JP2974370B2 - Sialidase inhibitors - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an antiviral agent having a sialidase inhibitory activity and useful for treating a viral disease such as influenza.

[Prior art and problems] Influenza diseases are respiratory infections caused by influenza viruses, and the characteristics to be summarized are as follows.
It is a severe epidemic in which the virus is represented by a severe antigenic variation and the world is one epidemic area.

At present, there are no satisfactory anti-influenza virus agents at the clinical application level, and the situation where influenza virus has been a major threat to humanity as before has not been improved.

Recently, it has been known that sialic acid plays an important role in the process of infecting cells with certain viruses such as influenza virus. In particular, it has been suggested that sialic acid hydrolase (sialidase) on the envelope of influenza virus is closely involved in the virus invasion into host cells or the release of virus from host cells.

Therefore, sialidase inhibitors are considered to be promising targets as next-generation anti-influenza virus drugs, and their development has been demanded.

[Means for Solving the Problems] The present inventors have intensively studied to develop an antiviral agent, particularly an anti-influenza virus drug, and have been searching for a substance having a sialidase inhibitory activity. The present inventors have found that certain types of flavone represented by the following formula I have a sialidase inhibitory action, and have completed the present invention. That is, the present invention is as described below.

Formula I below (In the formula, R represents a hydrogen atom, a hydroxyl group or a methoxyl group.) A sialidase inhibitor comprising a compound represented by the following formula (hereinafter, referred to as a compound of the formula) as an active ingredient:

The following compound can be obtained, for example, as follows.

That is, a compound-containing crude drug of the formula (Scutellariae Radix), its original plant Scutellari
a baicalensis or half-branch lotus (Scutellariae Herba),
The original plant, Scutellaria rivularis, or another congener plant is extracted with a lower alcohol such as methanol or ethanol, an organic solvent such as chloroform or water, and the solvent is distilled off.The residue is subjected to silica gel column chromatography. Can be obtained.

In some cases, after extraction with a water-containing organic solvent, defatting is performed using a hydrocarbon such as benzene, and the mixture is extracted with shaking using a solvent such as ether or ethyl acetate. After removal, the filtrate is partitioned with ether, ethyl acetate, or a mixed solvent of water and ether, or water and ethyl acetate, the solvent in the organic solvent-soluble portion is distilled off, and the residue may be subjected to silica gel column chromatography ( R
Is a methoxyl group or a hydrogen atom). Also,
After deleading the precipitate generated when adding the above-mentioned lead acetate,
Partitioning is performed with ethyl ether ether or a mixed solvent of water and ether, or water and ethyl acetate, the solvent in the organic solvent-soluble portion is distilled off, and the residue may be subjected to silica gel column chromatography (R is a hydroxyl group. Compound).

Further, if necessary, purification may be performed by recrystallization using methanol or the like.

Some of the compounds of the formula are commercially available (Funakoshi Pharmaceutical Co., Ltd.) and may be purchased.

Although the compound of the formula is a known substance, it has never been known that it has a sialidase inhibitory action.

 Next, specific examples of the production of the compound of the formula will be shown.

Specific Example 1 Among the compounds of the formula, a compound in which R is a hydroxyl group is called isoscutellarein.
It can be obtained as follows.

100 g of leaves of Scutellaria baicalensis were immersed in a small amount of water overnight and extracted with chloroform.
The solvent was distilled off from this extract, and the residue was extracted with hot 50% methanol or 50% ethanol 200m. A 50% methanol or 50% ethanol saturated solution of lead acetate was added to the extract until no precipitation occurred, followed by centrifugation. Precipitation
Suspend in 100m of 10% hydrochloric acid aqueous solution and add 3m in 30m of ethyl acetate.
Extracted by shaking twice. The ethyl acetate layer was washed with water, the solvent was distilled off, and 0.4 g of a residue was obtained. This was monitored by thin-layer chromatography (developing solvent; chloroform: methanol: water: acetic acid = 100: 20: 2: 1, coloring reagent; 10% sulfuric acid), and subjected to silica gel column chromatography using chloroform as a developing solvent. Then, the product was recrystallized from methanol to obtain 0.2 g of isoscutellarein.

Specific Example 2 Among the compounds of the formula, a compound in which R is a methoxyl group is called 8-O-methylisoscutellarein and can be obtained, for example, as follows.

