JPS5877817A - Antiviral agent - Google Patents

Abstract

PURPOSE:The titled drug having high antiherpes activity, high solubility in aqueous solvents, and low toxicity, comprising 1-beta-D-arabinofuranosyl-(E)-5-(2-halogenovinyl)uracil-5'-phosphoric acid as an active ingredient. CONSTITUTION:An antibiral agent comprising a 1-beta-D-arabinofuranosyl-(E)- 5-(2-halogenovinyl)uracil-5'-phosphoric acid shown by the formulaI(X is halogen) (hereinafter referred to as 5-halogenovinylara UMP) or its salt as an active ingredient. The 5-halogenovinylara UMP shown by the formulaIis obtained by reacting a compound shown by the formula II with a phosphorylating agent (e.g., phosphorus oxychloride) so that the hydroxyl group at 5'-position is selectively phosphorylated. The compound shown by the formulaIhas higher solubility in an aqueous solvent system than the compound shown by the formula II and enables increase in medication concentration when it is used as venoclysis, syrup, or ophthalmic solution.

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, compounds of the general formula: [wherein X represents a halogen such as bromine, chlorine, or iodine. ] 1-β-DT arabinofuranosyl-
(E)-5-(2-halogenovinyl)uracil (hereinafter referred to as
[Abbreviated as 5-halogenovinylara UJ. ) 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 prepared a compound of the general formula [I] which is a 5'-phosphoric acid form of 5-halogenovinylara U [wherein X represents a halogen such as bromine, chlorine, or iodine]. ] 1-β-D-arabinofuranosyl-(
E) -5-(2-halogenovinyl)uracil-5'-phosphoric acid and its pharmaceutically acceptable salts (hereinafter abbreviated as r5-halogenovinylaraLIMPJ) have strong antiviral activity and low toxicity. While retaining the characteristics, 5
- It was found that the solubility in an aqueous solvent system is significantly improved compared to Halogeno Vinylara U, and the present invention was completed.

That is, the present invention provides an antiviral agent containing 5-halogenovinylara IMF as an active ingredient.

5-halogenovinylara UMP is represented by the above general formula [1], 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. Examples include salts such as mo, ゛- or g.

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, for example, Hydrocarbons (hexane, cyclohexane, benzene, etc.), halogenated/hydrocarbons (dichloromethane, chloroethane, chlorhexane, lyalbenzene, etc.), phenols (phenol, o-, m-, or p-cresol, 0-chlorophenol, etc.) ), 111m esters (ethyl acetate, ethyl benzoate, methyl acrylate, etc.), tromethane for nitro compounds, etc.

(nitroethane, nitropropane, nitrobenzene, etc.)
, nitrile compounds (acetonitrile, propionitrile, benzonitrile, malonitrile, etc.), ethers (
Examples include ethylene glycol dimethyl ether, ethylene glycol diethyl ether, tetrahydrofuran, dioxane, etc.), trialkyl phosphoric acid (trimethyl phosphoric acid, triethyl phosphoric acid, etc.), and the like. Also present in these polar solvents are organic bases (e.g., pyridine, picoline, etc.), amine-inorganic acid salts (e.g., pyridine-hydrochloride, picoline-hydrobromide, etc.), phosphorus-type reagents, or tetrachloropyrophosphorus. Pyrophosphate type reagents such as acids 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.

Reaction M 5-halogenovinylara U M P (
1) The isolation and purification method is not particularly limited, and any method commonly used for purifying nucleotides may be appropriately employed. For example, known purification means such as silica gel, adsorption chromatography using an adsorption resin as a carrier, ion exchange chromatography, and recrystallization may be appropriately selected and used in combination.

The solubility in For example, 1-β
-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)°uracil (hereinafter BVAU, !: abbreviated)
The solubility in water is 0496, while 1-β
The solubility of the disodium salt of -D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil-5'-phosphate (hereinafter abbreviated as BVAUP) in water is 50
It is extremely high at around 96. Therefore, it is particularly advantageous that when used as an intravenous drug, syrup, or eye drops, an effective concentration can be maintained and the administered concentration can be increased.

