JP5103613B2 - Sialic acid thioglycoside polymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sialic acid thioglycoside polymer and to provide a polymer suppliable as a medicine in relation to a pharmaceutical comprising the polymer as an active ingredient. <P>SOLUTION: The sialic acid thioglycoside polymer is represented by the chemical formula (wherein, R<SB>1</SB>represents a hydroxy group, an amino group or an acetyl group; R<SB>2</SB>represents hydrogen, a metal atom or a methyl group; and m and n are each an integer of &ge;1 and may each be same or different) and a pharmaceutically acceptable salt thereof and a hydrate thereof. A pharmaceutical composition for prophylaxis and therapy of infectious diseases is provided. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a sialic acid thioglycoside polymer and a medicament containing the polymer as an active ingredient. More specifically, the present invention relates to a water-soluble polymer to which thioglycoside-type sialic acid is bound, which has an infection inhibitory activity by influenza virus, and a medicine containing the polymer as an active ingredient.

  Influenza viruses tend to introduce mutations into the eight single-stranded RNAs present therein, and as a result, resistant viruses to vaccines and the like that have been effective in the past tend to occur. Due to the nature of the influenza virus, infectious diseases caused by the influenza virus are prevalent every year, and many deaths are caused by this. Adhesion and detachment from host cells are important for influenza virus infection, and two different glycoproteins (hemagglutinin and sialidase) act on it. In addition, it is known that sugar chains are involved in the recognition of antigens present on the surface of host cells by these glycoproteins. And it is also known that the site | part which recognizes those sugar chains does not change.

  Focusing on the nature of such influenza viruses, several anti-influenza virus agents have been produced and have been effective in suppressing the epidemic of influenza infections. As these preparations, for example, an ion channel inhibitor of influenza membrane protein (Symmetrel (registered trademark) (amantadine)), an inhibitor of sialidase (Tamiflu (registered trademark) oseltamivir phosphate) and Relenza (registered trademark) (zanamivir) ) Is prescribed as a specific medicine for influenza. However, since these specific drugs are different from natural products, the emergence of resistant viruses is feared, and in recent years there are reports of cases where resistant viruses against Symmetrel (registered trademark) and Tamiflu (registered trademark) have emerged. .

  Up to now, the inventors have specifically adhered hemagglutinin present on the surface of viruses such as influenza viruses based on the knowledge about various sugar chain-containing carbosilane dendrimer compounds (Non-Patent Document 1), and virus infection to living bodies. A sialyl lactose-containing dendrimer has been disclosed as a substance capable of preventing the above (Patent Document 1). Furthermore, the dendrimer which couple | bonds a sugar chain through the amide bond excellent in the in vivo compatibility and safety | security is also performed (patent document 2).

Matsuoka et al., Bull. Chem. Soc. Jpn. 71: 2709-2713 1998 International Publication WO02 / 02588 JP2003-212893

An object of the present invention is to provide a water-soluble polymer that suppresses the emergence of resistant viruses and is effective in protecting against infection with influenza viruses.
Furthermore, this invention aims at provision of the pharmaceutical which contains this polymer as an active ingredient.

  In view of the above circumstances, the present inventors have conducted extensive research on the development of a polymer that suppresses the emergence of resistant viruses and is effective in protecting against infection with influenza viruses. As a result, sialic acid thioglycoside polymers were infected with influenza. Has been found to be able to be effectively inhibited, and the present invention has been completed.

That is, the present invention provides the following formula (I)
(I)
(Wherein R ₁ represents a hydroxyl group, an amino group or an acetyl group, R ₂ represents hydrogen, a metal atom or a methyl group, and m and n are integers of 1 or more, which may be the same or different). A sialic acid thioglycoside polymer is provided.

  The sialic acid thioglycoside polymer of the present invention is a compound having antiviral activity, particularly anti-influenza activity, and having an effect of suppressing the appearance of resistant viruses at a low rate.

In formula (I), R ₁ is most preferably a water-soluble compound such as a hydroxyl group, an amino group or an acetyl group, and R ₂ is hydrogen, a metal atom or a methyl group. M and n are integers of 1 or more and may be the same or different, preferably 1 or more and 10 or less, more preferably 1 or more and 7 or less, and may be the same or different.

  The compound of the formula (I) of the present invention can be produced, for example, according to the following reaction formula.

