RU2398767C2 - Agent representing 5-amino-6-methyluracil and exhibiting antioxidant activity and method for preparing thereof - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to an agent representing 5-amino-6-methyluracil of formulas (1) which exhibit antioxidant activity and to a method for preparing thereof. The agent can be used in medicine for preparing a drug capable to inhibit lipid peroxidation processes and to activate restorative processes in chemical intoxications.

The method for preparing 5-amino-6-methyluracil consists in hydrasine sulphate reduction of 5-nitro-6-methyluracil in ethanol medium at temperature 70-80°C with Raney-nickel added as a catalyst followed with product recovery.

EFFECT: invention allows for higher product yield.

2 cl, 5 tbl, 5 ex

Description

The invention relates to the pharmaceutical industry, and in particular to a tool representing 5-amino-6-methyluracil (1) of the formula:

exhibiting antioxidant activity.

The antioxidant activity of compound (1) is not described in the literature. The closest analogue in chemical structure is 5-hydroxy-6-methyluracil (oxymethyluracil):

It is known that 5-hydroxy-6-methyluracil has an antioxidant effect in model systems in vitro, under conditions of acute hypoxia modeled by the introduction of sodium nitrite, as well as on models of experimental intoxication with dichloroethane, a mixture of polychlorinated biphenyls (Sovtol), carbophos, tetrachloromethane [1- 6]. The disadvantage of 5-hydroxy-6-methyluracil is its low solubility in water and organic solvents.

The problem to which the claimed technical solution is directed is to expand the arsenal of pharmacological preparations with low toxicity and high antioxidant activity in conditions of hypoxia and chemical intoxication, and simplification of the method for producing compound (1).

A method is described for preparing compound (1) by nitration of 6-methyluracil (2) with excess HNO ₃ in the presence of H ₂ SO ₄ followed by reduction of the obtained 5-nitro-6-methyluracil (3) with hydrogen at 5 atm in the presence of Raney nickel in distilled water [7 ].

The total yield of compound (1) in two stages is 76.19%. The disadvantage of this method is the technical complexity of the synthesis, which consists in the use of special equipment, gaseous hydrogen and high pressure.

A known method of producing compound (1), which consists in the fact that 5-nitro-6-methyluracil (3) in an aqueous medium is treated with Na ₂ S ₂ O ₄ at 80-90 ° C, then it is boiled for 5 minutes with coal, filtered and cooled [8]. The yield of compound (1) is 74.5%.

This method does not require high pressure and special equipment, its disadvantage is the low yield of the target product.

In the claimed technical solution, the compound (1) is obtained by reduction of 5-nitro-6-methyluracil (3) with hydrazine sulfate in the presence of Raney nickel in ethanol at a temperature of 70-80 ° C. The yield of the target product is 95%.

The toxicity of compound (1) when administered once to the stomach was determined in mice and rats. The compound (1) in the range of doses from 1 to 10 g / kg does not cause visible signs of intoxication and death of animals within 14 days of observation. In accordance with GOST 12.1.007-76, compound (1), when introduced into the stomach, is a low-hazard substance (table 1) [9].

Table 1 Medium lethal doses (LD ₅₀ ) of compound (1) and 5-hydroxy-6-methyluracil for various routes of entry Compound Animal species LD ₅₀ with various routes of entry into the body, g / kg intragastrically intraperitoneally Compound (1) the mouse > 10 2.8 (1.9 ÷ 3.7) rats > 6 2.3 (1.7 ÷ 3.3) 5-hydroxy-6-methyluracil the mouse > 10 1.95 (1.5 ÷ 2.5) rats > 6 1.7 (1.2 ÷ 2.4)

When intraperitoneally administered to mice with compound (1), a dose of 2.8 g / kg causes 50% death of animals; according to the classification of toxicity of substances, compound (1) is practically non-toxic [10].

The in vitro study of the antioxidant activity (AOA) of compound (1) and 5-hydroxy-6-methyluracil was performed using 20% rat liver homogenate as a substrate for lipid peroxidation (LPO). LPO induction was induced by introducing into the 20% liver homogenate a system of the following composition: 0.5 mM ascorbate + 6 μM Mohr's salt. LPO activity was studied by the biochemical method according to the concentration of substances reacting with 2-thiobarbituric acid (TBA-RP) (TBA-reactive products) [11]. AOA of the studied compounds was calculated by the formula [12]:

- прирост ТБК-РП в контрольной пробе во время инкубации;

- an increase in TBA-RP in the control sample during incubation;

- прирост ТБК-РП в опытной пробе во время инкубации;

- an increase in TBA-RP in the experimental sample during incubation;

- оптическая плотность, измеренная до инкубации;

- optical density measured before incubation;

- оптическая плотность, измеренная после инкубации.