5 100g of root of Scutellaria baicalensis
After digestion three times with 300m of 0% methanol and concentrating the extract to about half volume, this was shake-extracted twice with 200m of benzene,
The soluble part was removed. The aqueous layer was extracted three times with 300 m of ether,
The solvent was distilled off to obtain an extract. Add this extract to methanol 10
Dissolved at 0 m, added a saturated solution of lead acetate in methanol until no more precipitate formed and centrifuged. Water 10 in the filtrate
After adding 0m and concentrating to about half volume, 3m with 50m of ether.
Extracted by shaking twice. The solvent was distilled off from the extract, and the residue was monitored by thin-layer chromatography (developing solvent; chloroform: methanol: water: acetic acid = 100: 10: 1: 0.5, coloring reagent; 10
% Sulfuric acid) and subjected to silica gel column chromatography using chloroform as a developing solvent, and recrystallized from methanol to obtain 0.4 g of 8-O-methylisoscutellarein.

Specific Example 3 Among the compounds of the formula, a compound in which R is represented by a hydrogen atom is called apigenin, which was purchased from Funakoshi Pharmaceutical Co., Ltd.

Next, the fact that the compound of the formula has a sialidase inhibitory activity and is useful as an anti-influenza virus agent will be described with reference to experimental examples.

Experimental Example 1 (Sialidase Inhibitory Activity) 10 μm of influenza HA vaccine (manufactured by Kitasato Institute) as an enzyme and p-nitrophenyl-N-acetyl-α-D-neuraminic acid (p-nitrophenyl-Na) as a substrate
cetyl-α-D-neuraminic acid, PNP-NeuAc) 25 nmol
And a citrate-phosphate buffer (pH
5.0) Add 110μ into a microplate, react at 37 ° C for 15 minutes, stop the reaction by adding 190μ of 0.2M borate buffer (pH9.8), and measure the absorbance of released PNP at 405nm. I asked.

 Table 1 shows the results.

From the above results, the sialidase inhibitory activity of the compound of the formula was confirmed.

Experimental Example 2 After adsorbing influenza A virus to MDCK cells or MDBK cells in a culture plate at 34 ° C. for 30 minutes, a paper disk containing a solution in which the compound of the formula was dissolved in methanol was removed. Alternatively, a solution dissolved in dimethyl sulfoxide (DMSO) was added, and the mixture was cultured at 34 ° C. under 5% carbon dioxide for 3 days. For the culture supernatant, the virus was quantified by hemagglutination, and the plate was washed, followed by Giemsa staining to observe the ratio of viable cells.

 Table 2 shows the results.

(However, in Table 2, A is 100-90%, B is 80-40%, C
Represents a cell viability of 30 to 10%. The hemagglutination titer was expressed as the highest dilution factor at which an agglutination reaction occurred was defined as 1 unit. The agglomeration value when the compound of the formula was not added was 64.
nd is not measured. From the above results, it was confirmed that the compound of the formula has anti-influenza virus activity in vitro.

Experimental Example 3 Influenza virus A / PR / 8/34 and the compound of formula 100μ
g / egg after dissolving a solution in dimethyl sulfoxide,
Incubated chicken eggs (10-day eggs) were inoculated into the allantoic cavity and incubated at 34 ° C. for 2 days. Serum was collected and influenza virus was quantified by hemagglutination and sialidase activity.

 Table 3 shows the results.

(However, 1 unit of the sialidase activity is the amount of the enzyme that hydrolyzes 1 μmol of PNP-NeuAc per minute.) From the above results, the compound of the formula was expressed in an anti-influenza virus in a system using incubated chicken eggs (in vivo). It was confirmed to have activity.

From the above results, it was confirmed that the compound of the formula has a sialidase inhibitory activity and anti-influenza virus activity also in vitro and in vivo.

The compound of the formula did not show any side effects such as cytotoxicity during the course of the experiment, and its safety was also confirmed.

Accordingly, the compound of the formula has a sialidase inhibitory action,
Because of its high safety, it is promising as a sialidase inhibitor such as an anti-influenza virus agent.

Further, the compound of the formula inhibits sialidase activity of influenza virus, but shows almost no inhibitory activity against sialidase present in mouse liver. The search for a substance that specifically acts on only influenza virus is very important in developing a specific anti-influenza virus agent. In order to simply carry out the method, using the sialidase inhibitory activity as an index as disclosed in the present invention has not been known until now, and has been performed for the first time by the present inventors.

Next, the dosage and formulation of the compound of the formula will be described.

The compounds of the formula can be administered to animals and humans as such or together with conventional pharmaceutical carriers. The administration form is not particularly limited and may be appropriately selected and used as needed. Examples thereof include oral preparations such as tablets, capsules, granules, fine granules and powders, and parenteral preparations such as injections and suppositories. Can be

In order to exert the initial effect as an oral preparation, it depends on the age, weight and degree of disease of the patient, but usually 50 mg to 5 g as the weight of the compound of the formula in adults is divided into several times a day. Seems appropriate.

Oral preparations are produced by a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like.

