RU2663836C1 - L-enantymer of 2-ethyl-6-methyl-3-hydroxypiridinium hydroxybutandioate possessing cerebroprotective activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the levorotatory enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxyethane butanedionate which possesses cerebroprotective activity and prolongs the period of bioelectrical activity of the myocardium.

.

EFFECT: new compound that can be used in medicine as a cerebroprotective agent.

1 cl, 11 tbl

Description

The invention relates to medicine, specifically to a new chemical compound - the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate having cerebroprotective activity.

The purpose of the invention is a new chemical compound having cerebroprotective and other types of activity.

The goal is achieved by a new compound, namely, the enantiomer - 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate.

State of the art

One of the promising areas developed by the well-known pharmacologists is the search and creation of innovative domestic drugs in the range of hydroxypyridine derivatives.

Among the derivatives of 2-ethyl-6-methyl-3-hydroxypyridine, a number of preparations are known having a wide spectrum of biological activity.

In the early 1980s at the State Research Institute of Pharmacology RAMS Smirnov L.D. and Kuzmin V.I. Mexidol - 2-ethyl-6-methyl-3-hydroxypyridine succinate was synthesized. Mexidol is a drug with a multicomponent spectrum of pharmacological effects and a multifactorial mechanism of action. The most important components of the mechanism of action of Mexidol are its antioxidant, membranotropic effects, the ability to modulate the functioning of receptors and membrane-bound enzymes and restore neurotransmitter balance. (Mexidol: main neuropsychotropic effects and mechanism of action. T.A. Voronin. State Institution "VV Zakusov Research Institute of Pharmacology". M., RAS)

Mexidol is widely used in medicine and veterinary medicine as an antioxidant and antihypoxic agent, characterized by a wide spectrum of pharmacological action and high efficiency (nootropic and tranquilizing effect - Pat. RU 2065299, anti-ischemic and anti-atherosclerotic action - Pat. RU 2144822, antianginal - Pat. RU. 2168993, hepatoprotective - US Pat. RU 2189817, antibacterial - US Pat. RU 2157686 and others).

Known compositions based on 2-ethyl-6-methyl-3-hydroxypyridine succinate in the form of various dosage forms: solution for parenteral administration (Order of the Ministry of Health of the Russian Federation dated 31.12.1996 No. 432 "Solution of Mexidol® 5% for injection", US Pat. RU 2205640 , 2380089, 2398583), tablets (Order of the Ministry of Health of the Russian Federation dated 01.26.1998 No. 21 “Mexidol® coated tablets, 0.125 g”), capsules (US Pat. RU 2144822, 2145855), dressings (US Pat. RU 2149648), etc. .

A pharmaceutical composition in the form of tablets for oral administration based on 2-ethyl-6-methyl-3-hydroxypyridine succinate (RU 2444359) is proposed.

The drug Ethoxidol is also known - 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate (racemate), which has anti-ischemic, cerebroprotective, neurotropic and lipid-regulated activities (RU 2377237), of the formula:

as well as pharmaceutical compositions containing ethoxidol: tablets, injectable solutions.

It was shown that ethoxidol at a dose of 10 mg / kg eliminates ventricular fibrillation during occlusion of the coronary arteries and reduces the number of animals that experience ventricular fibrillation to 2 out of 8 experimental animals at a dose of 10 mg / kg and 2 out of 9 at a dose of 23 mg / kg.

Ethoxidol significantly increases the threshold for cat ventricular fibrillation at a dose of 23.0 mg / kg from 5 to 60 minutes after occlusion of the coronary arteries, while Anaprilin has this effect in an isotoxic dose from 30 to 60 minutes.

In conditions of acute myocardial ischemia at a dose of 15 mg / kg, it has a distinct cardioprotective effect on cardiodynamics, bringing contractility (dP / dt), blood pressure, etc. to the level of intact control.

Ethoxidol at a dose of 1 mg / kg has the ability to reduce the intensity of manifestations of lipid peroxidation processes caused by rabbit immobilization for 30 days, as evidenced by a decrease in the level of malondialdehyde (MDA) in blood plasma by 46% and in kidney tissue. At the same time, the level of total cholesterol is reduced by 41.7%, and the atherogenic index is reduced by 82.9%.

The successes of stereopharmacology over the past decades have enriched science with an understanding of universal laws: structure - activity and structure - toxicity, which are subject to substances that are racemic mixtures of left- and dextrorotatory isomers, D- and L-enantiomers with different pharmacological properties were obtained. Studies have shown that levorotatory isomers, as a rule, are more toxic, but also more active, while dextrorotatory and racemates are inferior to levorotatory by the strength of the pharmacological effect.

A classic example is L-adrenaline: its intracardiac concentration is 11 times higher than the dextrorotatory isomer. High stereospecificity is characterized by receptor interactions, including the processes of hepatic metabolism, which are also determined by the nature of the drug-receptor relationship.

Clinical studies of S-amlodipine. The antihypertensive and antianginal effects of S-amlodipine are supported by clinical studies. It is shown that to achieve the optimal therapeutic effect of S-amlodipine, half the dose of the drug is required.

Currently, only 15% of drugs are chiral pure substances, that is, consisting of enantiomers of the same type. The use of optical isomers of known cardiovascular agents is a way to increase their effectiveness and tolerance (M.I. Lutay, A.F. Lysenko, O.I. Moiseenko. National Scientific Center "Institute of Cardiology named after academician ND Strazhesko "AMS of Ukraine, Kiev).

Disclosure of invention

The aim of the invention is a new chemical compound -L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate (compound 1), as well as the study of its antihypoxic activity, taking into account the requirements for preclinical studies of drugs:

.

.

1. Synthesis of the L-enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate.

Example 1

To 13.7 g (0.1 m) of 2-ethyl-6-methyl-3-hydroxypyridine, 13.41 g (0.1 m) of L-hydroxybutanedioic acid are sprinkled, the mass is thoroughly mixed and heated until melting at 100-110 ° FROM. To the obtained melt, 120 ml of water are gradually added with stirring, the resulting solution is filtered, 60-70% of water is distilled off in vacuo, 80 ml of acetone is added to the obtained residue, the mass is crystallized, the resulting precipitate is filtered off and dried in vacuum. Obtain 23.8 g of a white crystalline substance with high hygroscopicity, spreads in air.

C ₁₂ H ₁₇ NO ₆

Found,%: C 53.07; H 6.38; N, 5.11.

Calculated,%: C 53.14; H, 6.27; N, 5.17.

IR spectrum, ν cm ^-1 : 3510, 3230, 1660, 1610, 1560, 1292, 1161.

UV spectrum, nm: 228.291 (water)

The rotation angle [α] _D ²⁰ -13.6 (4.5% acetonitrile).

2. Biological research

General characteristics of laboratory animals

The studies were carried out on 69 white SHK mice males, 69 white SHK mice females weighing 20-22 g, obtained from the nursery of the Andreevka Branch, Federal State Budgetary Institution Scientific Center for National Medicine, FMBA, Russia.

Number of animals

The formation of animal groups of 6 mice of each sex for the experiment on the study of acute toxicity and antihypoxic activity is a representative sample and allows for many years of experience in toxicological studies to obtain statistically reliable data. During the experiment, the animals were kept under controlled conditions: ambient temperature 20-24 ° C, relative humidity 50-60%.

2.1. Determination of acute toxicity of compound 1

In the present study, acute toxicity was evaluated in mice of both sexes with a single intravenous administration of compound 1 and the reference drug.

White outbred mice (6 animals of each sex in the group) were injected with compound 1 or a comparative preparation in increasing doses of 300, 400, 500, 600, 700 mg / kg of animal weight in the form of a solution in water for injection with a total volume of 0.5 ml, permissible for this type of animal.

Observation of animals was carried out for at least 14 days after administration. The toxicity of the drugs was judged by the death of animals and the general picture of intoxication.

The general condition of the animals was recorded: the features of their behavior, the intensity and nature of motor activity, the presence and nature of seizures, coordination of movements, skeletal muscle tone, reaction to tactile, pain stimuli, the frequency and depth of respiratory movements, the condition of the hair and skin. Counted the number of dead animals during the experiment.

The data obtained were processed by the generally accepted method of variation statistics with the calculation of the mean value (M) and standard error of the mean value (m). The paired Student's t test with Bonferroni correction at a significance level of P <0.05 was used as a criterion for assessing the reliability of differences in average values at a significance level of P <0.05, the non-parametric chi-square test was used to determine the significance of differences in absolute values, with a sample size of less than 10, Fisher’s exact test Wilcoxon test was used to determine the significance of the differences before and after administration of the pharmacological agent.

Based on the results obtained, the characteristics of “acute” toxicity were determined using a probit analysis according to Miller and Taint [10] (Table 1-4).

The clinical picture of intoxication in mice with the introduction of both compared pharmacological agents was identical. With the introduction of toxic doses of compound 1 and the reference drug Ethoxidol, Russia, a decrease in the motor activity of animals was noted. With an increase in dose, a short-term (2-3 min) period of hyperkinesia with anxiety symptoms was observed. The coordination of movements was disturbed, the skeletal muscle tone was increased, tonic and clonic convulsions were observed. During this period, involuntary acts of defecation and urination were observed. Some animals died on the background of seizures. Surviving animals were in a "inhibited" state, which was expressed in a reduced response to light, sound, tactile and pain stimuli (tail root irritation), with convulsive muscle twitching, decreased appetite, diarrhea, or stupefaction against the background of increasing water consumption. Breathing was rapid and superficial; hyperthermia was recorded. In the above state, the animals were in the first two days, after which they gradually assumed their initial physiological position.

Over the next 14 days, intoxication phenomena in animals manifested themselves in a reduced response to stimuli. The hairline lost the luster characteristic of healthy animals, the skin remained unchanged. In the future, the condition of the animals gradually returned to normal.

