RU2365591C2 - MEDICATION POSSESSING ANTI-VIRAL ACTIVITY AND CONTAINING 2-METHYLTHIO-5-METHYL-6-NITRO-1,2,4-TRIAZOLO[1,5-a]PYRIMIDIN-7(3H)-ON - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: described is novel application of 2-methylthio-5-methyl-6-nitro-1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-on. Substance possesses anti-viral activity with respect to flue A (H5N1) virus, West Nile virus and other viral infections. Wide-spectrum anti-viral activity is discovered for the first time.

EFFECT: obtaining anti-viral substance with wide spectrum of action.

1 cl, 8 tbl, 4 ex

Description

The invention relates to the field of biologically active compounds and relates to a medicinal product containing 2-methylthio-5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one having antiviral action intended for the treatment and prevention of infectious viral diseases of animals and humans, and can be used in medical institutions, research laboratories, in animal husbandry and poultry farming.

The urgency of the problem of antiviral therapy, especially in conditions of rapid mutation of viruses, the identification of new pathogens of dangerous and slow viral infections, causes a constant need for new drugs that would have high activity, long-lasting effect and low toxicity. The literature describes the preparation of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one by nitration of 5-methyl-1,2,4-triazolo [1,5-a] pyrimidin-7-one (Y. Makisumi Synthesis of potential anti-cancer agents. VII. 6-Nitro- and 6-amino-s-triazolo [2,3-a] pyrimidines, Chemical & Pharmaceutical Bulletin, 1961, No. 9, p. 873-877; T.P. Koffman, T.A. Uvarova, G.Yu. Kartseva, T.L. Uspenskaya 6-Nitro and 6-bromo derivatives of 4,7-dihydro-1,2,4-triazolo [1,5-a] pyrimidin-7-one, Journal of Organic Chemistry, 1997, vol. 33, issue 12, p. 1784-1793); 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one sodium salt is also mentioned (M.N. Kushnir, V.L. Rusinov, E.N. Ulomsky, N.A. Klyuyev, S.V. Shorshnev, G.G. Aleksandrov, O.N. Chupakhin Nitroazines XXII Alkylation and prototropic tautomerism in the series 6-nitro-7-oxo-1,2,4-triazolo [1 5-a] pyrimidines, Journal of Organic Chemistry, 1993, v.29, issue 3, p.629-638); there is also evidence of anti-arbovirus activity of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one (Vinograd I.A., Plastunov V.A., Kozlovsky M .M., Benzel L.V., Bilecka G.V., Lozinsky I.M., Rogochiy E.G., Sholomey M.D. Microbiological Journal. 2001, vol. 63, No. 2, pp. 14-19 )

As the invention, there is provided a medicament containing an active component having an antiviral effect and representing 2-methylthio-5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) - he is of the formula (1). In addition to compound (1), the drug may contain pharmaceutically acceptable components: excipients, diluents and / or other excipients.

To assess the antiviral properties of the compound (1) and a medicine based on it, the known antiviral drugs, remantadine, arbidol and ribavirin, were used.

Compound (1) was obtained by condensation of 3-methyl-5-amino-1,2,4-triazole (2) with acetoacetic ester followed by nitration of the resulting 2-methylthio-5-methyl-1,2,4-triazolo [1,5 a] pyrimidin-7-one (3).

Compound (1) is a pale yellow crystalline substance, soluble in water, ethanol, dimethyl sulfoxide, insoluble in ethyl acetate, chloroform, hexane.

The data of elemental analysis, ¹ H NMR and IR spectroscopy of compound (1) fully correspond to the attributed structure (see example 1).

The drug can be used either orally or by parenteral injection in solution. It can be used alone, for example in the form of microcapsules, or with suitable adjuvants and / or excipients.

