RU2330036C1 - SODIUM SALT 5-METHYL-6-NITRO-1,2,4-TRIAZOLE[1,5-a]PYRIMIDINE-7-ONE DIHYDRATE - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention describes sodium salt 5-methyl-6-nitro-1,2,4-triazole[1,5-a]pyrimidine-7-one, dihydrate, formula

and the pharmaceutical composition, which contains an effective quantity of bonds (1).

EFFECT: increased antiviral action of the bonds.

2 cl, 1 ex, 9 tbl, 2 dwg

Description

1. The technical field to which the invention relates.

The invention relates to the field of biologically active compounds and relates to a new compound, the sodium salt of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one, an antiviral dihydrate intended for treatment and prevention of infectious viral diseases of animals and humans, and can be used in medical institutions, research laboratories, as well as in animal husbandry and poultry farming.

2. The level of technology

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 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] pyrimidine- 7-one (Y. Makisumi Synthesis of potential anticancer 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), the 5-methyl-6-nitro-1,2,4 sodium salt is also mentioned in the literature -triazolo [1,5-a] pyrimidin-7-one (M.N. Kushnir, V.L. Rusinov, E.N. Ulomsky, N.A. Klyuyev, S.V. Shorshnev, G.G. Aleksandrov , ABOUT .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, no. 3, p. 629-638).

3. The invention

Sodium salt of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one, a dihydrate of the formula (1),

possessing antiviral effect.

4. Information confirming the possibility of carrying out the invention

4.1. Synthesis of the 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one sodium dihydrate salt

Compound (1) was obtained by nitration of 5-methyl-1,2,4-triazolo [1,5-a] pyrimidin-7-one (2) followed by conversion of the obtained 5-methyl-6-nitro-1,2, 4-triazolo [1,5-a] pyrimidin-7-one (3) in the sodium salt, dihydrate.

The inventive compound is a pale yellow crystalline substance, soluble in water, methanol, dimethylformamide, dimethyl sulfoxide, sparingly soluble in ethanol, insoluble in benzene, chloroform, and most aprotic solvents.

The data of elemental analysis, ¹ H NMR and IR spectroscopy fully correspond to the attributed structure. The 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one sodium salt, the dihydrate has the following characteristics. Found (%): C 28.65, H 3.26, N 27.47. The gross formula is C ₆ H ₈ N ₅ NaO ₅ . Calculated (%): C 28.47, H 3.19, N 27.66.

Spectrum ¹ H NMR (DMSO-d ₆ , δ, ppm): 2.47 (3H, C, CH ₃ ), 8.05 (1H, C, 2-H).

IR spectrum (VM), ν, cm ^-1 : 1325 (NO ₂ ), 1680 (C = 0).

The presence in the claimed compound of two water molecules is confirmed by the data of derivatography (figure 1, 2).

Figure 2. The decrease in the mass of the sodium salt of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one dihydrate when heated

m ₁ = 0.1361 g .; m ₂ = 0.1168 g .; 253 cu;

m ₁ is the initial mass of the sample;

m ₂ is the mass of the sample after heating;

M ₁ is the molecular weight of the starting compound (5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one sodium salt dihydrate);

M ₂ is the molecular weight of the compound after heating (sodium salt of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one);

ν is the amount of substance;

ν = (m ₁ / M ₁ ); M ₂ = (m ₂ / ν) = ((m ₂ · M ₁ ) / m ₁ ) = ((0.468 · 253 / 0.1361) = 217.12;

M ₂ = 217 cu; ΔM = (M ₁ -M ₂ ) = (253-217) = 36 cu, which corresponds to two water molecules.

The substance can be administered 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.

The invention also relates to pharmaceutical compositions or preparations comprising the compound (1) in addition to pharmaceutically acceptable excipients, diluents and / or other 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 monoatomic and polyatomic alcohols, for example glycerin.

Example 1. Synthesis of the sodium salt of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one, dihydrate (1)

5-Methyl-1,2,4-triazolo [1,5-a] pyrimidin-7-one (2). A mixture of 84 g (1 mol) of 3-aminotriazole and 140 ml (1.1 mol) of acetoacetate in 200 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) 120 g (80%).

