RU2632005C1 - N-(acetylsalicyloyl)pyrazol with cerebroprotector action in case of cerebral circulation insufficiency - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention can be used to obtain effective cerebroprotective agents based on pyrazole and 2-acetoxybenzoic acid (acetylsalicylic acid, aspirin). A pyrazole derivative with the formula given below is proposed.

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EFFECT: derivative has a cerebroprotective effect, effectively and in small doses improves cerebral circulation in the model of one-stage irreversible bilateral occlusion of common carotid arteries, surpasses the action of pyracetam.

3 tbl, 2 ex

Description

As world practice shows, interest in acetylsalicylic acid (aspirin) and its derivatives does not decrease. On the basis of acetylsalicylic acid, a huge number of substances with a wide spectrum of pharmacological activity have been synthesized. So N-acetylsalicyloyl derivatives of amino acids and peptides are promising nootropic agents, some amides of salicylic (2-hydroxybenzoic) acid and heterocyclic compounds (N-salicyloyl substituted) and their salt forms have antitumor activity, and are also non-nucleoside 24 transcriptase inhibitors, 326557, F. Chen et.al, 2012; T. Laxmi et.al, 2007; F. Liu et.al, 2010].

The invention relates to a new derivative of salicylic acid containing a heterocyclic fragment, and its use as a cerebroprotective agent. The purpose of the invention is to obtain an effective derivative of acetylsalicylic acid, combining the main effects inherent in cerebroprotective agents and superior to those of piracetam.

The invention consists in the synthesis of N- (acetoxisalicyloyl) pyrazole of the formula

and its use as a cerebroprotective agent for cerebrovascular insufficiency.

Example 1. N- (acetoxisalicyloyl) pyrazole

To a solution of 100 mmol of pyrazole in dry diethyl ether (50 ml), a solution of acetylsalicylic acid chloride (50 mmol) in diethyl ether (30 ml) was slowly added dropwise over 15 minutes, the mixture was stirred for 1 hour at room temperature. Then the solution is quickly washed with cold water (3 times 10 ml). The washed product is dried over anhydrous magnesium sulfate. The residue was distilled in vacuo at a residual pressure of 3-5 mm Hg, collecting the fraction — a light yellow oily liquid boiling at 103–105 ° C, R _f 0.350 (ethyl acetate: hexane 3: 1), d ²⁰ 1.250, n _D ²⁰ 1.594. The yield was 70%, M + 230 (Saturn-2100 gas mass spectrometer, Varian). NMR ¹ N, ppm (DMSO): 7.77-7.72 m, 7.63-7.60 m, 7.04-7.00 m (3H, pyrazole); 7.68-7.70 m, 7.50-7.56 m, 7.40-7.35 m, 7.31-7.28 m (4H, aromatic ring); 2.19 s (3H, -CH ₃ ). For C ₁₂ H ₁₀ N ₂ O ₃ found,%: C (62.61), H (4.38), N (12.16); calculated,%: C (62.60), H (4.38), N (12.17).

Example 2. The cerebroprotective effect of substances with irreversible bilateral occlusion of the carotid arteries

The experiments were performed on sexually mature outbred male rats 180-200, 3.5-4 months old (nursery FSUE PLZH "Rappolovo", Leningrad Region). After delivery from the cattery, all animals were kept in standard vivarium conditions and underwent 14-day quarantine, at the end of which they were included in the study. In the course of the experiments, the following groups were formed: 1) LO (falsely operated) - animals that received physiological saline, and during surgery on the common carotid arteries, ligatures were placed under the vessels, but were not delayed; 2) control - animals of this group received physiological saline and both common carotid arteries (OSA) were ligated; 3) N- (acetoxisalicyloyl) pyrazole - animals that received a patented compound at a dose of 10 mg / kg intraperitoneally before the OSA ligation; 4) piracetam - animals that received piracetam at a dose of 800 mg / kg intraperitoneally before OSA ligation. To assess the cerebroprotective effect of the patented compound in cases of cerebrovascular insufficiency, the model of simultaneous irreversible bilateral occlusion of the OSA was used [R. Mirzoyan Guidelines for the preclinical study of drugs for the treatment of cerebrovascular accidents and migraines // Guidelines for the preclinical study of drugs / Under. ed. A.N. Mironova. Part one. - M .: Grif and K, 2012. - 944 p.]. The cerebroprotective effect of the compound was judged by increasing the survival of animals after bilateral OCA and reducing the severity of neurological deficit according to the McGraw scale in the modification of I.V. Gannushkina.

A comprehensive study of the neurological picture in diseases of the central nervous system of various etiologies involves an assessment of behavioral abnormalities, the analysis of which allows us to judge the extent of brain damage with the possibility of extrapolation to some extent in clinical practice. Therefore, in order to assess the neurological state of animals, animal behavior was studied, as well as their cognitive functions, for which standard psychopharmacological tests were used: Open Field (OP), Conditional Passive Avoidance Reaction (passive avoidance reaction), and Extrapolation Relief Test (TEI).

