound having a polyglutamic acid residue introduced can be produced. . As a method for introducing polyglutamic acid, for example, a method described in JP-A-11-147951 can be employed.

The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, or a hydrate or a solvate thereof, used in the present invention may be hemagglutinin, a membrane-bound protein of influenza virus. And sialidase have an inhibitory action, and a similar inhibitory action can be expected for membrane-bound proteins of other viruses. Also,
Since the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, or a hydrate or a solvate thereof used in the present invention is resistant to sialidase hydrolysis, Their administration is an effective treatment for diseases in which the sialic acid recognition reaction is in the early stage of infection.

Therefore, the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is useful as an active ingredient of a medicament for preventing and / or treating a viral infection such as an influenza virus infection. is there. The pharmaceuticals of the present invention can be applied to, for example, influenza, viral hepatitis (type A, B, C, or E), viral pneumonia, viral bronchitis, herpes infection, polio, AIDS, Adult T-cell leukemia, papilloma, measles,
Rubella, exanthema rash, infectious erythema, viral encephalitis, viral meningitis, cytomegalovirus infection, epidemic parotitis, chickenpox, rabies, viral enteritis, etc., and particularly preferably influenza Can be mentioned.

The active ingredient of the medicament provided by the present invention includes a compound represented by the formula (I) in free form, a physiologically acceptable salt thereof, a hydrate thereof and a solvate thereof. A substance selected from the group can be used. Generally, the medicament of the present invention is provided in the form of a pharmaceutical composition containing the above-mentioned active ingredient and a pharmaceutical additive (carrier, excipient, etc.).

The route of administration of the medicament of the present invention is not particularly limited, and is orally or parenterally administered (for example, intramuscular administration,
Intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to the nasal cavity, or inhalation administration). The form of the medicament of the present invention is not particularly limited, and examples of formulations for oral administration include tablets, capsules, fine granules, powders, granules, solutions, syrups, and the like. Preparations include, for example, injections, drops, suppositories, inhalants, transmucosal absorbents, transdermal absorbents, nasal drops, ear drops and the like.

Those skilled in the art can appropriately select the form of the medicament of the present invention, the additives for the preparation to be used, and the method for producing the preparation. The dosage of the medicament of the present invention depends on the sex of the patient,
It can be appropriately selected in consideration of the age or weight, the severity of the symptoms, the purpose of administration such as prevention or treatment, the presence or absence of other complications, and the like. The dose is generally 0.001 μg / kg body weight / day to 1000 μg.
/ Kg body weight / day, preferably 0.001 μg / kg body weight / day to 100 μg / kg body weight / day. The present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited by the examples.

[0027]

EXAMPLES Test compounds: The compounds used in the tests are the following sialic acid derivatives 1 to 9 and 24 (the synthesis of these compounds is described in JP-A-11-343295 and the like). )

[0028]

Embedded image

Test Example 1: Sialidase hydrolysis resistance and sialidase inhibitory activity of sialic acid derivatives p-nitrophenyl-α-O-glycoside of sialic acid optionally having a substituent at the 3-position (compounds 2 to 5) And 2
Using 4) as a test compound, (1) acid hydrolysis resistance, (2) enzyme (sialidase) hydrolysis resistance,
(3) sialidase inhibitory activity (kinetic analysis), (4)
The inhibitory activity on hemagglutinin and (5) the inhibitory effect on influenza virus infection of MDCK cells were examined. (1) Acid hydrolysis resistance test The acid hydrolysis resistance test was performed for 4 to 10 mM compounds 2 to 5
And 24 (0.6 mL of 1.0 N D ₂ SO ₄ / D ₂ O
Medium (pD1.37)) and ¹ H NMR (270 MHz) at 50 ° C. on a JEOL EX-270 spectrometer.
The hydrolysis was monitored over time. Using a 2 second acquisition time and 20 scans, spectral data was collected at 3 hour intervals until hydrolysis had progressed 50%. The reaction yield was calculated from the relative integration of the signals corresponding to the aromatic protons of the substrate (H-3 ', 5' and H-2 ', 6') with respect to the signal of p-nitrophenol.

(2) Enzyme hydrolysis (sialidase-catalyzed hydrolysis) resistance test 50 mM acetate buffer (pH 5.5 for Arthrobacter Ureafaciens sialidase; Clostridium perfring
pH 5.0 for ens sialidase; and Vibrioch
olerae sialidase, pH 5.5), 0.5 m
M-sialic acid p-nitrophenyl-α-O-glycoside, test compounds (compounds 2 to 5 and 24) and sialidase (20 mU Arthrobacter Ureafaciens sialidase: 15 mU Clostridium perfringens sialidase: and 50 mU Vibrio cholerae sialidase; Was also purchased from Boehringer Mannheim GmbH), kept at 37 ° C., aliquoted (approximately 10 μL) of the reaction solution, and stopped with 10 μL of 200 mM Na ₂ CO ₃ aqueous solution. Electrophoresis was performed as follows.