In this type of preparation, in addition to the above-mentioned excipients, a binder,
Disintegrant, surfactant, lubricant, fluidity enhancer, flavoring agent,
Coloring agents, fragrances and the like can be used. Specific examples are as follows.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, melt cellulose,
Sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low-substituted hydroxypropylcellulose.

[Surfactant] Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80.

[Lubricant] Talc, wax, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

[Fluidity promoter] Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The compounds of the formula may also be administered as suspensions, emulsions, syrups and elixirs, and these various dosage forms may contain flavoring and coloring agents.

In order to achieve the desired effect as a parenteral drug, it depends on the age, weight, and degree of disease of the patient, but it is usually 0.1 mg to 1 g per day as an adult compound by intravenous injection and drip in adults. Intravenous, subcutaneous and intramuscular injections may be appropriate.

This parenteral preparation is produced according to a conventional method, and generally uses distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. as a diluent. be able to. Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. Further, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, water can be removed by a usual freeze-drying technique, and a liquid preparation can be prepared from the freeze-dried product immediately before use. Further, if necessary, an isotonic agent, a stabilizer, a preservative, a soothing agent, and the like may be appropriately added.

Examples of other parenteral preparations include liquid preparations for external use, ointments such as ointments, suppositories for rectal administration, and the like, and are produced according to a conventional method.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Corn starch 44 g Crystalline cellulose 40 g Carboxymethyl cellulose calcium 5 g Light anhydrous silicic acid 0.5 g Magnesium stearate 0.5 g Compound obtained in Example 1 10 g Total 100 g Then, compression molding was performed to obtain a tablet of 200 mg per tablet.

Each tablet contains 20 mg of the compound obtained in Example 1, and 10 to 25 tablets per day for an adult are divided into several doses.

Example 2 84.5 g of crystalline cellulose 0.5 g of magnesium stearate 0.5 g of carboxymethyl cellulose calcium 5 g of the compound obtained in the specific example 2 10 g in total 100 g According to the above formula, and a part of the mixture was uniformly mixed, compression-molded, and then pulverized. The remaining amounts of and were added and mixed, followed by compression molding with a tableting machine to obtain a tablet of 200 mg per tablet.

One tablet contains 20 mg of the compound obtained in Example 2, and 10 to 25 tablets per day for an adult are divided into several doses.

Example 3 Crystalline cellulose 49.5 g 10% hydroxypropyl cellulose ethanol solution 35 g Carboxymethyl cellulose calcium 5 g Magnesium stearate 0.5 g Compound obtained in Example 3 10 g Total 100 g
The mixture was wetted by a conventional method, granulated by an extrusion granulator, dried and crushed, mixed with each other, and compression-molded with a tableting machine to obtain a tablet of 200 mg per tablet.

One tablet contains 20 mg of the compound obtained in Example 3, and 10 to 25 tablets per day for an adult are taken in several doses.

Example 4 Cornstarch 34.5 g Magnesium stearate 50 g Carboxymethyl cellulose calcium 5 g Light anhydrous silicic acid 0.5 g Compound obtained in Specific Example 1 10 g Total 100 g The mixture was pulverized with a crusher and sieved to obtain granules.

1 g of this granule contains 100 mg of the compound obtained in Example 1, and 2 to 5 g of an adult is taken several times a day.

Example 5 55 g of crystalline cellulose 35 g of a 10% hydroxypropyl cellulose ethanol solution 35 g of the compound obtained in the specific example 2 10 g in total 100 g was uniformly mixed according to the above-mentioned formulation, and was mixed. After granulation by an extrusion granulator, the granules were dried and sieved to obtain granules.

1 g of this granule contains 100 mg of the compound obtained in Example 2, and 2 to 5 g of an adult is taken several times a day.

Example 6 89.5 g of corn starch 0.5 g of light anhydrous silicic acid 10 g of the compound obtained in the specific example 3 Total 100 g According to the above-mentioned formula, was mixed uniformly, and 200 mg was filled in a No. 2 capsule.

One capsule of this capsule contains 20 mg of the compound obtained in Example 3, and 10 to 25 capsules per day for an adult are divided into several doses.

Example 7 5 g of soybean oil 5 g of distilled water for injection 89.5 g of soybean phospholipid 2.5 g of glycerin 2 g of the compound obtained in Example 1 1 g of total amount 100 g was dissolved in and dissolved according to the above formula, and the resulting solution was added and emulsified. An injection was obtained.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-3-101623 (JP, A) Chem Pharm, Bull, Vol. 38, No. 5, p. 1329-1932 (1990) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 31/00-31/35 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] (1) The following formula I (In the formula, R represents a hydrogen atom, a hydroxyl group or a methoxyl group.) A sialidase inhibitor comprising a compound represented by the following formula: