JPS637528B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antiviral agent. It has been known that various pyrimidine nucleoside analogs have antiviral activity. Among these pyrimidine nucleoside analogs, the general formula [] [In the formula, X represents a halogen such as bromine, chlorine, or iodine. ] 1-β-D-arabinofuranosyl-(E)-5-(2-halogenovinyl)
Uracil (hereinafter referred to as “5-halogenovinylara U”)
It is abbreviated as. ) is currently being considered for practical use as a medicine because it has the most powerful anti-herpes activity and low toxicity. The present inventors have discovered that 5-halogenovinylara U
General formula that is 5′-phosphate form [] [In the formula, X represents a halogen such as bromine, chlorine, or iodine. ] 1-β-D-arabinofuranosyl-(E)-5-(2-halogenovinyl)
Uracil-5'-phosphate and its pharmaceutically acceptable salts (hereinafter referred to as "5-halogenovinylaraUMP")
It is abbreviated as. ) was found to have significantly improved solubility in aqueous solvent systems compared to 5-halogenovinylara U while retaining strong antiviral activity and low toxicity, leading to the completion of the present invention. Ivy. That is, the present invention provides 5-halogenovinylara
The present invention provides an antiviral agent containing UMP as an active ingredient. 5-halogenovinylara UMP is represented by the general formula [], and its pharmaceutically acceptable salts include alkali metals such as sodium, potassium, and lithium, alkaline earth metals such as calcium and magnesium, and ammonium salts. Mono- or di-salt is punched. 5-halogenovinylara UMP can be prepared by selective phosphorylation of the hydroxyl group at the 5' position of 5-halogenovinylara U. The phosphorylation reaction is carried out by employing a method commonly applied as a selective phosphorylation method for the 5'-hydroxyl group of general nucleosides. That is, a method in which a phosphorylating agent is allowed to act on 5-halogenovinylara U in an organic solvent suitable for selective phosphorylation reaction can be applied. Various organic solvents can be used, such as hydrocarbons (hexane, cyclohexane, benzene, etc.), halogenated hydrocarbons (dichloromethane, chloroethane, chlorhexane, chlorobenzene, etc.), phenols (phenol, o -, m-, or p
-cresol, o-chlorophenol, etc.), organic acid esters (ethyl acetate, ethyl benzoate, methyl acrylate, etc.), nitro compounds (nitromethane, nitroethane, nitropropane, nitrobenzene, etc.), nitrile compounds (acetonitrile,
propionitrile, benzonitrile, malonitrile, etc.), ethers (ethylene glycol dimethyl ether, ethylene glycol diethyl ether, tetrahydrofuran, dioxane, etc.),
Examples include trialkyl phosphoric acid (trimethyl phosphoric acid, triethyl phosphoric acid, etc.). These polar solvents also contain organic bases (e.g. pyridine, picoline, etc.), amine-inorganic acid salts (e.g. pyridine-hydrochloride, picoline-hydrobromide, etc.).
may exist. Phosphorus oxidizing agents include phosphorus halogenated reagents such as phosphorus oxychloride, phosphorus trichloride, phenyl phosphoric acid dichloride, morpholinophosphoric acid dichloride, partially hydrated phosphorus oxychloride, or pyrophosphoric acid type reagents such as tetrachloropyrophosphoric acid. Reagents etc. can be used. The combination of the phosphorylating agent and the reaction solvent may be determined depending on each type. for example,
Suitable examples include a combination of phosphorus oxychloride and trialkyl phosphoric acid, or a combination of tetrachloropyrophosphoric acid and phenol. The reaction temperature is preferably around 0° C. to around room temperature, and the reaction time is preferably several hours to several tens of hours. The method for isolating and purifying 5-halogenovinylara-UMP from the reaction solution is not particularly limited, and any method commonly used for purifying nucleotides may be suitably employed. For example, known purification means such as adsorption chromatography using silica gel or adsorption resin as a carrier, ion exchange chromatography, and recrystallization may be appropriately selected and used in combination. The thus obtained 5-halogenovinylara
UMP has significantly improved solubility in aqueous solvents compared to 5-halogenovinylara-U. For example, 1-β-D-arabinofuranosyl-(E)-5
-(2-bromovinyl)uracil (hereinafter abbreviated as BVAU) has a solubility of 0.4% in water, whereas 1-β-D-arabinofuranosyl-(E)
The disodium salt of -5-(2-bromovinyl)uracil-5'-phosphoric acid (hereinafter abbreviated as BVAUP) has an extremely high solubility in water of about 50%. Therefore, effective concentration can be maintained when used as an intravenous drug, syrup, or eye drops.