The measurement was performed using a Water Quanta 4000E type capillary electrophoresis apparatus. Fused silica capillary (inside diameter 75μm, effective length 53cm)
The sample was washed with 0.1 M KOH for 2 minutes before sample introduction, and the column was replaced with a running solution. The sample was injected at a height difference of 10 cm for 30 seconds (3 seconds) by the siphon effect.
8.4 nL). For electrophoresis, a 50 mM borate buffer (pH 10.2) was used as an electrolyte, and a voltage of 20 kv was applied.
The test was performed at a constant temperature of 37 ° C. Detection was performed at 7.5 cm from the anode side by an ultraviolet absorption (405 nm) method. Mi
The results were analyzed using a llienium 2010 system (Millipore).

(3) Sialidase Inhibitory Activity (Kinetic Analysis) First, the IC _{50 of the} test compound (Compounds 2 to 5 and 24)
After the measurement, using the buffer used in the above hydrolysis test,
4-Methylumbelliferyl-α-DN-acetyl
Using neuraminic acid (compound 1) (0.4 mM) as a substrate, compounds and sialidase (1.
25 mU of Arthrobacter Ureafaciens sialidase:
0.1 mU Clostridium perfringens sialidase:
And 2.0 mU of Vibrio cholerae sialidase) to make a final volume of 30 μL, and kept at 37 ° C. for 10 minutes. The reaction was stopped by adding 30 μL of a 200 mM Na ₂ CO ₃ aqueous solution, and the measurement was carried out using capillary electrophoresis as follows.

For the measurement, a Backman P / ACE 5010 type capillary electrophoresis apparatus and a fused silica capillary were used.
He-Cd Laser (Kimmon Electro Co., Ltd, Ja.
(pan), excitation at 325 nm, using fluorescence at 375 nm, performed 7.0 cm from the positive electrode. The sample was injected by a pressurization method (1.0 sec., 300 nL). The electrophoresis was performed at 50 ° C. and a voltage of 35 ° C. using a 50 mM borate buffer (pH 10.2) as an electrolyte. Ki value is
Assuming that the inhibition mode was antagonistic inhibition, it was estimated from the following formula based on the IC ₅₀ (%) value. The IC ₅₀ value was obtained using the inhibitor concentration as a variable.

[0034]

(Equation 1)

(Where i is the inhibition rate (50%), [I] is the inhibitor concentration, [S] is the substrate concentration, and Km is the Michaelis constant.) The results obtained in the above three tests are shown in Table 1. Shown in

[0036]

[Table 1]

As can be seen from the results in Table 1, p-nitrophenyl-α-O-glycoside (compound 2), a natural sialic acid, was completely hydrolyzed by sialidase within 30 minutes, whereas position 3 was The modified sialic acid p-nitrophenyl-α-O-glycoside (compounds 3-5) was not hydrolyzed under similar conditions for 24 hours. On the other hand, in sialidase inhibition experiments by capillary electrophoresis,
In particular, p-nitrophenyl-α-O-glycoside of sialic acid in which a hydroxyl group or a fluorine atom is introduced in the 3-position axial configuration (compounds 4, 5 and 24) is obtained from Clostridium perf.
It showed a strong inhibitory activity against ringens sialidase of about Ki = 10 ^-6 M.

Test Example 2: Inhibitory Activity of Synthetic C-3 Modified Sialyl DSPE on Sialidase Activity of Human Influenza A Virus Influenza virus sialidase of sialyl DSPE derivative (compound 7-9) modified with C-3 of sialic acid The effects were examined. As a substrate for viral sialidase
PNP-Neu5Ac (compound 2) was used. Virus (A / PR / 8/34
(H1N1) or A / Aichi / 2/68 (H3N2) strain, 5 μg / μl) was incubated with 250 μM of each derivative at 37 ° C. for 1 hour, and then the viral sialidase activity was measured. As a result, viral sialidase was completely inhibited by Neu5Ac3αF-DSPE, but not by the other derivatives tested. Neu5Ac3αF-DSPE (9) has an IC ₅₀ (concentration of inhibitor required for 50% inhibition of viral sialidase) of 6 against A / PR / 8/34 (H1N1) sialidase.
2.50 μM, 31.25 μM for sialidase of A / Aichi / 2/68 (H3N2).