In the above formula, 4 can be synthesized according to the following scheme 1.
Scheme 1

  That is, a fully protected body was obtained by acetylating the hydroxyl group of sialic acid (1) and methylating the carboxyl group. Subsequently, it induced | guided | derived to (beta) sialic acid chloride (2) with hydrogen chloride gas. By treating (2) with a thioacetate anion, the stereochemistry of the anomer was reversed (3). S-glycoside derivative (4) was synthesized by sulfide formation reaction between (3) and mesylate derived from 10-undecen-1-ol. On the other hand, O-glycoside derivative (5) was prepared by silver-catalyzed glycosylation of (2) and 10-undecen-1-ol.

(7) and (8) were synthesized according to Scheme 2 below.
Scheme 2

  The polymer was derived from a radical copolymerization reaction of sialic acid derivative (4) and vinyl acetate (6) (7). A water-soluble polymer (8) from which a protecting group was removed by deacetylation and demethylation was synthesized.

By containing the compound of the present invention as an active ingredient, a prophylactic and therapeutic agent for viral infection, particularly influenza virus infection, can be prepared.
The drug may contain one or more kinds of sialic acid thioglycoside polymers represented by the general formula (I), pharmaceutically acceptable salts thereof or hydrates thereof. Further, when the compound of the present invention represented by the general formula (I) is formulated, it is usually contained in the preparation so as to be 0.1 to 50% by mass, preferably 0.5 to 20% by mass. Is done.

The compounds of the present invention can be used as therapeutic agents for specific diseases in the form of pharmaceutical compositions that do not adversely affect the living body. Such compositions typically include a pharmaceutically acceptable carrier in addition to the compound of the present invention.
“Pharmaceutically acceptable carriers” are those suitable for pharmaceutical administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, agents that act isotonically to delay adsorption and the like. Examples of preferred for diluting the carrier and the carrier include, but are not limited to, water, saline, finger solution, dextrose solution, collagen, human serum albumin, organic solvent, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, Carboxyvinyl polymer, sodium alginate, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol , Lactose and the like. Non-aqueous media such as liposomes and non-volatile oils are also used. In addition, certain compounds that protect or promote the activity of the compounds of the present invention may be included in the composition.

The pharmaceutical composition according to the present invention includes intravenous, intradermal, subcutaneous, oral (including inhalation, etc.), transdermal and transmucosal administration, and is formulated to be suitable for a therapeutically appropriate route of administration. It becomes. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application include, but are not limited to, sterile diluents such as water for injection, saline solutions, non-volatile oils, polyethylene glycols, Glycerin, propylene glycol, or other synthetic solvents, benzyl alcohol or other preservatives such as methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, soothing agents such as benzalkonium chloride, procaine hydrochloride, ethylenediaminetetraacetic acid Chelating agents such as (EDTA), buffering agents such as acetate, citrate, or phosphate, and agents for osmotic pressure adjustment such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations are contained in ampoules, glass or plastic disposable syringes or multiple dose vials.

  Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water soluble) or dispersion media and sterile powders for the preparation of sterile injectable solutions or dispersion media at the time of use. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). When used as an injection, the composition must be sterile and must be fluid enough to be administered with a syringe. The composition must be stable to chemical changes, corrosion, and the like during formulation and storage, and must prevent contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures. For example, using a coating agent such as lectin, maintaining a required particle size in the dispersion medium, and maintaining a proper fluidity by using a surfactant. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal can be used to prevent microbial contamination. In addition, polyalcohols such as sugar, mannitol, sorbitol, and agents that maintain isotonicity such as sodium chloride may be included in the composition. Compositions that can delay adsorption include agents such as aluminum monostearate and gelatin.

  Sterile injectable solutions are prepared by adding the required ingredients alone or in combination with other ingredients to the required amount of the active compound in a suitable solvent and sterilizing. Generally, a dispersion medium is prepared by incorporating the active compound into a sterile medium that contains a basic dispersion medium and the other necessary ingredients described above. Methods for preparing a sterile powder for preparing a sterile injectable solution include vacuum drying and lyophilization to prepare a powder containing the active ingredient and any desired ingredients derived from the sterile solution. .

Oral compositions include inert diluents or carriers that are not harmful when incorporated into the body. Oral compositions are, for example, contained in gelatin capsules or compressed into tablets. For oral treatment, the active compound is incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a flowable carrier, and the composition in the flowable carrier is applied orally. In addition, pharmaceutically compatible binding agents, and / or adjuvant materials may be included.
Tablets, pills, capsules, troches and the like may contain any of the following components or compounds with similar properties: excipients such as microcrystalline cellulose, binding such as gum arabic, tragacanth or gelatin Agents; bulking agents such as alginic acid, PRIMOGEL or corn starch such as starch or lactose; lubricants such as magnesium stearate or STRROTES; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl A fragrance additive such as salicylic acid or orange flavor.