Suitable solid or liquid dosage forms include, for example, granules, powders, coated tablets, microcapsules, suppositories, syrups, elixirs, suspensions, emulsions, drops or injection solutions, as well as preparations for the targeted delivery of the active substance in the production of which are usually used excipients such as fillers, disintegrants, binders, shelling agents, disintegrants, lubricants, perfumes or sweeteners. Suitable excipients are, for example, titanium dioxide, lactose, mannitol and other sugars, talc, milk albumin, gelatin, flour, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents such as sterile water, and monohydric and polyhydric alcohols e.g. glycerin.

Example 1. Synthesis of 2-methylthio-5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one (1)

Stage 1: 2-Methylthio-5-methyl-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one (2). A mixture of 130 g (1 mol) of 3-methylthio-5-amino1,2,4-triazole (3) and 140 ml (1.1 mol) of acetoacetate in 300 ml of acetic acid is boiled for 2 hours. After cooling the reaction mass, the precipitate formed is filtered off and recrystallized from water and dried in air at room temperature. Yield (2) 125.5 g (64%).

Stage 2: 2-Methylthio-5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one (1). To a mixture of 76 ml (1.25 mol) of concentrated nitric acid (d 1.4 g / ml) and 250 ml of concentrated sulfuric acid (d 1.83 g / ml), cooled to 0 ° C, 98 g (0 , 5 mol) 2-methylthio-5-methyl-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one (3) so that the temperature of the reaction mass does not exceed 40 ° C. After adding all the substance, the reaction mass is stirred for 3 hours at room temperature and poured into 500 ml of ice water. The resulting solution was neutralized with concentrated aqueous ammonia and the precipitated ammonium salt precipitate was filtered off. The ammonium salt is suspended in 40 ml of water, cooled to 5 ° C, and concentrated hydrochloric acid is carefully added to pH 1. The precipitate formed is filtered off and dried in air. Yield (1) 67.5 g (54%).

2-Methylthio-5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one has the following physicochemical characteristics:

T _PL > 280 ° C; ¹ H NMR spectrum in DMSO-d ₆ δ, ppm: 2.57 (3H, t, C-CH ₃ ), 3.02 (3H, s, SCH ₃ ), 10.2 (1H, br .s, NH). Found: C - 34.62, H - 3.07, N - 29.22. The gross formula is C ₇ H ₇ N ₅ O ₃ S. Calculated: C - 34.85, H - 2.92, N - 29.03%. IR spectrum, ν, cm ^-1 : 1365, 1558 (shaft, NO ₂ ), 1720 (shaft, C = O), 3420 (shaft, NH).

Example 2. Assessment of the toxicity of the compound (1)

1. Assessment of cytotoxicity.

Cytotoxicity was evaluated in relation to cell culture GMK-AN-1 (D) and SPEV. The cell density was 250 thousand / ml. A cell suspension of 1 ml was introduced into glass tubes, incubated for 48 h at 37 ° C to form a monolayer. Compound (1) prepared on maintenance medium at a concentration of 500 to 2 μg / ml was introduced into test tubes with a monolayer of cell culture. For each dose of compound (1), 4 tubes with a monolayer were used. Visual recording using a light microscope was performed daily. The initial stages of the cytopathic action (CPD) were characterized by a change in the morphology of the cells, then they were rounded and detached from the glass, and the destruction of the monolayer. Depending on the concentration of the drug, CPD occurred 1-7 days after the introduction of the drugs.

Destructive changes in 50% of SPEV cells were observed when 500 μg / ml of compound (1) was introduced into the nutrient medium. In 25% of cases, the destruction of the cell monolayer was caused by the compound at a dose of 250 μg / ml. In a GMK-AH- (D) cell culture, 50% monolayer destruction was caused by the introduction of 500 μg / ml of the compound into the nutrient medium. When the compound was added at a concentration of 250 μg / ml, cytopathic changes in the cell culture of SPEV and GMK-AH-1 (D) were not detected.