It is particularly advantageous that the dosage concentration can be increased. The drug of the present invention can be used to treat the herpesvirus group, including Herpes simplex, Varicella-Zoster, in mammals, such as herpetic keratitis in rabbits, humans, and herpetic encephalitis in mice. It is effective against DNA virus infections such as The drug of the present invention is a pharmaceutical composition comprising 5-halogenovinylara UMP or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier selected according to the administration method and form of the drug. prepared as a product. That is, the drug of the present invention is administered orally or parenterally depending on the subject to be treated for internal virus diseases or external tissue virus diseases, and can be administered as powder, granules, or injections with an appropriate drug carrier depending on the administration method. It can be prepared as a liquid for oral or internal use, tablets, suppositories, petals, ointments, creams, aerosols, and drops. For internal diseases in the body, 5-halogenovinylara UMP is administered orally or parenterally at a dose of 0.5 to 10 g/body/day. For oral administration, finely divided powders or granules which may contain diluents, separating agents and/or surfactants; solutions in water or syrup; or suspensions which may contain suspending agents; binders and lubricants. Tablets: Administered in dry form, in non-aqueous solutions or suspensions, and in capsules or capsules. Flavoring agents, preservatives, suspending agents, diluents, thickening agents, emulsifying agents, and excipients can also be contained in these pharmaceutical compositions as required.
Particularly for purposes of oral administration, tablets and granules are preferred, and these may be coated with any coating agent. When administering parenterally such as injections or as drops for eye diseases, 5-halogenovinylara UMP should be administered as an aqueous solution at a concentration of approximately 0.1 to 10 (W/V)%. Can be done. Preservatives, buffers, etc. can also be included in this solution. For diseases of tissues external to the body such as the eyes, mouth and skin, 5-halogenovinylara UMP can be administered topically in the form of an ointment using a water-soluble ointment base or a cream using an oil-in-water cream base.
It can be administered at a concentration of ~20 (W/V)%. 5-halogenovinylara UMP has low toxicity,
For example, the LD ₅₀ of 5-bromovinylara-UMP in mice was 1728 mg/Kg when administered intraperitoneally and 1610 mg/Kg when administered intravenously. Examples of the synthesis of 5-halogenovinylara-UMP and test examples showing its antiviral activity are shown below. Synthesis Example 1 3.49 g of BVAU was dissolved in 50 ml of trimethyl phosphoric acid, cooled on ice, 2.8 ml of phosphorus oxychloride was added, and the mixture was left overnight. 500 ml of the reaction solution was poured into ice water, and the pH was adjusted to 7.0 with 40% sodium hydroxide. After the neutralized solution was concentrated to dryness under reduced pressure, the residue was dissolved in 1 part of water and adsorbed on a 500 ml column of adsorption resin Diaion HP20 (trade name, manufactured by Mitsubishi Chemical Industries, Ltd.). After washing with water, elute with 5% methanol aqueous solution,
Eluate ion exchange resin Amberlite IRA68
(formic acid type) (trade name, manufactured by Rohm and Haas) and eluted with 2N formic acid. The eluate was concentrated to dryness under reduced pressure, and the residue was recrystallized from water to obtain 1.86 g of BVAUP (free form) (yield: 43.4
%) Melting point 190-192℃ (decomposed) Ultraviolet absorption spectrum λ ^0.01N-NaOH _nax 256nm, 284nm (sh) λ ^0.01N-HCI _nax 251nm, 293nm (sh) Elemental analysis C ₁₁ H ₁₄ N ₂ O ₉ BrP・１ /2H ₂ O Calculated value: C, 30.16; H, 3.45; N, 6.39% Experimental value: C, 30.19; H, 3.18; N, 6.30% Synthesis example 2 1-β-D-arabinofuranosyl-( E)-5-
3.05 g of (2-chlorovinyl)uracil (hereinafter abbreviated as CVAU) was dissolved in 30 ml of trimethyl phosphoric acid, cooled on ice, 1.9 ml of phosphorus oxychloride was added, and the mixture was left overnight. The reaction solution was poured into 500 ml of ice water, and the pH was adjusted to 7.0 with 40% sodium hydroxide. After concentrating the above neutralized solution to dryness under reduced pressure, the residue was dissolved in water.
Dissolved in 300ml and adsorbed resin Diamond Ion HP20,
It was adsorbed onto a 150ml column. After washing with water, elution was performed with a 5% methanol aqueous solution. A part of the water washing solution and the 5% methanol eluate were combined and adsorbed on a 100ml column of ion exchange resin Amberlite IRM68 (formic acid type).