  The compounds of the present invention can be prepared as sustained release formulations such as delivery systems encapsulated in implantable tablets and microcapsules using a carrier that can prevent immediate removal from the body. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Useful liposomes are prepared as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanol (PEG-PE) through a filter of appropriate pore size to obtain a size suitable for use, and reverse phase. Purified by evaporation.

In the prevention or treatment of a specific disease with the compound of the present invention, the appropriate dosage level depends on the condition of the patient to be administered, the administration method, etc., but can be easily optimized by those skilled in the art. It is.
In the case of injection administration, for example, it is preferable to administer about 0.1 μg / kg to about 500 mg / kg of the patient's body weight per day, and it will generally be possible to administer a single dose or divided into multiple doses. Preferably, the dosage level is about 0.1 μg / kg to about 250 mg / kg per day, more preferably about 0.5 μg to about 100 mg / kg per day.
For oral administration, the composition is preferably provided in the form of a tablet containing 1.0 to 1000 mg of active ingredient, preferably 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750. 0, 800.0, 900.0 and 1000.0 mg. The compounds are administered on a regimen of 1 to 4 times daily, preferably once or twice daily.

  A pharmaceutical composition or formulation must consist of uniform unit doses to ensure a constant dose. A unit dose is a unit formulated with a pharmaceutically acceptable carrier, including a single dose effective for treating a patient. The unit dosage of the present invention is determined by the physical and chemical characteristics of the compound to be formulated, the expected therapeutic effect, and the formulation considerations specific to the compound. .

  The pharmaceutical composition of the present invention can be included in the form of a kit in a container or pack together with instructions for administration. When the pharmaceutical composition according to the present invention is supplied as a kit, different constituents of the pharmaceutical composition are packaged in separate containers and mixed immediately before use. The reason why the components are packaged separately in this way is to enable long-term storage without losing the function of the active component.

  Reagents contained in the kit are supplied in any type of container in which the components remain active for an extended period of time, are not adsorbed by the container material, and are not subject to alteration. For example, a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents. Examples of other suitable containers include simple bottles made from similar materials such as ampoules, and packaging materials that are internally lined with foil such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. The container has a sterile access port such as a bottle having a stopper that can be penetrated by a hypodermic needle.

  The kit also includes instructions for use. Instructions for using the kit comprising the pharmaceutical composition are printed on paper or other material and / or floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as an electrically or electromagnetically readable medium. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.

Furthermore, the present invention also includes a method for preventing or treating viral infections, particularly mammals infected with or at risk of infection with influenza viruses.
Here, “treatment” means to prevent or alleviate the progression of the pathology of the infectious disease in a mammal that is likely to be infected with a virus or is infected. It is used as a broad meaning including.
The “mammal” to be treated means any animal classified as a mammal, and is not particularly limited. For example, in addition to humans, pet animals such as dogs and cats, cows, pigs, sheep, horses It is a domestic animal. Particularly preferred “mammals” are humans.

  Examples are shown below, but the present invention is not limited thereto.

ω-undecylenyl methanesulfonate (10-undecenyl mesylate) (11)
10-Undecen-1-ol (10) (77.1 mg, 0.453 mmol) was dissolved in pyridine (0.771 mL), and methanesulfonyl chloride (70.1 μL, 0.906 mmol) was added. After argon substitution, the mixture was stirred for 3 hours under ice cooling. After confirming the completion of the reaction by TLC, the reaction mixture was diluted with chloroform (1 mL), water (0.5 mL) was added, and methanesulfonyl chloride was crushed. Extraction was performed using chloroform in the order of 1 M sulfuric acid, saturated aqueous sodium hydrogen carbonate solution, and saturated brine. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated and 10-undecenyl mesylate (11) was quantitatively synthesized (Scheme 3).
R _f 0.87 [20: 1 (v / v) chloroform-methanol]

Scheme 3

Methyl- (ω-undecylenyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-α-D-galacto-2-nonuropyranoside) onate (12)
The sialic acid thioacetyl derivative (9) (80.0 mg, 0.145 mmol) was dissolved in methanol (0.83 mL), purged with argon, and stirred under ice cooling. 10-undecenyl mesylate (11) (70.5 mg, 0.302 mmol) and potassium carbonate (20.9 mg, 0.151 mmol) were sequentially added, and the mixture was stirred overnight at room temperature. After confirming the completion of the reaction by TLC, acetic acid (17.3 μL, 0.302 mmol) was added and stirred, followed by concentration. After concentration, the residue was dissolved in pyridine (1.54 mL), acetic anhydride (0.285 mL, 3.02 mmol) was added, and the mixture was stirred overnight at room temperature. Extraction was performed using chloroform in the order of ice-cold water, 1M sulfuric acid, saturated aqueous sodium hydrogen carbonate, and saturated brine. Anhydrous magnesium sulfate was added to the organic layer and dried, followed by filtration after drying. The filtrate was concentrated and purified by silica gel column chromatography [chloroform-methanol, 70: 1 (v / v)] to give a novel sialic acid-S-glycoside derivative (12) (88.0 mg, 92.0% %) Was synthesized (see Scheme 4, Table 1).