- optical density measured after incubation.

The experimental results are presented in table 2.

table 2 Antioxidant activity of compound (1) in the dynamics of rat liver homogenate oxidation Compound (concentration, μM) Induction * The duration of the oxidation period, in hours one 3 four Compound (1) (100) BUT 20.8 59.0 63.3 ASKT 31.8 51.3 67.3 5-Hydroxy-6-methyluracil (100) BUT 11.1 57.1 55.5 ASKT 18.5 6.8 32.9 Note: A - auto-oxidation; ASKT - a system containing: 0.5 mm ascorbate + 6 μm Mora salt; * - average data of 5 experiments.

Under the conditions of autooxidation, the addition of compound (1) and 5-hydroxy-6-methyluracil to the incubation medium in the first 3 hours of the experiment suppressed the accumulation of TBA-RP in approximately the same degree, after 4 hours, compound (1) was less effective.

AOA of compound (1) upon induction of lipid peroxidation by ascorbate after 1 hour of incubation was more pronounced compared to 5-hydroxy-6-methyluracil. After 3 hours of incubation, this difference in the AOA of the two preparations was maximal and persisted after 4 hours of oxidation.

Thus, the AOA of compound (1) with induced lipid peroxidation was significantly higher than that of the reference drug, 5-hydroxy-6-methyluracil.

The antioxidant activity of compound (1) in vivo was studied on a model of acute hemic hypoxia (OHeG) with determination of the content of ketodienes (CTD) in the organs of mice.

OGeG was created by subcutaneous administration to mice of a 4% aqueous solution of sodium nitrite at a dose of 100 mg / kg. Compound (1) was administered to experimental mice once in the abdominal cavity in the form of 0.25% aqueous-twin solutions at a dose of 100 mg / kg 1 hour before the administration of sodium nitrite. The control animals were injected with a solvent in a similar volume. As the reference preparation, 5-hydroxy-6-methyluracil was used, which was used according to a scheme similar to compound (1) [3].

The content of CTD in the lipid extracts of the brain and liver of mice was determined by spectrophotometric method [13, 14]. Testing was performed 6 hours after the administration of sodium nitrite.

The results of the experiments are presented in table 3.

The introduction of sodium nitrite into animals increases the content of CTD in the hemispheres of the brain and liver by 1.75 and 1.67 times, respectively, compared with the control, which indicates an increase in the activity of LPO in organs. Under these conditions, compound (1) and 5-hydroxy-6-methyluracil prevent the accumulation of CTD, but to a different extent. 5-Hydroxy-6-methyluracil reduces the amount of CTD in the brain increased by hypoxia by 54.7% and in the liver by 44.1%. When using compound (1), the amount of CTD in the organs of experimental mice practically does not differ from the control. A statistically significant difference was found in the content of CTD in the brain and liver of animals treated with compound (1) and 5-hydroxy-6-methyluracil.

The antioxidant activity of compound (1) was also studied in a model of acute toxic hepatitis caused by the administration of tetrachloromethane (TCM) to rats. TCM in the form of a 50% solution in olive oil was injected once into the stomach at a dose of 0.2 ml / kg [6]. The antioxidant efficacy of compound (1) (at a dose of 50 mg / kg) and the reference drug 5-hydroxy-6-methiouluracil (at a dose of 50 mg / kg) was evaluated by the change in the content of TBA-RP in the liver 48 hours after administration of TCM to rats. The content of TBA-RP products in the liver of rats during intoxication of TCM was determined by the method of Steel I.D., Gurashvili T.G. [eleven].

The results of the experiment are presented in table 4.

The administration of TCM to animals increases the content of TBA-RP in rat liver by more than 2 times in comparison with the control. A single injection of compound (1) and 5-hydroxy-6-methyluracil reduced the amount of TBA-RP in the liver of poisoned rats by 1.9 times (P <0.001) and 1.69 times (P <0.05), respectively. Compound (1) is more effective than 5-hydroxy-6-methyluracil: the difference in the ratios between groups of rats is 5-hydroxy-6-methyluracil + TCM and compound (1) + TCM is statistically significant (P <0.05).

Table 4 The effect of compound (1) on the content of TBA-RP in rat liver after intoxication with carbon tetrachloride Groups of animals (n = 8) Statistical indicator TBA-RP in the liver, nmol / g of tissue Control - Intact Animals M ± m% 0.51 ± 0.04 100 TXM M ± m% 1.05 ± 0.07 * 205.8 5-Hydroxy-6-methyluracil + TXM M ± m% 0.62 ± 0.06 ** 121.5 Compound (1) + TXM M ± m% 0.55 ± 0.04 ** *** 107.8 Note: * - the difference is significant (P <0.05) compared with the control; ** - the difference is significant (P <0.05) compared with TXM; *** - the difference is significant (P <0.05) compared with 5-hydroxy-6-methyluracil + TCM.

Thus, the compound (1) has a higher antioxidant activity and low toxicity with intraperitoneal and intragastric administration than the reference drug 5-hydroxy-6-methyluracil.

The essence of the technical solution is illustrated by the following examples.

Example 1. Synthesis of compound (1).

In a two-necked flask equipped with a reflux condenser, 0.029 mol (5 g) of 5-nitro-6-methyluracil, 200 ml of ethyl alcohol and 0.087 mol (11.32 g) of hydrazine sulfate were mixed. The mixture was heated to 70-80 ° C and an alcoholic suspension of Raney nickel was added in small portions. The beginning of the reaction was noticed by the release of nitrogen. Each subsequent portion of the catalyst (Raney nickel) was added after gas evolution decreased. The progress of the reaction was monitored by thin layer chromatography (eluent chloroform-methanol, 8: 2). At the end of the reaction, a catalyst residue was added to completely decompose the hydrazine sulfate. The hot solution was filtered off from the catalyst, the filtrate was cooled. The precipitated crystals were filtered off, recrystallized from ethanol. Yield of compound (1): 3.89 g (95%).

Mp 253-255 ° C. IR spectrum, υ, cm ^-1 : 1000, 1080 (ω _{C = C} ; 1180 (= N-); 1276, 1296 (υ _{C = O} ); 1624, 1648, 1712 (υ _{C = C} , υ _{C = O} , = NC = O); 2920, 3400 (-NH ₂ ). ¹ H NMR spectrum (DMSO-d ₆ , δ ppm): 2.25 (s, 3H, CH ₃ C ⁶ ); 3.7 (br s , 2H, NH ₂ ); 10.2 (s, 1H, N ¹ H); 10.95 (s, 1H, N ³ H). ¹³ C NMR spectrum (DMSO-d ₆ , δ ppm): 13.93 (CH ₃ C ⁶ ), 117.77 (C ⁵ ), 127.69 (C ⁶ ); 149.40 (C ² = O); 161.29 (C ⁴ = O). Mass spectrum, m / z: 140 (M-H) ^- , 142 ( MN) ⁺ . Found,%: С 43.00; Н 5.09; N 29.35. С ₅ Н ₇ N ₃ O _2. Calculated,%: С 42.55; Н 4.99; N 29.77.

Example 2. The study of acute toxicity of the compound (1).

a) The acute toxicity of compound (1) was investigated in mice and rats. A group of animals (n = 10) was once injected into the stomach through a probe compound (1) at doses of 5 g / kg, 7.5 g / kg, 10 g / kg in 2% starch mucus. Control animals received starch mucus. Observations of animals were carried out for 14 days. The absence of animal death at the above doses indicates the low toxicity of compound (1) in accordance with GOST 12.1.007-76 [9]. The dose of compound (1), causing 50% death: for mice - more than 10 g / kg, for rats - more than 6 g / kg.

b) Toxicity with a single intraperitoneal administration of compound (1) in doses of 1.5 g / kg, 2 g / kg, 3 g / kg was studied in mice and rats. The statistical group consisted of 10 animals. The effect of compound (1) was observed for 14 days after administration, was evaluated by the lethal effect, and the data were processed using probit analysis. As a result of the study, the dose of compound (1) was determined, causing 50% death: for mice - 2.8 g / kg, for rats - 2.3 g / kg. The comparison drug has a 5-hydroxy-6-methyluracil dose that causes 50% death of animals: for mice - 1.9 g / kg, for rats - 1.7 g / kg (table 1).