Test "Open Field" (OP) [Khaunina R.A., Lapin I.P. 1976, Voronina T.A. 1982] is intended to study the behavior of rodents in new conditions. The technique was as follows: an animal was placed with the tail to the experimenter in the center of a round white arena, after which visual behavior was monitored for 3 minutes, fixing the following indicators: the number of sectors crossed, number of racks, number of holes examined for minks. Orientation and research activity was judged by the sum of racks and peeps into holes, and by the number of sectors crossed - spontaneous motor activity.

The methodology for assessing the conditioned reflex of passive avoidance ”(passive avoidance reaction) is the basic model for assessing cognitive function in animals. It is used to study the effect of substances on the formation, preservation and reproduction of a memorable trace in normal conditions of brain pathology. This technique was performed as follows:

1) the formation of the passive avoidance reflex - the animal was placed in the central zone of the illuminated compartment (100 W lamp), tail to the hole in the dark compartment. If a rat turned into a dark one after examining the bright compartment, then a current of 40 V, 3 pulses of 1 s each, with an interval of 0.5 sec and an animal was fed through the electric field of the latter, resulting in electric pain irritation and left the dark compartment. The animal was observed for 180 seconds. Rats that did not enter the dark compartment or entered the dark compartment with electric pain stimulation more than 2 times were excluded from the experiment. The following indicators were recorded: the latent period (PL) of the first entry into the dark compartment (the time from the moment the animal was placed in the middle of the platform to the first entry into the dark compartment) and the number of entries into it;

2) control of the development of the reflex - to be performed 24 hours after the first session. All animals that entered the dark compartment of the installation were excluded from the experiment;

3) reproduction of the skill of passive avoidance - at this stage, in contrast to the training stage, if the animal entered the dark compartment, then no pain pain was applied. The same indicators were recorded as during training.

The “extrapolation disposal test” (TEI) is designed to study the cognitive functions of rodents in an aversive environment and allows you to evaluate: individual differences in the cognitive style of solving the problem (finding a way to actively get rid of the aversive environment). A diving medication was recorded (the time from the moment the animal was immersed in water to diving under the edges of the cylinder). Like the passive avoidance reaction test, the technique includes the stage of learning the skill (developing the reflex of deliverance) and the stage of its reproduction after 24 hours. Animals that do not solve the problem during the observation at the training stage are excluded from experience. The training and the first reproduction in animals in the passive avoidance reaction and TEI tests were performed 48 and 24 hours, respectively, before modeling bilateral OSA, and the second reproduction, 72 hours after the operation.

Irreversible occlusion of the OSA caused a pronounced disturbance in cerebral circulation, which led to the death of some animals. The patented compound significantly increased the survival of rats after ligation of the carotid arteries compared with the control group (control-ischemia) (table 1). The cerebroprotective effect exerted by him exceeded that of piracetam.

When assessing neurological deficit according to the McGrawy scale of animals with bandaged OCA, it was noted that the administration of N- (acetoxysalicyloyl) pyrazole prevented neurological symptoms in rats, as evidenced by the lowest neurological deficit in animals treated with the patented compound (Table 2).

The motor activity of animals with bandaged OSA in the test "OP" was reduced relative to the false-operated animals (table 2). In animals treated with the patented compound, the indices of motor and orientational research activity were significantly higher than the control animals.

When performing and analyzing the results of passive avoidance reaction and TEI tests, it was noted that surviving animals with OCA receiving N- (acetoxysalicyloyl) pyrazole in the TEI test needed less time to solve the problem of extrapolation disposal, and in the passive avoidance reaction they did not enter the dark chamber, which indicates on the preservation of a memorial trail associated with aversive effects (table 2).

Thus, taking into account that N- (acetoxisalicyloyl) pyrazole increases the survival of animals and reduces the severity of neurological disorders after ligation of the carotid arteries, the claimed compound can be used to create new drugs for the treatment of cerebrovascular disorders of ischemic origin.

Example 3. Determination of acute toxicity

Acute daily toxicity with a single administration was studied on male mice weighing 20-24 g. The compound was administered intraperitoneally in distilled water to animals in various increasing doses once. Observation of the animals was carried out during the day, noting the number of dead animals. Calculation of LD ₅₀ was carried out by the Litchfield and Wilcoxon method.

Studies of acute toxicity showed that according to the classification of toxicity of substances when they are introduced into the abdominal cavity of animals (K.K. Sidorova, 1973), the claimed substance belongs to the class of moderately toxic according to the classification proposed by I. Sanotsky. and Ulanova I.P. (1975) (table 3).

Claims (3)

N- (acetylsalicyloyl) pyrazole of the formula

with cerebroprotective effect in case of cerebrovascular insufficiency.