Scheme 4

Copolymerization by vinyl acetate - Synthesis novel sialic acid thioglycoside derivative (12) of the sugar chain polymer (14) (50mg, 7.58 × 10- 2 mmol) of (12) with respect to 10eq vinyl acetate (69.9MyuL, 7.58 × 10 ⁻¹ mmol) or 20 eq vinyl acetate (139 μL, 15.2 × 10 ⁻¹ mmol) (Table 2). AIBN (1.0 mg to 1.3 mg, 6.09 μmol to 7.92 μmol) was added, and the mixture was stirred at a predetermined temperature while refluxing. The progress of the reaction was confirmed by TLC, and after completion of the reaction, it was dissolved in methanol and concentrated. The sialic acid thioglycoside polymer (14) was synthesized by purification by gel filtration chromatography [Sephadex LH-20] (see Scheme 5, Table 3 and FIG. 1).

Scheme 5

Synthesis of vinyl alcohol-sugar chain polymer (15) by deprotection Sialic acid polymer (14) (22 mg) was dissolved in 1 M sodium methoxide-methanol solvent (1 mL). After stirring at room temperature for 3 hours, 0.5 M aqueous sodium hydroxide solution (1 mL) was added and stirred overnight. After neutralization with strong acid cation exchange resin IR-120B (H ⁺ type), cotton filtration was performed. The filtrate was concentrated and the structure was confirmed by NMR. Since the methyl ester was not removed, a 1M sodium methoxide-methanol solution (1 mL) and a 0.5M aqueous sodium hydroxide solution (1 mL) were simultaneously added and dissolved again, followed by stirring overnight. After neutralization with strong acid cation exchange resin IR-120B (H ⁺ type), cotton filtration was performed. After cotton filtration, the filtrate was concentrated and the structure was confirmed by NMR. After confirming that the acetyl group and the methyl group were removed, purification by gel filtration chromatography [Sephadex G-50] was followed by freeze-drying to obtain a water-soluble sialic acid polymer (15) (see FIG. 2).

Experiment on Inhibitory Activity of Influenza Virus Sialidase A well of a 96-well plate was mixed with various concentrations of sample solution (4 μL) and influenza A virus (4 μL) and incubated at 4 ° C. for 1 hour. To each well, 0.4 mM 4-MUNEuAc solution (2 μL) was added and cultured at 37 ° C. for 0.5 hour. Subsequently, reaction stop solution (200 μL) was added to each well to stop the hydrolysis reaction. 100 μL of each well was transferred to a separately prepared 96-well plate for fluorescence measurement, and the fluorescence intensity at excitation light of 355 nm and emission of 460 nm was measured (Table 4 and FIG. 3).

  The present invention can be used for the development of pharmaceuticals for inhibiting influenza virus infection.

FIG. 1 shows an NMR chart of the monomer (12) and the sugar chain polymer (14). FIG. 2 shows an IR chart of the sugar chain polymers (14) and (15). FIG. 3 shows the results of measurement of influenza virus sialidase inhibitory activity with compound (15).

Claims (5)

Formula (I)
(I)
(In the formula, R ₁ is a hydroxyl group, an amino group or an acetyl group, R ₂ represents hydrogen, a metal atom or a methyl group, and m and n are integers of 1 or more and may be the same or different). And pharmaceutically acceptable salts thereof and hydrates thereof. The sialic acid thioglycoside polymer according to claim 1, R ₁ is a hydroxyl group, and R ₂ is hydrogen, pharmaceutically acceptable salts thereof, and hydrates thereof.   The sialic acid thioglycoside polymer and pharmaceutically acceptable salt thereof and hydrates thereof according to claim 2, wherein m is 1 and n is an integer of 1 to 9.   A thiosialic acid thioglycoside polymer according to any one of claims 1 to 3, a pharmaceutically acceptable salt and hydrate thereof, and a pharmaceutically acceptable carrier, and A pharmaceutical composition for treatment. The pharmaceutical composition according to claim 4, wherein the infectious disease is an influenza virus infection.