Example 3. The study of the antioxidant activity of the compound (1) in vitro.

A 20% rat liver homogenate was subjected to spontaneous and induced oxidation at 37 ° C. LPO induction of 0.05 ml of rat liver homogenate was induced by introducing a system containing 0.5 mM ascorbate + 6 μM Mora salt. The level of LPO activity in rat liver homogenate was determined by the concentration of substances reacting with TBA.

The studied preparations — compound (1) and 5-hydroxy-6-methyluracil — were dissolved in absolute alcohol, introduced into the system at the same concentration of 100 μM, and the content of TBA-RP was determined spectrophotometrically.

AOA of the studied compounds in each series of experiments and for each duration of the oxidation period of 1 hour, 3 hours and 4 hours was calculated by the formula, the result was expressed as a percentage:

The arithmetic mean value was calculated for 5 experiments (table 5).

Table 5 The effect of compound (1) and 5-hydroxy-6-methyluracil on the content of TBA-RP in rat liver homogenate No. p / p The content of TBA-RP in rat liver homogenate after 4 hours of incubation, in mmol / mg protein Compound (1) 5-hydroxy-6-methyluracil BUT ASKT BUT ASKT TO experience TO experience TO experience TO experience one 0.170 0,064 0.272 0,112 0.170 0,077 0.272 0,200 2 0.185 0,067 0.280 0,080 0.185 0,080 0.280 0.198 3 0.180 0,065 0,305 0,083 0.180 0,075 0,305 0.196 four 0.195 0,060 0.289 0,101 0.195 0,090 0.289 0.182 5 0.172 0,074 0.285 0,100 0.172 0,080 0.285 0.183 M 0.180 0,066 0.285 0,093 0.180 0,080 0.289 0.191 m 0.0031 0.003 0.01 0.007 0.005 0.0031 0.001 0.003 % 63.3 67.3 55.5 32.9 Note: A - auto-oxidation; ASKT - a system containing: 0.5 mm ascorbate + 6 μm Mora salt; To - control.

Example 4. The study of the antioxidant activity of the compound (1) in a model of acute hemic hypoxia (OHG).

To study AOA, mice were used, groups of 8 animals. The OGeH model was created by subcutaneous administration to mice of a 4% aqueous solution of sodium nitrite at a dose of 100 mg / kg [14]. Compound (1) was administered to experimental mice once in the abdominal cavity in the form of 0.25% aqueous-twin solutions at a dose of 100 mg / kg 1 hour before the administration of sodium nitrite. Control animals were injected with a 0.25% aqueous twin solution in the same volume. As the reference preparation, 5-hydroxy-6-methyluracil was used, which was used according to a scheme similar to compound (1). The antioxidant activity of compound (1) was evaluated by the content of CTD in the organs (brain, liver) of mice (table 3).

Example 5. The study of the antioxidant activity of the compound (1) in a model of acute toxic hepatitis.

Acute toxic hepatitis in rats was caused by a single intragastric administration of a 50% solution of TXM in olive oil at a dose of 0.2 ml / kg. Compound (1) (at a dose of 50 mg / kg) and the reference preparation 5-hydroxy-6-methyluracil - (at a dose of 50 mg / kg) were administered once into the abdominal cavity in the form of a 0.25% aqueous tween solution per 1 hour before the administration of TXM. The antioxidant activity of compound (1) and 5-hydroxy-6-methyluracil was evaluated by the content of TBA-RP in rat liver 48 hours after administration of TCM (table 4).

Thus, the compound (1) has a higher antioxidant activity and low toxicity when introduced into the stomach and intraperitoneally.

Literature

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7. Auth. St. USSR 130044, 07.15.1960. / Adamovich A.I., Vitenberg A.G. The method of obtaining 5-amino-4-methyluracil - (R.Zh. Khim., 1961, 22L223).

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Claims (2)

1. The agent is a 5-amino-6-methyluracil of the formula:

showing antioxidant activity. 2. The method of producing 5-amino-6-methyluracil by reduction of 5-nitro-6-methyluracil in a solvent at elevated temperature, followed by isolation of the product, characterized in that hydrazine sulfate is used as a reducing agent, ethyl alcohol is used as a solvent, and the process is carried out at a temperature of 70-80 ° C in the presence of a catalyst, which is used as Raney nickel.