RU2294936C1 - 2-methylthio-6-nitro-1,2,4-triazolo[5,1-c]-1,2,4-triazine-7(4h)-one sodium salt dihydrate possessing antiviral activity - Google Patents

Abstract

FIELD: organic chemistry, medicine, virology.

SUBSTANCE: invention relates to biologically active compounds and concerns the development of a novel substance - 2-methylthio-6-nitro-1,2,4-triazolo[5,1-c]-1,2,4-triazine-7-(4H)-one sodium salt dihydrate of the formula:

. This compound is designated for treatment and prophylaxis of diseases caused by viruses that are pathogenic form humans and animals. Proposed compound protects against infections caused by Rift Valley fever virus. Also, it shows activity against viruses of WEE(West Equine Encephalomyelitis), parainfluenza, respiratory-syncytium, Aujeszky's disease virus, avian infectious laryngotracheitis virus, avian influenza virus - totally against above 10 RNA- and DNA-containing viruses. The proposed compound is active in curative schedule of its using that is especially valuable.

EFFECT: valuable medicinal properties of compound.

1 cl, 6 tbl, 2 dwg, 7 ex

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-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one, dihydrate possessing an antiviral effect intended for the treatment and prevention of infectious diseases of the viral nature of humans and animals. 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 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.

2. The prior art.

Among the condensed azoloazines, there is evidence of the antiviral effect of 6-nitro-4,7-dihydro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-ones in relation to the Aujeszky's disease virus (WBA) [Rusinov V.L., Ulomsky E.N., Chupakhin O.N. et al. Pharmaceutical Chemistry Journal. 1990, No. 96, p.41-44]. The antiviral effect of 4-alkylthio-5,7-dihydro-7-oxo-1,2,4-triazolo [4,3-c1] -1,2,4-triazines is also known [Cristesku C., Derivati de 5-oxo -5,6-dihidro-s-triazolo (4,3-d) -as-triazina si procedeu pentru prepararea lor. Pat. CPP, class 12 (C 07 d 55/10), No. 56269, 03/13. 1974], as well as the antitumor effect of imidazo [2,1-s] -1,2,3,5-tetrazines [Yongfeng W., Stevens M.F.G. Antitumor Imidazotetrasines. 35. New Synthetic Routesto to the Antitumor Drug Temozolomide. J. Org. Chem. 1997, vol. 62, No. 21, p. 7288-7294]. As a prototype, the most suitable compound is the sodium salt of 6-nitro-4,7-dihydro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one, which showed an antiviral effect against in relation to WBA [Rusinov V.L., Ulomsky E.N., Chupakhin O.N. et al. Pharmaceutical Chemistry Journal. 1990, No. 96, p.41-44].

3. Disclosure of the invention.

The technical result of the invention is the discovery of a new chemical compound having a wide spectrum of antiviral action, effective against RNA and DNA viruses.

The specified technical result is achieved by the fact that according to the invention, a new compound is proposed - the sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazine-7 (4H ) -one, a dihydrate having antiviral activity and having the formula 1

The sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one dihydrate (1) is obtained by condensation of the diazonium salt (3 ) obtained by diazotization of 3-amino-5-methylthio-1,2,4-triazole (2) with ethyl nitroacetate in an alkaline medium.

The inventive compound is a yellow crystalline high melting point, soluble in water, methanol, acetone, dimethylformamide, dimethyl sulfoxide, sparingly soluble in methanol, insoluble in benzene, chloroform and most aprotic solvents.

The data of elemental analysis and NMR spectroscopy fully correspond to the attributed structure. The presence in the claimed compound of two water molecules is confirmed by the data of derivatography (figure 1, 2).

Figure 1. Thermogram of the sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7 (4H) -one, dihydrate.

Figure 2. A decrease in the mass of the sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7 (4H) -one, dihydrate when heated.

To remove water from the sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7 (4H) -one, dihydrate, the substance must be heated for a long time at 150 ° C in vacuo over P ₂ O ₅ . An anhydrous preparation in the air instantly absorbs moisture and under ordinary conditions and in 2-3 hours completely turns into dihydrate.

Example 1. The synthesis of the sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one, dihydrate (1)

A solution of 8 g is added to a solution of 13 g (0.1 mol) of 5-amino-3-methylthio-1,2,4-triazole in 16 ml of nitric acid (d = 1.4) and 100 ml of water, cooled to 0 ° C. sodium nitrite in 50 ml of water for 15 min, incubated for 10 min and portionwise bring a chilled mixture of 11 ml (0.1 mol) of ethyl nitroacetate in 150 ml of a 2 M sodium carbonate solution. It is stirred for 2 hours at 0-5 ° C, the precipitate formed is filtered off, successively crystallized from 20% acetic acid, water and dried in air. Yield: 21.2 g (74%).

Sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7 (4H) -one, dihydrate has the following physicochemical characteristics: T _PL > 300 ° C; ¹ H NMR spectrum in DMSO-d ₆ δ, ppm: 2.52 (3H, s, SCH ₃ ), 4.76 (4H, br s, 2 H ₂ O). Found: C - 20.83, H - 2.62, N - 29.21, S - 11.54%. The gross formula is C ₅ H ₇ N ₆ NaO ₅ S. Calculated: C - 20.98, H - 2.47, N - 29.36, S - 11.20%.

Example 2. The study of the antiviral effect against the virus of Venezuelan encephalomyelitis in horses (VEL) and Sindbis

The antiviral activity and cytotoxic effect of the claimed compound was studied by a micromethod in 96-well micropanels in a transplantable Vero cell culture against VEL viruses strain 230 and Sindbis strain EgA2-339, belonging to the genus alphaviruses. 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).

Antiviral activity was studied by reducing the infectious titer (IT) of the VEL-230 virus and Sindbis virus in a Vero cell culture 24 hours after infection with an infectious dose of 0.01 PFU / cell. The results are shown in table.2. The inventive compound at a concentration of 200, 12.5 μg / ml reduced I.T. VEL-230 virus by 4.0, 1.9 IgBOE / ml, respectively, and I.T. Sindbis virus at 3.9, 1.8 lg PFU / ml, respectively. The prototype compound at a concentration of 125, 6.25 μg / ml reduced I.R. Sindbis virus at 3.0, 1.0 lg PFU / ml, respectively, and I.T. VEL-230 virus at 3.3, 1.5 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: described - 20, prototype - 12, remantadine - 2.

Example 3. The study of antiviral activity against the virus of Western encephalomyelitis in horses (ZEL)

The inventive compound was administered to outbred mice weighing 10-12 g orally according to 2 schemes: 1 - prophylactic in 24 hours, 2, 24, 48, 72, 96 hours after infection with 10 LD ₅₀ viruses, II scheme - therapeutic - the virus was administered 2, 24, 48, 72, 96 hours after infection with the same infectious dose of the virus.

A single dose of compounds of 50 mg / kg of mass corresponded to 1/32 LD ₅₀ for these compounds. The course dose of both compounds was 300-250 mg / kg weight, depending on the scheme used. The infectious dose of the virus ZEL 10 LD ₅₀ in a volume of 0.1 ml intramuscularly. The method of administration of the compounds is oral. As can be seen from table 3, with the treatment-and-prophylactic administration schedule (I) of the described compound, the protection was 65%, the average life span of the animals was extended by 10 days (Table 3). The percentage of protection when applying the treatment regimen was 60%, and the average life expectancy increased by 5.9 days. The prototype compound was ineffective against ZEL.

Example 4. The study of the antiviral effect against viruses of fever Rift Valley fever (LDR)

The inventive compound was administered to outbred 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. As can be seen from table 4, with the treatment-and-prophylactic regimen of administration of the claimed compound, the protection was 90%, the average life expectancy of the treated animals increased by 12.2 days. The percentage of protection when applying the treatment regimen was slightly higher and amounted to 80%, and the average life expectancy increased by 13.4 days (Table 4).

Example 5. The study of antiviral activity 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) test tubes with tissue culture, in which instead of the test compound was introduced a nutrient medium.

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 the respiratory syncytial virus (Table 5).

Example 6. The study of the antiviral effect against the virus Aujeszky's disease, bird flu and infectious laryngotracheitis of birds

Tests were carried out with avian influenza virus (AHV) and infectious bird laryngotracheitis (ILT) on 10-day-old chicken embryos (CE) and Aujeszky's disease virus (WBA) on chicken fibroblast (CF) cell culture.

When determining the viral-static (inhibitory) action, CEs were infected with ILT virus at a dose of 50-100 PFU ₅₀ , 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 TBE and CF.

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

It was found that in experiments on TBE with ILT virus, the claimed compound at a dose of 500 μg / TB inhibited the development of HAO plaques by 30% 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 CE with AHP, the claimed compound at a dose of 500 μg / ml and after exposure for 24 hours intacted AHP by 3.6 ELD ₅₀ .

Example 7. Determination of toxicity of the claimed compounds

Toxicity was determined on outbred white mice weighing 7-8 g in a concentration range of 50-200 mg / kg (Table 6). From table 7 it is seen that the LD _{50 of the} sodium salt of 2-methylthio-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-one, the dihydrate is 1400 mg / kg

The study of the antiviral effect of the claimed compound (sodium salt of 2-methylthio-1,2,4-triazolo [5,1-s] -1,2,4-triazine-7 (4H) -one, dihydrate) and its comparison with the prototype - the sodium salt of 1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-one and the remantadine drug used in medical practice showed the following.

1. With respect to the VEL and Sindbis viruses, the claimed compound is significantly superior to remantadine. In experiments on laboratory animals, the claimed compound has a stronger protective effect than the prototype.

2. The inventive compound has a wider spectrum of antiviral activity than the prototype. Sodium salt of 2-methylthio-1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -one, dihydrate protects animals from infections caused by Rift Valley fever viruses. It is especially valuable that the compound is active in the treatment regimen.

The claimed compound, in contrast to the prototype compound and remantadine, is active against the ZEL virus, parainfluenza, respiratory syncytial virus, Aujeszky's disease virus, infectious laryngotracheitis of birds, avian influenza virus.

Thus, the sodium salt of 2-methylthio-1,2,4-triazolo [5,1-c] -1,2,4-triazin-7 (4H) -one, the dihydrate has a more pronounced antiviral effect than the prototype and rematadin. The inventive compound has a wider spectrum of biological activity than the prototype compound and the remantadine preparation used in medical practice.

Table 1
Antiviral activity of the claimed compound, prototype and remantadine against VEL and Sindbis viruses compound MIC, μg / ml VEL-230 Sindbis ED ₅₀ , mcg / ml HTI ED ₅₀ ; mcg / ml HTI R The claimed 200 10.0 twenty 10.0 twenty 0.01 prototype 125 10.0 12 10.0 12 0.01 remantadine fifty 25 2 25 2 0.01 IPC - maximum tolerated concentration; ED ₅₀ - the minimum concentration that delays the development of cytopathic effet (CPE) by 50%;
CTI - chemotherapeutic index (IPC / ED ₅₀ ); P is the level of confidence. table 2
Antiviral activity of the claimed compound, prototype and remantadine against VEL and Sindbis viruses compound MIC, μg / ml concentration, mcg / ml VEL-230 Sindbis virus titer, lg PFU / ml decline titer, lg PFU / ml HTI virus titer, lg PFU / ml decline titer, lg PFU / ml HTI R 200 5.5 4.0 ± 0.06 6.0 3.9 ± 0.14 0.01 claimed 200 12.5 7.6 1.9 ± 0.08 16 7.9 1.8 ± 0.11 16 0.01 0 9.3 9.5 fifty 6.0 3.3 ± 0.16 5.2 3.0 ± 0.14 0.01 prototype 125 5.12 7.8 1.5 ± 0.06 12 8.0 1.0 ± 0.08 12 0.01 0 9.3 9.5 fifty 7.3 2.0 ± 0.11 7.5 2.0 ± 0.08 0.01 remantadine fifty 25 7.9 1.4 ± 0.16 2 8.0 1.5 ± 0.14 2 0.01 0 9.3 9.5 IPC - maximum tolerated concentration; CTI - chemotherapeutic index (IPC / ED ₅₀ ); P is the level of confidence.

Table 5
Antiviral activity of the claimed compounds against viruses of parainfluenza and respiratory syncytial virus dose of compound, mcg / ml virus dose, TCD ₅₀ experience the control total tubes of which contain the virus protection index,% total tubes of which contain the virus parainfluenza 500 1-10 twenty four 60 twenty 19 10-100 twenty 16 fifteen twenty 13 respiratory 60 1-10 10 3 55 10 10 syntial 10-100 10 10 10 10 10 Table 6
Determination of LD _{50 of the} claimed compound when administered orally to white mice weighing 7-8 g drug concentration, mg / kg fifty one hundred 200 400 800 1400 2000 relative mortality ^a 0/6 0/6 0/6 0/6 0/6 3/6 4/6 and - is expressed as the ratio of the number of fallen white mice to the number of white mice in the experiment.

Claims (1)

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 and having the formula

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