Thus, as a result of the studies, it was found that the maximum tolerated dose (MPD) of compound (1) for SPEV cell culture is 125 μg / ml, GMK-AH-1 (D) is 25 μg / ml.

2. Acute toxicity for laboratory animals

The acute toxicity of compound (1) was evaluated for uninfected 2-week-old white sucker mice with a single oral administration of compound (1). Dilutions of compound (1) in the range from 30 mg / kg to 2000 mg / kg were prepared on physical. solution and was administered to animals at 0.05 ml once. At least 20 animals were used for each breeding, which were monitored for 14 days. We calculated LD ₅₀ according to Kerber in the modification of I.P. Ashmarin. Controlled changes in animal behavior, appearance, weight, as well as the absence or presence of animal death. The toxicity results shown in table 1 indicate that the compound (1) in a concentration of from 30 to 250 mg / kg is not toxic to white mice weighing 4-5 g with a single oral administration. At the maximum dose used, 100% animal death with a single use of compound (1) was also absent.

Thus, it is not possible to calculate LD ₅₀ for white mice. At higher concentrations in a small volume of solvent, the compound (1) is poorly soluble, their thick consistency does not allow the use of gaiters for administration to animals.

Table 1
Assessment of the toxicity of drugs with a single oral administration to white mice - suckers A drug The concentration of the drug, mg / kg animal weight 2000 1000 500 250 125 62 31 number of animals killed / number of animals in the experiment Compound (1) - 2/20 1/20 0/20 0/20 0/20 0/20

Example 3. The study of the effectiveness of compound (1) in cell cultures

1. Regarding West Nile Virus

The antiviral efficacy of compound (1) against West Nile virus (ZN) was studied in GMK-AN-1 (D) cell culture. To study antiviral efficacy, compound (1) was added to the support medium 1 hour after infection. At least 4 tubes with a monolayer of two-day cell culture were used for each dose of the drug. The infectious dose of the virus was 0.01 PFU / cell. After the adsorption of the virus for 60 minutes at a temperature of (37.0 ± 0.5) ° C, the monolayer was washed three times with PS-4 nutrient medium in a Hanks solution containing 2% cattle serum and 100 IU / ml penicillin and streptomycin. Then, fresh medium was introduced containing the studied doses of compound (1), and incubated for 2 days at a temperature of (37.0 ± 0.5) ° С. At the end of the incubation, the cells were destroyed by cryodestruction: threefold quick freezing (in a cryostat at a temperature of minus 30 ° С) and quick thawing (water bath at room temperature). The level of pathogen accumulation in the studied samples was determined by titration of the samples by the method of obtaining negative virus colonies in a monolayer of GMK-AH-1 (D) cell culture under solid agar coating.

The results of the evaluation of antiviral efficacy are presented in table 2. When used in the maximum tolerated concentration of compound (1), the suppression of virus reproduction was 60.0%.

table 2
The study of the effect of drugs on the reproduction of the virus ZN, strain Eg 101 in cell culture GMK-AH-1 (D) A drug Dose, mcg / ml The level of accumulation of the virus, lg PFU / ml The level of suppression of virus reproduction, Δ, lg Inhibition rate, percent 2 3 four 5 6 Compound (1) one hundred 6.5 0.4 60.0 10 6.7 0.2 37.5 one 6.8 0.1 18.8 Ribavirin one hundred 5,0 1.9 98.6 Virus dose control - 6.9 - -

2. In relation to influenza A virus (H5N1)

Evaluation of the effectiveness against influenza virus, strain A / chicken / Kurgan / 2/05 (H5N1) was carried out in vitro in an MDSC cell culture. The allantoic fluid of infected RCEs with a biological activity of 6.5 lg of a CPD of ₅₀ / ml was used as an infectious preparation.