Test Example 3: Effect of Neu5Ac on Influenza A Virus Sialidase Activity Infected by Different Hosts
Inhibitory activity of 3αF-DSPE Ne against various influenza virus sialidases
The effect of u5Ac3αF-DSPE (9) was examined. In this case, human,
Sixteen strains isolated from duck and pig were examined. Compound 9 inhibited the sialidase activity of all influenza viruses tested (Table 3). The 50% inhibitory concentration (IC ₅₀ ) of Neu5Ac3αF-DSPE (9) against the tested viral sialidase was 1 when 4-MU-Neu5Ac was used as a substrate.
It was 5.6 to 500 μM, and 31.3 to 250 μM when PNP-Neu5Ac (compound 2) was used as a substrate.

Test Example 4: Influenza virus hemag
Inhibitory activity against rutinin Influenza virus A / PR / 8/34 (H1N
1) and 15 strains of A / Aichi / 2/68 (H3N2)
After culturing at 35 ° C. for 48 hours in aged chicken eggs, sucrose density gradient
Purified by the heart method. House rabbit anti flu virus
Antibodies were collected from chicken eggs as described above.
Irs A / PR / 8/34 (H1N1) and A / Aich
It was prepared using the i / 2/68 (H3N2) strain. TLC
Plate (Polygram Sil G; Macherey-Nagel, German
y), spot the test compounds (compounds 6 to 9)
Roform / Methanol / 12mM MgCl _Two(5:
4: 1, v / v / v) and then the plate
Egg albumin (crystallized, Taiyo Kagaku Compan
y, Ltd., Japan) and PBS containing 1% polyvirpyrrolidone
After blocking with a solution (solution A) at room temperature for 2 hours,
Remove the blocking solution by pulling, wash three times with PBS,
2⁸HAunits concentration (titer; Suzuki,
Y et al., Virology, 1992,189, 121-131) A / PR / 8
/ 34 (H1N1) and A / Aichi / 2/68 (H3
N2) up to 1 at 4 ° C with virus-containing PBS turbidity
It was kept warm for 2 hours. After washing 5 times with PBS, TLC plate
Was blocked with solution A at 4 ° C. for 30 minutes, and then PBS
And 3 times at 4 ° C together with the above antibody solution.
Warmed. The antibody was dissolved in PBS containing 3% polyvirpyrrolidone.
Anti-influenza diluted 1: 1000 with liquid (solution B)
The antibody was used. Plate washed with PBS with solution A
Block at 4 ° C for 30 minutes and remove blocking solution.
And then washed with PBS, horseradish peroxida
-Separated Protein A (Organon Teknika N.V. Cappel
Products) and a 1: 1000 dilution of solution B and 2 at 4 ° C.
Incubated for hours. Thereafter, the plate is washed with PBS, and the substrate solution (1
0 mM Tris-HCl buffer (pH 7.2), 0.3% 4-
Chloronaphthol, 3% H_TwoO_Two(5: 1: 0.02, v
/ V / v) and kept at room temperature for 20 minutes. Various
The binding activity of the compound to the virus is determined by dual-wavelength chromatography.
620n by Nah (CR-910, Shimazu, Kyoto)
Colorimetry was performed with m color development (control 430 nm). Got
The results are shown in Table 2 below.

The virus hemagglutination inhibitory activity was determined as follows. Hemagglutination inhibition activity was measured using a 96-well microplate, using a 0.01% (w / v) gelatin-PBS solution (pH 6.5) as a dilution buffer.
Performed using human erythrocytes. Various test compounds (compound 6
To 9) PBS solution containing 0.01% (w / v) gelatin (initial concentration 2 mM, serially diluted twice), add the virus turbid solution, and incubate at 4 ° C for 1 hour. The erythrocyte-PBS turbid solution was added dropwise (0.5 mL / well), and the mixture was further kept at 4 ° C. for 1 hour. Aggregation inhibitory activity was expressed as the highest dilution of the compound that completely inhibited aggregation. The results obtained are shown in Table 2 below.

[0042]

[Table 2]

As can be seen from the results in Table 2, Compounds 7 to 9 having a substituent at the 3-position showed H3 type virus HA affinity similar to that of Compound 6 having no substituent at the 3-position.
Affinity increased to 144% for compound 7 having a substituent in the equatorial configuration. In addition, these compounds are H
No affinity was shown for the type 1 virus HA. further,
As can be seen from the results in Table 2, in the HA inhibitory activity test,
Compounds 6 to 9 exhibited a strong inhibitory activity (HAI value on the order of μM) against H3 virus HA.