5-Methyl-6-nitro-1,2,4-triazolo [1,5-a) pyrimidin-7-one (3). To a mixture of 76 ml (0.55 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, 75 g (0 , 5 mol) 5-methyl-1,2,4-triazolo [1,5-a] pyrimidin-7-one (2) so that the temperature of the reaction mass does not exceed 40 ° C. After adding all the substance, the reaction mass is stirred for 2 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 poured to pH 1. The precipitate formed is filtered off and dried in air. Yield (3) 65.5 g (67%).

5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one sodium salt, dihydrate (1). To 100 ml of a 4 M sodium hydroxide solution, 80 g (0.41 mol) of triazolopyrimidine (3) are added with stirring, the mixture is stirred for 30 minutes and the precipitated sodium salt (1) is filtered off. The resulting product is recrystallized from distilled water, dried in air at room temperature. The yield (1) is 56.5 g (66%).

4.2. The biological effect of the sodium salt of 5-methyl-6-nigro-1,2,4-triazolo (1,5-a] pyrimidin-7-one, dihydrate

The study of the biological effect of the described compound, as well as comparison with the basic object - remantadine - was carried out in experiments on cell culture, chicken embryos and outbred white mice.

4.2.1. Antiviral effect against the Sindbis virus. The antiviral activity and cytotoxic effect of the described compound was studied by micromethod in 96-well micropanels in a Vero transplantable cell culture against Sindbis virus strain EgA2-339. The multiplicity of virus infection (mi) was 0.01 TCD ₅₀ . The experience was taken into account 72 hours before the onset of 100% cytopathic effect (CPE) in the wells of the virus control. CPE was determined by a 4-cross pattern; 4 + -100% destruction of the cell monolayer, 3 + -75%, 2 + -26% destruction (Table 1, 2).

In a second test, antiviral activity was studied by reducing the Sinbis virus infectious titer (IT) in a Vero cell culture 24 hours after infection with an infectious virus dose of 0.01 PFU / cell. The results are shown in table 1. The inventive compound at a concentration of 200, 12.5 μg / ml reduced I.T. Sindbis virus at 3.5, 1.6 lg PFU / ml, respectively. Remantadine at a concentration of 50, 25 mct / ml reduced I.T. VEL-230 virus at 2.0, 1.4 lg PFU / ml, respectively. CTI (chemotherapeutic index) of the compounds: the claimed - 16, remantadine - 2.

Table 1 Antiviral activity of the claimed compounds and remantadine against Sindbis viruses compound MIC, μg / ml ED ₅₀ , mcg / ml HTI claimed 200 12.5 16 remantadine fifty 25 2 IPC - maximum tolerated concentration; ED ₅₀ - the minimum concentration that delays the development of the cytopathic effect (CPE) by 50%; HTI - chemotherapeutic index (IPC / ED ₅₀ )

table 2 Antiviral activity of the claimed compounds and remantadine against VAL and Sindbis viruses compound MIC, μg / ml concentration, mcg / ml virus titer, lg PFU / ml decline titer, lg PFU / ml HTI claimed 200 200 6.0 3.5 ± 0.14 16 12.5
0 7.9
9.5 1.6 ± 0.11 remantadine fifty fifty 7.5 2.0 ± 0.08 2 25
0 8.0
9.5 1.5 ± 0.14

4.2.2. Antiviral Effects Against Rift Valley Fever Viruses (VLDR)

The inventive compound was administered to mongrel white mice weighing 6-8 g orally according to 2 schemes: 1. Scheme - therapeutic and prophylactic (24 hours and 1, 2, 3, 6, 7, 8, 9. 10 days after infection with the same infectious dose of the virus). A single dose of the compounds is 50 mg / kg of mass, which corresponds to 1/32 LD _{50 of} these compounds. The course dose of both compounds was 500-450 mg / kg of weight, depending on the scheme used. An infectious dose of 10 LD ₅₀ virus in a volume of 0.2 ml was administered subcutaneously. In the treatment-and-prophylactic regimen of administration of the described compound, protection was 70%, the average life expectancy of treated animals increased by 11.1 days. The percentage of protection when applying the treatment regimen was slightly higher and amounted to 80%, and the average life expectancy increased by 12.8 days. (Tables 3, 4).