To study the antiviral efficacy, compound (1) and remantadine were added to the support medium 1 hour after infection. At least 10 tubes with a monolayer of two-day cell culture were used for each dose of the drug. The infectious dose of the virus was 0.1 CPD ₅₀ / cell. After the adsorption of the virus for 60 minutes at a temperature of (37.0 ± 0.5) ° C, the monolayer was washed three times with PS-4 nutrient medium in a Hanks solution containing 2% cattle serum and 100 IU / ml penicillin and streptomycin, then fresh medium containing various concentrations of the studied drugs, and incubated for 3 days at a temperature of (37.0 ± 0.5) ° C. At the end of the incubation, the cytopathic effect caused by the virus in the cell culture was visually taken into account using a light microscope (lens x 8-10, eyepiece x 7-10).

The results of a study of the suppression of the cytopathic activity of the influenza virus by drugs in the MDCK cell culture, presented in Table 3, indicate that when using the compound (1) 1 hour after infection of the cell culture, the level of suppression of the virus CPD was 40%.

Table 3
Evaluation of the effectiveness of drugs against influenza virus, strain A / chicken / Kurgan / 2/05 (H5N1) in MDCK cell culture to suppress the cytopathic effect of the virus A drug Dose, mcg / ml The frequency of detection of the CPP, X CPD suppression, percent, X ± σ _x Compound (1) 100.0 19/30 36.6 ± 3.3 10.0 25/30 16.7 ± 3.3 1,0 10/10 0 Remantadine 25 3/30 86.7 ± 3.3 Dose control - 30/30 - Environmental control - 0/10 -

Table 4 shows the results of a study of the suppression of virus reproduction in an MDCK cell culture. The level of virus accumulation was assessed by titration of samples according to the death of RCE. Compound (1) at maximum concentration also statistically significantly inhibits the reproduction of influenza virus. The inhibition rate was 98.2%. Comparison drug remantadine almost completely suppressed the reproduction of the virus.

Table 4
Evaluation of the effectiveness of drugs against influenza virus, strain A / chicken / Kurgan / 2/05 (H5N1) in MDCK cell culture to suppress virus reproduction A drug Dose, mcg / ml Scheme of drug administration The level of accumulation of the virus, lg ELD ₅₀ / ml The level of suppression of virus accumulation, Δ, lg Inhibition rate, percent Compound (1) one hundred +1 h 5.3 1.7 98.2 Remantadine 25 + 2 h 2.0 5,0 99,99 The control - - 7.0 - -

Example 4. The study of the effectiveness of drugs in laboratory animals 1. The study of the effectiveness of drugs in relation to influenza A virus (H5N1)

Evaluation of the effectiveness against influenza virus, strain A / chicken / Kurgan / 2/05 (H5N1) was carried out in vivo using white mice weighing 12-15 g. Allantoic fluid of infected RCEs with biological activity of 6.5 lg CPD was used as an infectious preparation. ₅₀ / ml, 9.01g ELD ₅₀ / ml, 5.0 lg LD ₅₀ / ml.

The compound (1) and the comparison compound were administered orally to white mice according to the prophylactic and therapeutic regimens, as well as according to the emergency prophylaxis regimen.

The results of a study of the prophylactic efficacy of the compound in relation to the experimental form of influenza in white mice infected intranasally with the influenza virus strain A / chicken / Kurgan / Russia / 02/05 (H5N1) are presented in tables 5. The protection against death when using compound (1) was an average of 20%.

Table 5
The study of the prophylactic efficacy of drugs in relation to the experimental form of influenza in white mice intranasally infected with the influenza virus, strain A / chicken / Kurgan / Russia / 02/05 (H5N1) A drug Scheme of drug administration Dose of the drug
mg / kg The number of animals in the group Death of animals Protective efficiency from death, percentage The average life time of animals in the group, days Increase in average life expectancy, Δ, days -120 h ... 9,4 0.7 Compound (1) -24 h, -1 h fifty twenty 16 twenty 60 twenty eleven 47 11.3 2,8 Arbidol -24 h, -1 h thirty twenty eleven 45 11.6 2.5 Dose control - - twenty twenty - 8.7 - Herd control - - twenty 0 - fourteen -

The results of a study of the effectiveness of the compound (1) and comparison drugs when applied according to the emergency prevention and treatment regimen presented in tables 6 and 7, indicate the effectiveness of the claimed compounds.