During dynamic observation of animals for 14 days, death among surviving animals was not noted.

The results of the study of mortality in mice after administration of compound 1 and the reference drug "Ethoxidol" are presented in tables 1-4.

The calculation of the parameters of acute toxicity gave the following results:

DL ₅₀ = 419.74 (364.2 ÷ 474.3) mg / kg, DL ₁₆ = 304.3 mg / kg, DL ₈₄ = 523.8 mg / kg.

The calculation of the parameters of acute toxicity gave the following results:

DL ₅₀ = 498.93 (414.4 ÷ 643.9) mg / kg, DL ₁₆ = 391.2 mg / kg, DL ₈₄ = 622.3 mg / kg.

The calculation of the parameters of acute toxicity gave the following results:

DL ₅₀ = 428.81 (291.8 ÷ 554.4) mg / kg, DL ₁₆ = 313.4 mg / kg, DL ₈₄ = 574.7 mg / kg.

The calculation of the parameters of acute toxicity gave the following results:

DL ₅₀ = 499.36 (350.1 ÷ 628.3) mg / kg, DL ₁₆ = 367.3 mg / kg, DL ₈₄ = 628.0 mg / kg.

The dynamics of the weight of surviving mice treated with toxic doses of FS and the reference drug Ethoxidol did not significantly differ from those of the control groups. When analyzing the dynamics of water and food consumption, intravenous administration of toxic doses of compound 1 and the comparison drug in the subsequent significant difference in comparison with the control groups of mice was also not detected (table. 5-10).

 * Differences in comparison with control are significant at p <0.05 (chi-square test).

 * Differences in comparison with control are significant at p <0.05 (chi-square test).

* Differences in comparison with control are significant at p <0.05 (chi-square test).

* Differences in comparison with control are significant at p <0.05 (chi-square test).

 * Differences in comparison with the outcome (Wilcoxon test) and the corresponding values in the control (t student test) are significant at p <0.05.

* Differences in comparison with the outcome (Wilcoxon test) and the corresponding values in the control (t student test) are significant at p <0.05.

Thus, the analysis of the results allows us to conclude that compound 1 in terms of "acute" toxicity belongs to toxicity class 3 (moderately toxic) [11] according to GOST 12.1.007-76.

2.1. Determination of antihypoxic activity

Experiments to determine the antihypoxic activity of substances were performed on white mice weighing 20-22 g, anesthetized with thiopent sodium (50 mg / kg ip).

Compound 1 and the reference drug were administered intravenously (in the tail vein) at a dose of 5% of the LD ₅₀ for 3 minutes using an electronic dispenser (Kent Scientific, USA) 5 minutes before tracheal ligation. The results of the study are presented in table 11.

As shown by the results of experiments, the reference drug "Ethoxidol", used in the highest therapeutic dose, led to an increase in the life time of the experimental animals - the shortest bioelectric activity conservation time was 592 s, and the longest - 610 s, which significantly differed from similar parameters in mice of the control group . In addition, under the influence of the substance, there was no noticeable decrease in heart rate (Table 11).

^a difference from the control group is significant, (Gehan’s survival criterion with Yates correction); ^b The difference from the Ethoxidol group is statistically significant at p <0.05 (one-dimensional analysis of variance, Newman-Keyles test); * The difference from the control is statistically significant at p <0.05 (one-dimensional analysis of variance, Newman-Cales test).

Compound 1 prolonged the period of myocardial bioelectric activity, being administered to mice at a dose of 5% of the LD ₅₀ value to 696 ± 10 s, which is statistically significantly greater than in the control and in the comparison drug group. In addition, under the influence of the analyte, myocardial contractility was maintained up to 381 ± 9 beats / min.

A study of the acute toxicity of the enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium L-hydroxybutanedioate (compound 1) in comparison with the preparation "Ethoxidol" showed that when the intravenous single administration of SHK to mice, the DL ₅₀ value of compound 1 for male mice was 419, 7 mg / kg, for mice of females - 428.8 mg / kg; the DL ₅₀ value of the ethoxidol reference preparation for male mice was 498.9 mg / kg, for female mice - 499.4 mg / kg. Compound 1 belongs to the 3rd class of toxicity for parenteral routes of administration according to GOST 12.1.007-76.

When studying the antihypoxic activity of compound 1 in comparison with the finished dosage form of the drug “Ethoxidol”, it was found that the levorotatory isomer, with a single prophylactic injection of tail into the vein of mice 5 minutes before reproducing hypoxia at a dose of 5% of DL ₅₀ , has antihypoxic activity. In this case, compound 1 is statistically significantly superior to the comparison drug for this type of action, as judged by the duration of the bioelectric activity of the heart. Neither the reference drug nor compound 1 has a bradycardic effect.

Claims (3)

The L-Enantiomer of 2-ethyl-6-methyl-3-hydroxypyridinium hydroxybutanedioate of the formula:

possessing cerebroprotective activity and a prolonged period of myocardial bioelectrical activity, in comparison with the racemate.