2-methylsulphanyl-6-nitro-7-oxo-1, 2, 4-triazolo [5, 1-C] [1, 2, 4] triazinide L-arginine dihydrate active toward west nile virus

Abstract

The claimed invention relates to the field of biologically active compounds and concerns 2-methylsulphanyl-6-nitro-7-oxo-1,2,4-triazolo[5,1-c][1,2,4]triazinide L-arginine dihydrate, which exhibits an antiviral effect and is intended for the treatment and prophylaxis of human and animal viral diseases, primarily West Nile Virus, and can be used in the chemical and pharmaceutical industry, in scientific research laboratories and medical facilities, and also in veterinary science. The claimed invention is directed toward achieving the technical result of creating a novel effective drug of the azoloazine variety which exhibits antiviral activity toward a group of RNA-containing viruses, and reducing the dependence of the active compound on cell metabolism. This technical result is achieved in the creation of the novel drug 2-methylsulphanyl-6-nitro-7-oxo-1,2,4-triazolo[5,1-c][1,2,4]triazinide L-arginine dihydrate, which exhibits an aniviral effect and has the formula (I). This technical result is achieved in that a method for producing 2-methylsylphanyl-6-nitro-7-oxo-1,2,4-triazolo[5,1-c][1,2,4]triazinide L-arginine dihydrate includes mixing arginine, dissolved in water, and 2-methylthio-6-nitro-7-oxo-1,2,4-triazolo[5,1-c][1,2,4]triazinide sodium dihydrate, dissolved in a (1:1) water-ethanol mixture, whereupon the resultant mixture is boiled then cooled, and the precipitate is filtered off and dried.

Claims

1. A compound which is 2-methylsulfanyl-6-nitro-7-oxo-1,2,4-triazolo[5,1-c] [1,2,4]triazinid L-arginine dihydrate, the compound having the formula: ##STR00004##

2. A method for the production of 2-methylsulfanyl-6-nitro-7-oxo-1,2,4-triazolo[5,1-c] [1,2,4]triazinid L-arginine dehydrate according to claim 1, which comprises mixing arginine dissolved in water and 2-methylthio-6-nitro-7-oxo-1,2,4-triazolo [5,1-c] triazinid sodium dehydrate dissolved in a water-ethanol mixture (1:1), heating the resulting mixture, and cooling the mixture such that a precipitate is formed; and filtering off and drying the formed precipitate.

3. A method of treating West Nile fever in a patient in need thereof, the method comprising administering an effective amount of the compound of claim 1 to the patient, such that the West Nile fever is treated in the patient.

Description

EXAMPLE 1

(1) Evaluation of the cytotoxicity of the test compound using a constant cell culture GMK-AH (1D).

(2) To form a continuous monolayer, 1 ml of cell suspension with a density of 200 thousand/ml was added into each tubes and incubate at a temperature (37.0±0.5)° C., 5% CO.sub.2.

(3) Then the growth medium was removed and fresh medium containing various concentrations of test drugs (from 1000 to 6.25 g/ml) was added. The tubes were then incubated for 5 days at a temperature (37.0+0.5)° C., 5% CO.sub.2.

(4) Using a light microscope, the state of the monolayer cells was observed: partial or complete destruction of the monolayer cells, damage to individual cells, cell syncytium formation. Control samples were tubes with a monolayer, which included maintenance medium without a drug.

(5) Results of the evaluation of cytotoxicity of the test compound indicate that the active substance in a concentration of 500 μg/ml did not cause any visible changes in the cell culture (see Table No.1).

(6) TABLE-US-00001 TABLE 1 Study of cytotoxicity of the claimed compound in the cell culture GMK-AH (1D) The frequency of the cytopathic effect of the claimed compound when used in a concentration mg/mL TCID50, MIC, ½ MIC Drug 1000.0 500.0 250.0 125,0 62.5 31.3 15.5 7.8 μg/ml μg/ml μg/ml Claimed 2/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 1000.0 500.0 250.0 compound

EXAMPLE 2

(7) Study of the effectiveness of the claimed compound in cell culture GMK-AH (1D) against West Nile virus.

(8) Monolayer of the cell culture GMK-AH (1D) was infected with West Nile virus in a dose of 0.05 PFU/cell. Virus was adsorbed at 37° C., 5% CO.sub.2, for 60 minutes. At the end of the incubation, the monolayer was washed three times with maintenance medium containing 100 u/ml penicillin and streptomycin to remove non-adsorbed virus, and test compounds were added at a concentration of ½ MIC. The substance was dissolved in maintenance medium to the desired concentration. For each compound, not less than 10 tubes with a monolayer of cells were used. Incubation was performed at 37° C., 5% CO.sub.2 for 48 hours. Next, the cryodestruction of cells was performed and the samples were combined. The infectious titer in the combined samples was determined by titration by forming negative colonies under the agar coating in the cell culture GMK-AH (1D).

(9) Results of the study of the antiviral activity of the claimed compounds indicate that the drug inhibits the reproduction of West Nile virus by 2.31 g, at the same time inhibiting factor is 99.5% (See table No.2).

(10) TABLE-US-00002 TABLE 2 Study of the effectiveness of the claimed compound in cell culture GMK-AH (1D) against West Nile virus Concentration Level of Reduction Ratio of of the the virus of virus claimed accumulation the virus reproduction compound, of, accumulation, inhibition, Drug μg/ml lg PFU/ml lg PFU/ml percent Claimed 250.0 4.2 2.3 99.5 compound K dose — 7.8 — —

(11) Thus, the claimed chemical compound is nontoxic in the concentrations used. It has significant antiviral activity in vitro against West Nile virus.