The drug of the invention can be used to treat herpetic keratitis in mammals such as rabbits, herpetic keratitis in humans and herpetic encephalitis in mice.
simplex), Parisella intestinal sister (Var
Herpesvirus group (Herpesv@rus > etc. 17) DNA including
Effective against viral infections.

The drug of the present invention is a drug comprising 5-halogenovinylara UMP or a pharmaceutically acceptable salt thereof as an active main ingredient, and a pharmaceutically acceptable carrier selected according to the administration method and form of the drug. Prepared as a composition.

In other words, the drug of the present invention can be administered to C1 depending on the target for treatment of internal virus diseases or external tissue virus diseases.
It is administered either intravenously or parenterally, and is prepared as a powder, granules, injection or oral liquid solution (2 tablets, waste tablets, pessaries, ointment) with an appropriate drug carrier depending on the method of administration.

It can be prepared as a cream, aerosol, or drops.

For internal diseases in the body, 5-halogenovinylara UM
P is administered orally or parenterally at a dose of 0.5 to 10 g/bOdy/day.

For oral administration, a diluent: 1. a fine powder or granules which may contain dispersants and/or surfactants; a solution in water or syrup; 2. a suspending agent which may contain a suspending agent: a binder and a lubricant. Liquid tablets: Administered in the dry state, in the form of a non-aqueous solution or suspension, and in the form of a capsule or a capsule. Flavoring agents, preservatives, suspending agents, diluents, thickening agents, emulsifying agents, and excipients can also be included in these pharmaceutical compositions as necessary. Particularly for purposes of oral administration, tablets and granules are preferred, and these may be coated with any coating agent.

In the case of parenteral administration such as an injection or as a drip for eye diseases, 5-logenovinylara UMP is administered as an aqueous solution at a concentration of about 0.1 to 10 (W/V)96. can do. Preservatives, buffers, etc. may also be included in this solution.

For diseases of tissues external to the body such as the eyes, mouth and skin, 5-halogenovinylara UMP 05 may be administered locally 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)96.

5-halogenovinylara-UMP has low toxicity, for example, 5-bromovinylara-UMP is administered to mice by intraperitoneal administration. 281rf1
kg, 1610~/ky by intravenous administration.

Below, a test example showing the synthesis of 5-halogenovinylara-UMP IFT1 and its antiviral activity will be shown.

Synthesis Example I 8.49 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. Pour the reaction solution into a 500-proof ice water bottle and
% sodium hydroxide to pH 7.0.

After the neutralized solution was concentrated to dryness under reduced pressure, the residue was dissolved in water 11 and adsorbed on a 500 ml column of adsorption resin Diaion HP5120 (trade name, manufactured by Mitsubishi Chemical Industries, Ltd.).

After washing with water, the eluate was eluted with a 5% aqueous methanol solution, and the eluate was adsorbed on an ion exchange resin Annotite RA6B (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 BV.
Obtained 1.86 g of AUP (free form) (yield 48.49
6) @ Melting point 190-192°C (decomposition) Elemental analysis C11H14N209BrP -, calculated value as H2O: C,8G, 16; H,8,45i
N, 6.8996 Experimental value: C, 80,19; H
,8,1a; N, 6.8096 Synthesis Example 2 1-β-D-arabinofuranosyl-(E)-5-(2-
After dissolving 8.05 g of (chlorovinyl) uracil (hereinafter abbreviated as -CVAUJI) in 30 ml of trimethyl phosphoric acid, the solution was 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 800 g of water.
Dissolved in xtt and adsorbed resin Diamond Ion HP20°15
It was adsorbed onto a 0 W/column. After washing with water, it was eluted with a 59b methanol aqueous solution. Combine part of the water washing solution and 5$ methanol eluate, and add ion exchange resin Amberlite IRA6.
8 (formic acid type) was adsorbed onto a 100 ml column and eluted with 2N formic acid. The eluate was concentrated to dryness under reduced pressure, and the residue was recrystallized from water to give 1 to β-D1 (free form) 2.18
9 was obtained (yield 55.4%).