Table 6
The study of the effectiveness in relation to the experimental form of influenza in white mice intranasally infected with the influenza virus, strain A / chicken / Kurgan / Russia / 02/05 (H5N1) when it is introduced according to the emergency prevention scheme A drug Scheme of drug administration Dose, mg / kg Animal death rate Protective efficiency from death, percentage The average life time of animals in the group, days Increase in average life expectancy, Δ, days Compound (1) +1 h, +24 h, +48 h, +72 h, +96 h, +120 h, 144 h fifty 10/20 fifty 11.7 3,5 Arbidol thirty 16/20 twenty 9,4 1,2 Dose control - - 20/20 - 8.2 Herd control - - 0/20 - 14.0 -

Table 7
The study of the therapeutic efficacy of drugs in relation to the experimental form of influenza in white mice intranasally infected with the influenza virus, strain A / chicken / Kurgan / Russia / 02/05 (H5N1) A drug Scheme of drug administration Dose, mg / kg Animal death rate Protective efficiency from death,% The average life time of animals in the group, days Increase in average life expectancy, Δ, days Compound (1) +24 h, +48 h, +72 h, +96 h, 15/20 25 8.4 1.7 +120 h, +144 h fifty Arbidol +24 h, +48 h, +7 135 18/20 10 7.1 0.4 2 h, +96 h Dose control - - 20/20 - 6.7 - Herd control - - 0/20 - 14.0 -

2. Study of efficacy against West Nile virus

To evaluate the protective efficacy of white mice, they were infected subcutaneously at a dose of 10 LD ₅₀ . The drugs were administered orally at a dose of 50 mg / kg according to the following schemes: prophylaxis - within 6 days before infection once a day and 1 hour before infection; emergency prevention - 1 hour after infection and then for 5 days; treatment - 24 hours after infection and then for 6 days. The results of the effectiveness assessment, presented in table 8, indicate that the compound (1) is effective against the experimental form of LUS in white mice and protects 20% of infected animals from death. In this case, the extension of the SHF was 2.3 days. In emergency prophylaxis, protective efficacy was 20%.

Table 8
The results of evaluating the effectiveness of drugs in relation to the experimental form of West Nile fever in white mice A drug Scheme Coefficient
ent
protection,% Average life time, day Elongation of the average lifetime, Δ day The level of virus accumulation in the brain of dead animals lg PFU / ml Δ, lg -144 h, -120 h, -96 h, 20,0 14.5 2,3 7.4 0.9 -72 h, -48 h, -24 h, -1 h Compound +1 h, +24 h, +48 h, +72 h, 20,0 14.0 1.8 7.5 0.8 (one) +96 h, +120 h, +144 h +24 h, +48 h, +72 h, +9 0 12.1 0 8.5 0 6 h, +120 h, +144 h -72 h, -48 h, -24 h, -1 h 50,0 17.1 4.9 6.1 2.2 +1 h, +24 h, +48 h, +72 h, 85.0 21.0 8.8 0 8.3 Ribavirin +96 h, +120 h, +144 h +24 h, +48 h, +72 h, +9 10.0 12.3 0.1 7.8 0.5 6 h, +120 h, +144 h Dose control - - 12,2 - 8.3 - Herd control - - 21.0 - -

Thus, a drug containing as active component the compound (1) - 2-methylthio-5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one has a high antiviral effect that exceeds the action of known drugs, and can be used as an antiviral agent.

Claims (1)

An antiviral drug containing 2-methylthio-5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7 (3H) -one of the formula