It was eluted with 2N formic acid. The eluate was concentrated to dryness under reduced pressure,
The residue was recrystallized from water to obtain 1-β-D-arabinofuranosyl-(E)-5-(2-chlorovinyl)uracil-5'-phosphate (hereinafter abbreviated as CVMUP).
(Free form) 2.13g was obtained (yield 55.4%). Melting point 226-228℃ (decomposed) Ultraviolet absorption spectrum λ ^0.01N-NaOH _nax 253nm, 285nm (sh) λ ^0.01N-HCI _nax 247nm, 293nm Test example 1 Sidwell et al.'s method (Applied Microbiology) Applied
microbiology) 22, (5), 797 (1971)).
We investigated the antiviral activity of BVAUP and CVAUP using embryonic lung fibroblasts (HEL-F).
BVAU, CVAU and 5-iodo-2'-deoxyuridine (IDU) were assayed as controls. The results were as shown in the table below. [Table] Test Example 2 32LD ₅₀ in the brain of mouse ICR/JCR strain (4 weeks old)
herpes simplex virus (HSV)
Mice were infected with Type 1 VR-3 strain, and 100 mg/Kg of the test drug was intraperitoneally administered to mice every 24 hours starting 4 hours after infection, five times. As a control, phosphoric acid mild saline was administered. The number of surviving mice on the 21st day after infection and the average survival days of mice that died within the 21st day were as shown in the table below. [Table] Test Example 3 1 A colored domestic rabbit (weight 2.0 to 3.0 kg) was anesthetized with an intravenous injection of 30 mg/Kg of sodium pentobarbital, and then 0.5 ml of 1% procaine was injected retrobulbarly into both eyes. A suspension of HSV-1-RE strain was inoculated at 25 locations on the cornea of the dislocated eyeball using a glass capillary (British Journal of Ophtalmology,
63(6)P425-428 (1979)). Thirty seconds after completion of the inoculation, the eyelids were closed and fixed with sellotape. Both eyes were inoculated in the same manner, and the sellotape was removed when the rabbits emerged from anesthesia and began to move. For each group of 3 rabbits, the right eye was treated with 1% BVAUP ointment and the left eye was treated with 1% BVAU ointment, or the left and right eyes were treated in reverse. Three eyes in the other group received no treatment as controls. These treatments started 48 hours after virus inoculation, and the eye drops were applied every 2 hours during the day, 5 times a day, for 4 days. 1 every 48 hours after virus inoculation and every 24 hours after starting treatment
One drop of % Rose Bengal solution was instilled into the eye, and the lesion was observed using a slit lamp. The severity of symptoms at each inoculation site was scored based on the state of the infected circle. The total score for 25 inoculation sites was determined, and the value was expressed as a percentage of the value immediately before the start of treatment. The therapeutic effects of BVAUP and BVAU on ulcerative herpes keratitis were compared by calculating the average of their percentage values. The results were as shown in the table below. [Table] 2. 0.3% BVAUP ointment and 3% BVAUP ointment were prepared and tested in the same manner as in the previous section. The results were as shown in the table below. [Table] Formulation example 1 (oil-in-water cream base) BVAUP-2Na 5.0g White petrolatum 25.0g Stearyl alcohol 25.0g Propylene glycol 12.0g Sodium lauryl sulfate 1.0g Ethyl paraoxybenzoate 0.025g Propyl paraoxybenzoate 0.015g Purified water Total amount 100g Formulation example 2 (Water-soluble ointment base) BVAUP-2NH ₄ 10.0g Polyethylene glycol 4000 50.0g Polyethylene glycol 400 50.0g Formulation example 3 (Tablet) BVAUP-Ca 250mg Lactose 191mg Starch 50mg Polyvinylpyrrolidone 5mg Magnesium stearate 4mg Total weight 500mg Formulation example 4 (Eye drops) BVAUP-2Na 4g Benzalkonium chloride 1:50000 Purified water Total volume 100ml Formulation example 5 (Injection) BVAUP-2K 20g Sodium chloride 4.5g Purified water Total volume 1000ml

Claims (1)

[Claims] 1. General formula [] [In the formula, X represents a halogen. ] 1-β-D-arabinofuranosyl-(E)-5-
An antiviral agent containing (2-halogenovinyl)uracil-5'-phosphate or a pharmaceutically acceptable salt thereof as an active ingredient.