Melting point 226-228 IC<decomposition) Ultraviolet absorption spectrum 2g: 1. N~NNaOH258n, 285nm
(sh)λ0.01N-HCI 2471m, 2
98 nmaX Test Example 1 Method of Sidwell et al.
QicrObi010gy]22°+51. 797
(1971)).
The antiviral activities of BVAUP and CVAUP were evaluated using BVAUP and CVAUP (HEL-F)
AU.

CVAU and 5-iodo-2'-deoxyuridine (
IDU) was tested as a control. The results are shown in the table below.

*MIC: Minimum inhibitory concentration for cell degeneration (CPE)**
vR: Virus Rating 1) To HSV-1-VR-87 J repes simplex vi
rus) y Eve IVR-8 strain 2) HSV-2-MS: Herpes simplex virus type 2 MS strain test example 2 82 LD in the brain of mouse ICR/JCR strain (4 weeks old)
Mice were infected with 5Q herpes simplex virus (HSV) Thailand 7'l VR-8 strain, and from 4 hours after infection, the test drug was intraperitoneally administered at a dose of 100 μ/kq 5 times every 214 hours. Phosphate buffered saline was administered as a control. The average number of survival days for mice that died within the 21st day after infection and the number of surviving mice for 21,100 days are shown in the table below.

*Significant difference at P<0.087 using direct probability calculation method*
*Example of test with significant difference (P 0.001)
8 1) After anesthetizing a colored domestic rabbit (body weight 2.0-8.0 kti) with an intravenous injection of bentoparbital sodium 8011 g/kQ, 0.5 ml of 1% protonin was injected into both eyes.
A retrobulbar injection was given. A suspension of the HSV-1-RE strain was inoculated at 25 locations on the cornea of a dislocated eyeball using a glass capillary (f3ritish Journal of Qph
talmolog), 68 (6) p425-4
28 (1979)).

80 seconds after completion of the inoculation, the eyelids were closed and fixed with sellotape. Both eyes were inoculated in the same way, and the Sellotape was removed when the rabbit woke up from anesthesia and began to move.

For each group of 8 rabbits, the right eye was treated with 1% BVAUP ointment and the left eye was treated with 196BVAU ointment, or the left and right eyes were treated in reverse. Three eyes in other groups 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. One drop of 1% Rose Bengal solution was instilled into the eyes 48 hours after virus inoculation and every 24 hours after the start of treatment, and lesions were observed using a slit lamp. The severity of symptoms at each inoculation site was scored according to the state of infection. The total score of 25 inoculation sites was determined, and the value was compared with the value immediately before the start of treatment and expressed as a percentage value.

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.

2) 0.8% BVAUP ointment and 8% BVAUP ointment were prepared and tested in the same manner as in the previous section. The results are shown in the table below.

Formulation example 1 (oil-in-water cream base) BVAUP-2Na 5. OQ white petrolatum 25. OQ stearyl alcohol 25.0g propylene glycol
12.0 (i'Sodium lauryl sulfate
1.0g ethyl paraoxybenzoate 0.
025g Propyl paraoxybenzoate 0.015g
Total amount of purified water 100g Formulation example 2 (water-soluble ointment base) BVAUP-2NH41o, o q Polyethylene glycol 4000500g Polyethylene glycol 400 50. Og formulation example 8 (tablet) BVAUP-Ca 250a+y
lactose
191 1mg starch
501g polyvinylpyrrolidone 5 ■ Magnesium stearate 4 mg total weight 500 my Formulation example 4 (eye drops) BVAUP-2Na 4q benzalkonium chloride 1 : 50000 purified water -=----1-total amount 10
0 ml Formulation example 5 (injection) BVAUP-2K 20! /Sodium chloride 4.5s Purified water
- Total amount 1000 a/

Claims (1)

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