Test Example 5: Influenza A on MDCK Cells
Influenza virus infection of sialyl DSPE derivatives
The effect on the was investigated. Influenza in MDCK cells
During infection with derivatives of A / Aichi / 2/68 (H3N2)
The sum response was examined using the cytopathic effect of the virus as an index.
Derivative inhibition of influenza virus infection
The activity is determined by the lactate dehydrogenase (LD released from infected cells)
H) Activity was measured. Synthetic sialyl DSPE derivative
 (Compound 6-9) is MDCK cells of A / Aichi / 2/68 (H3N2)
Infection-dependently (IC ₅₀, 5 μM to 200 μ
M). Neu5Ac3αF-DSPE (Compound 9)
A / Aichi / 2/68 (H3N2) infection
Strongly suppressed. Compound 9 against A / Aichi / 2/68 (H3N2)
IC₅₀Was 5.6 μM. Compound 9 is another compound
(Neu5Ac-DSPE (compound 6), Neu5Ac3βOH-DSPE (compound
7) and 8 compared to Neu5Ac3αOH-DSPE (compound 8)).
It showed twice the activity.

To investigate the mechanism of inhibition, two stages of drug treatment were set before and after infection.
The virus and the derivative were pre-incubated for 2 hours, and the mixture was infected and contacted with MDCK cells for 1 hour. Thereafter, the solution was removed and washed, and the cells treated with MEME medium without serum were further maintained for 20 hours. Was measured. Neu5Ac
The inhibitory effect of 3αF-DSPE (compound 9) was reduced, but for the other derivatives it was similar to the case without removing the mixture. Add derivative immediately after virus infection and LD 20 hours later
When H activity was measured, Neu5Ac3αF-DSPE (compound 9)
Only inhibited the cytopathic effect due to viral infection, whereas the other derivatives tested did not show that activity. When these are combined, Neu5Ac3αF-DSPE (compound 9)
Was shown to suppress infection not only in the early stage of infection but also in the late stage. On the other hand, the other derivatives suppressed only the stage related to the adsorption of the virus to the cell membrane.

[0046]

[Table 3]

[0047]

Industrial Applicability According to the present invention, there is provided a novel drug having an inhibitory effect on both hemagglutinin and sialidase which are membrane-bound proteins of influenza virus.
The medicament of the present invention is useful as a prophylactic and / or therapeutic agent for viral diseases such as influenza virus infection.

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Claims (9)

[Claims] (1) The following formula (I): [Wherein, R ¹ represents a hydrogen atom or a C _1-6 alkyl group;
² is a phenyl group which may have a substituent, a C _1-20 alkyl group, a C _1-20 alkenyl group,-(CH ₂ ) _k- [NH-
_{(CH 2) m -NH] n} -Y, - (CH 2) k - [NH-
_{(CH 2 CH 2 O) m} -1 -CH 2 CH 2 NH] n -Y (Y represents a phosphatidylethanolamine residue or polyglutamic acid residue, 2 to k and m each independently 10
And n represents 0 or 1, provided that when n represents 1, Y represents a phosphatidylethyl group); R ³ represents a hydrogen atom or a hydroxyl group which may have a substituent; R ⁴ Represents a hydrogen atom, a halogen atom, an azide group,
R ⁵ , R ⁶ , R ⁷ , and R ⁸ each independently represent a hydroxyl group which may have a substituent; an amino group which may have a substituent, or a hydroxyl group which may have a substituent; Or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof as an active ingredient. 2. The medicament according to claim 1, which is a prophylactic and / or therapeutic agent for a viral disease. 3. The medicament according to claim 1, which is a prophylactic and / or therapeutic agent for influenza virus infection. 4. The medicament according to any one of claims 1 to 3, which has an influenza virus infection and / or growth inhibitory activity. Wherein R ² is _{- (CH 2) k - [} NH- (CH 2)
_m -NH] _n -Y or _{- (CH 2) k - [} NH- (CH 2 C
During _{H 2 O) m-1 -CH} 2 CH 2 NH] n -Y ( wherein, Y represents a phosphatidylethanolamine residue or polyglutamic acid residue, k and m to from 2 each independently represent an integer of 10 , N represents 0 or 1, provided that when n represents 1, Y represents a phosphatidylethyl group).
The medicament according to any one of claims 1 to 4. 6. The method according to claim 1, wherein R ² is — (CH ₂ ) _k —Y, wherein Y represents a phosphatidylethanolamine residue and k represents an integer of 2 to 10. 1
The medicament according to item. 7. One of R ³ and R ⁴ is a hydrogen atom,
The medicament according to any one of claims 1 to 6, wherein the other is a halogen atom or a hydroxyl group which may have a substituent. 8. The medicament according to claim 1, wherein R ³ is a hydrogen atom, and R ⁴ is a halogen atom or a hydroxyl group which may have a substituent. 9. The method according to claim ¹ , wherein R ¹ is a hydrogen atom.
The medicament according to any one of the above.