Table 3 The antiviral activity of the claimed compounds in infection of white mice with Rift Valley fever virus compound application pattern course dose, mcg / ml relative mortality ^a protection,% s.p.zh., ^b day Increase s.p.zh., day claimed preventive 500 6/20 70 16.3 11.1 medical 450 4/20 80 18.0 12.8 the control 20/20 5.2 a - is expressed as the ratio of the number of fallen white mice to the number of white mice in the experiment;
b - average life expectancy; c - significance of differences

4.2.3. Antiviral effect against parainfluenza virus and respiratory syncytial virus. The study of antiviral activity against these viruses was carried out in experiments on tissue culture using parainfluenza virus type 3 (strain HA-1) and respiratory syncytial virus (strain Long).

Each experiment included 2 groups:

1) tissue culture tubes into which the test compound was introduced;

2) tissue culture tubes into which culture medium was introduced instead of the test compound.

It was found that inhibition of parainfluenza virus was observed when using a dose of 500 μg / ml, while a concentration of 60 μg / ml was sufficient to inhibit respiratory syncytial virus (Table 5).

4.2.4. Antiviral effect against Aujeszky's disease virus, avian influenza and avian infectious laryngotracheitis. Tests were performed with avian influenza virus (strain H5N1) and infectious bird laryngotracheitis (IL1) on 10-day-old chicken embryos (CE) and Aujeszky's disease virus (WBA) on chicken fibroblast (CF) cell culture.

When determining the virus-static (inhibitory) action, CEs were infected with ILT virus at a dose of 50-100 PFU50, and CF-WBA cell culture at a dose of TCD ₅₀ (tissue cytopathic doses). 1-1.5 hours after infection with CE and CF, the test compound was introduced in doses of 50, 100, 250 μg / CE and incubated in an incubator at 37.5 ° C CE for 120 hours to 100% plaque formation on the chorioallantoic membrane (CAO ), and CF - until a clear cytopathic effect in the control. For each dose of the studied compound, 10 CE or 4 vials with CF were taken. The experiments were carried out in 3 replicates.

Virusstatic effect was determined by the difference in the titers of the virus in the experiment and in the control in CF and in the suppression of plaque formation in the experiment and control with CE. The inhibitory effect is expressed in TCD ₅₀ in CF and in% suppression of plaques with EC. The control was TBE and CF, infected with the virus, as well as intact CE and CF.

When studying the virucidal effect (inactivating), the claimed compound was mixed with material containing avian influenza virus (strain H5N1) and incubated at 37.5 ° C for 24 hours, after which it was titrated on CE in parallel with the control. A virus-containing material was used as a control, in which, instead of the claimed compound, saline solution (placebo) and intact CEV were added. Virulicidal action was determined by the difference in titers in the experiment and control and was expressed in ELD ₅₀ (embryonic detailed doses).

It was found that in experiments on CE with the ILT virus, the described compound at a dose of 500 μg / CE inhibited the development of HAO plaques by 40% with 100% formation in the control. In experiments on CE with BWA, the claimed compound at a dose of 100 μg / ml inhibited the reproduction of the virus by 2.75, and at a dose of 50 μg / ml - 1.5 TCD ₅₀ . In experiments on TBE with avian influenza virus (strain H5N1), the claimed compound at a dose of 500 μg / ml and after exposure for 24 hours in) activated the virus at 4.2 ELD ₅₀ .

4.2.5. Determination of toxicity of the claimed compound. Toxicity was determined on outbred white mice weighing 7-8 g in a concentration range of 50-2000 mg / kg (Table 6). It was found that LD _{50 of the} sodium salt of 5-methyl-6-nitro-7-oxo-4,7-dihydro-1,2,4-triazolo [1,5-a] pyrimidine, dihydrate is equal to 1600 mg / kg.

The study of the antiviral effect of the claimed compounds (sodium salt of 5-methyl-6-nitro-7-oxo-4,7-dihydro-1,2,4-triazolo [1,5 ~ a] pyrimidine, dihydrate) and its comparison with that used in In medical practice, the drug "remantadine" showed the following: The claimed compound is active against the virus, parainfluenza, respiratory syncytial virus, Aujeszky's disease virus, infectious laryngotracheitis of birds, avian influenza virus (strain H5N1) and has a more pronounced antiviral effect than rematadin.

Claims (2)

1. Sodium salt of 5-methyl-6-nitro-1,2,4-triazolo [1,5-a] pyrimidin-7-one dihydrate

2. A pharmaceutical composition having an antiviral effect and containing an effective amount of a compound according to claim 1 as an active agent, a pharmaceutically acceptable excipient, diluent and / or other excipients.

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