RU2493158C2 - SODIUM SALT OF 2-METHYL-6-FLUORO-1,2,4-TRIAZOLO[5,1-c]-1,2,4-TRIAZIN-7(4H)-ONE DIHYDRATE, HAVING ANTIVIRAL ACTIVITY - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: described is a sodium salt of 2-methyl-6-fluoro-1,2,4-triazolo[5,1-c]-1,2,4-triazin-7(4H)-one

, having antiviral activity with respect to influenza virus A(H1N1).

EFFECT: improved properties of the compound.

1 cl, 1 tbl

Description

1. The technical field to which the invention relates.

The invention relates to the field of biologically active compounds and relates to the sodium salt of 2-methyl-6-fluoro-1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -one dihydrate, possessing an antiviral effect, intended for the treatment and prevention of infectious diseases of animals and humans of viral nature. The invention can be used in medical institutions, research laboratories, as well as in livestock and poultry.

The urgency of the problem of antiviral therapy, especially influenza viruses, causes a constant need for new drugs that would have high activity, long-term action and low toxicity.

2. The prior art.

There is evidence of the antiviral effect of 6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -ones (Synthesis and antiviral activity of 6-nitro-7-oxo -4,7-dihydroazolo [5,1-c] [1,2,4] triazines V.L. Rusinov, E.N. Ulomsky, O.N. Chupakhin, M.M. Zubairov, A.B. Kapustin, N.I. Mitin et al. / Chemical-Pharmaceutical Journal. No. 9, 1990, pp. 41-44).

The sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -one (1) dihydrate, which belongs to to a number of compounds exhibiting antiviral effects (O. N. Chupakhin, V. L. Rusinov, E. N. Ulomsky, V. N. Charushin, A. Yu. Petrov, O. I. Kiselev. Sodium salt of 2-methylthio- 6-nitro-1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -one, a dihydrate having antiviral activity. RF Patent No. 2294936 of 03/10/2007) and can be used in medical practice, livestock, poultry.

The sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -one dihydrate (1) is a close structural analogue of the claimed compounds and therefore used as a prototype.

3. The invention.

The technical result of the invention is a new chemical compound having antiviral activity against influenza A virus (H1N1).

The specified technical result is achieved by the fact that according to the invention a new compound is proposed - the sodium salt of 2-methyl-6-fluoro-1,2,4-triazolo [5,1-c] -1,2,4-triazine-7 (4H) -one, a dihydrate of the formula (2) having an antiviral effect.

4. Information confirming the possibility of carrying out the invention.

4.1. Synthesis of sodium salt of 2-methyl-6-fluoro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one dihydrate (2)

The chemical scheme of the synthesis of the claimed compounds

To a solution of 9.8 g (0.1 mol) of 3-amino-5-methyl-1,2,4-triazole (3) (0.1 mol) in 50 ml of water was added 22 ml (0.26 mol) of concentrated of hydrochloric acid. The reaction mixture was cooled to -10 ° C and a solution of sodium nitrite (7 g, 0.1 mol) in 10 ml of water was added at -10 ÷ -5 ° C. After adding a sodium nitrite solution and holding (15 + 30 min), the reaction mass was added to a solution of 12.5 ml (0.1 mol) of ethyl 2-fluoroacetoacetate in a mixture of 93 ml (0.28 mol) of 3 M sodium acetate and 100 ml of ethanol cooled to 0 ° C. The reaction mass was kept for 1 h at 5 ° C and 2 h at room temperature. The precipitated product (5) was filtered off, washed with water and recrystallized from ethanol.

The intermediate, without drying, was dissolved in a solution of 8.2 g (0.1 mol) of sodium acetate in 70 ml of 70% aqueous ethanol and boiled for 5 hours. At the end of the reaction, the solvent was evaporated, and the product was dried. The dried product was dissolved in 20 ml of ethanol and 32 ml (0.1 mol) of a saturated solution of sodium hydroxide in ethanol was added. The precipitated product, after a half-hour exposure of the reaction mixture, was filtered off, crystallized from water, and dried. Yield: 12.1 g (47.6%).

The sodium salt of 2-methyl-6-fluoro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one dihydrate (2) has the following physicochemical characteristics: _Mp > 300 ° C; ¹ H NMR spectrum (DMSO-d ₆ + CCl ₄ , internal standard tetramethylsilane), δ, ppm: 2.34 (3H, s, CH ₃ ); IR spectrum, v, cm ^-1 : 1652 cm ^-1 (CO), 1294 cm ^-1 (CF). ¹⁹ F NMR spectrum (DMSO-d ⁶ + CCl ₄ , internal standard hexafluorobenzene) δ, ppm: -114.98; Found,%: C - 23.37; H 2.82; N 32.82; C ₅ H ₇ N ₆ NaO ₅ ; Calculated,%: C - 23.63%; H - 2.78%; N - 33.07.

The inventive compound is a pale yellow crystalline high-melting substance (mp. More than 300 ° C), soluble in water, methanol, dimethylformamide, dimethyl sulfoxide, insoluble in benzene, chloroform and most aprotic solvents.

4.2. Antiviral properties of 2-methyl-6-fluoro- sodium salt

1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -one dihydrate (2) Example 1. Determination of antiviral activity of compounds against influenza A virus (H1N1)

Viruses and cells. Studies were conducted on primary dog kidney cell culture (MDCK). To assess the antiviral activity of the drugs used reference virus A (H1N1) A / Brisbane / 59/07. This strain has sufficient hemagglutinating (1: 128) and infectious activity (6.5 lg ID 50/20 μl).

The study of the toxicity of drugs. MDCK cells were seeded in 96-well plates and cultured at 37 ° C. in MEM medium supplemented with 10% cattle serum to a monolayer state. From the studied preparations, mother solutions of a concentration of 10 mg / ml in dimethyl sulfoxide were prepared, after which a series of twofold dilutions of the preparations in MEM medium from 500 to 1.92 μg / ml was prepared. Dissolved preparations were added to the wells of the plates and incubated for 48 hours at 37 ° C. The degree of damage to the cell monolayer was evaluated visually.

Based on the data obtained, the minimum toxic (1000 μg / ml) and working dose of the drugs were determined.

Table 1 Reducing the infectious activity of influenza virus under the influence of the studied compounds No. Formula The minimum toxic dose of MTD, mcg / ml Working concentration, mcg / ml Antiviral activity (decrease in infectious activity in lg ID50 / 20 μl) one.

1000 fifty 1,5 2.

1000 fifty 0.5

Evaluation of the antiviral effect of the compounds. The antiviral activity of the preparations against influenza A (H1N1) A / Brisbane / 59/07 virus was determined on MDCK cells in 96-well cell culture plates. A series of ten-fold dilutions from 10 ^-1 to 10 ^-7 were prepared from the initial virus-containing allantoic fluid and introduced into the corresponding wells with a cell monolayer. After an hour of contact at 36-37 ° C, the drug was added to the wells at a working concentration. The results were taken into account after 48 hours according to the hemagglutination reaction (GA) with a suspension of 0.5% chicken red blood cells. Virus inhibitory effect of the drug was evaluated by reducing the titer of the virus in the experiment compared with the control. The research results are presented in table 1.

From the data given in table 1 shows that the prototype compound (1) is practically not active against influenza virus A (H1N1) A / Brisbane / 59/07. At the same time, the claimed compound (2) has moderate antiviral activity with toxicity comparable with Triazavirin.

Claims (1)

Sodium salt of 2-methyl-6-fluoro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one dihydrate

possessing antiviral effect.