RU2559875C1 - Disodium salt of 3-hydroxyhippuric acid, having antihypoxic and cerebroprotective action - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a novel water-soluble disodium salt of 3-hydroxyhippuric acid (disodium salt of N-(3-hydroxybenzoyl)glycine) of formula

, having antihypoxic and cerebroprotective action. The invention can also be used in the chemical and pharmaceutical industry to obtain effective antihypoxic and cerebroprotective agents.

EFFECT: improved properties of compounds.

5 tbl, 4 ex

Description

The invention relates to new water-soluble derivatives of 3-hydroxy-hippuric acid - disodium 3-hydroxy-hippurate (disodium salt of N- (3-hydroxybenzoyl) glycine) and its use as an antihypoxic and cerebroprotective agent.

Optimization of the treatment of neurodegenerative diseases is one of the urgent problems of medicine, the significance of which is due to the widespread prevalence, high percentage of mortality and disability of patients [L. Borodkina Neuroprotective properties and mechanism of action of new derivatives of gamma-aminobutyric acid analogues. Dis. Doct. honey. sciences. - Volgograd. - 2009. - 379 p.]. Despite the huge costs of the state for the treatment and rehabilitation of patients with neurodegenerative diseases, the effectiveness of therapy remains low and the choice of drugs that could positively affect the prognosis, stop the progression of neurodegenerative changes, is very limited (Burchinsky S.G., 2008; Gusev E.I. Skvortsova V.I. 2001; Eliseev E.V., Koryukova I.V., Rumyantseva S.L. et al., 2008; Kadykov A.S., Shakhparonova N.V., 2009; Muresanu DF, 2003, 2007).

Domestic drug Mexidol (ethylmethylhydroxypyridine succinate) exhibits nootropic, neuroprotective, anxiolytic, antihypoxic, anti-stress, anti-alcohol, anti-Parkinsonian and anticonvulsant activity, etc. [Voronina T.A. Domestic drug of the new generation Mexidol: main effects, mechanism of action, application. - M .: 2004. - 248 p.]. In animal studies, it was found that mexidol at a dose of 50 mg / kg orally once does not have an antistress effect. However, this effect appears with prolonged use of the drug (30-95 days) or with an increase in dose to 125 and 250 mg / kg (once). When Mexidol was administered by injection, the antistress effect was observed with a single administration of the drug at a dose of 50 mg / kg. With prolonged use of Mexidol, an increase in the aggressiveness of animals was recorded (RU 2065299). In medicine, sodium oxybutyrate (CAS 502-85-2) is used to treat psychopathic neurosis, prevent hypomanic and manic states, and in narcology in the treatment of alcohol and opiate addiction, it is a hypnotic of metabolic action and an indispensable drug in the standard for the treatment of convulsive status, it has a pronounced antihypoxic effect . At present, the level of sodium oxybutyrate consumption has significantly increased for non-medical purposes: as a doping agent and psychoactive substance, which is a serious social problem, since this substance causes drug dependence and also has significant side effects. An overdose of sodium oxybutyrate causes a deep depression of the central nervous system with respiratory arrest, which, without the timely provision of qualified medical care, leads to the death of the victim [Shurygin I.A. Sodium oxybutyrate: undocumented properties of the drug (what Mashkovsky is silent about) / Shurygin E.A. // Journal. Emergency medicine. 2008. - No. 2. - S. 15.]. The reference nootropic, which is used to restore impaired intellectual functions in patients with cerebrovascular accident, is piracetam (2-oxo-1-pyrrolidinacetamide, CAS 932-17-2). The disadvantage of piracetam as a reference preparation is its low activity: the level of effective doses of action is 200-800 mg / kg, the effect of piracetam is often poorly reproduced (RU 2050851). Glycine (aminoacetic acid, CAS 56-40-6) - a tool that improves mental performance and reduces psycho-emotional stress [Mashkovsky, M. D. Medicines: a manual for doctors. - T. 2. - M .: New wave. - 2002. - S. 124].

Acyl derivatives of glycine are metabolites of fatty acids in the body. N- (3-hydroxybenzoyl) glycine (3-hydroxy-hippuric acid, CAS 1637-75-8) is a metabolite of rutin and other polyphenols [Liebich H. M., Forst C. Basic profiles of organic acids in urine. J Chromatogr. 1990; 525 (1): 1-14. Pubmed: 2338430] - not used in medicine. Derivatives of a 3-hydroxy-hippuric acid with a heterocyclic fragment act as stearyl-CoA desaturase inhibitors (US 20110046134). Derivatives of 3-hydroxybenzoic acid and aminoadamantane (CID 4030213) exhibit antiviral activity [Martoglio B, Graf R, Dobberstein B. Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin. EMBO J., 1997; 16 (22): 6636-45].

The purpose of the invention is to obtain a highly effective water-soluble derivative of 3-hydroxyhippuric acid, combining the main effects inherent in antihypoxic and cerebroprotective agents, not inferior to or superior to those of sodium oxybutyrate, glycine, piracetam and mexidol.

The invention consists in the synthesis of disodium 3-hydroxyhippurate of the formula

and using it as an antihypoxic and cerebroprotective agent.

Example 1. Disodium salt of N- (3-hydroxybenzoyl) glycine (disodium 3-hydroxyhippurate)

Glycine and 50 ml of DMF are placed in a reactor equipped with a stirrer. 3-Hydroxybenzoic acid chloride is poured dropwise over 1.5 hours. The reaction is carried out under cooling for 1.5 hours. Then, the resulting reaction mixture was stirred for another 2.5 hours (with cooling), controlling the pH of the medium (pH> 7). A 6 N sodium hydroxide solution is added to maintain the medium. The resulting mixture was poured into ice and acidified with hydrochloric acid to pH 5, the precipitated crystals of N- (3-hydroxybenzoyl) glycine were filtered off and dried. Yield 73%. Mp = 167-170 ° C, Rf = 0.785 (propanol-1: water = 7: 3). M + 195 (Chromatomass spectrometer "Saturn-2100", Varian). Then, the obtained N- (3-hydroxybenzoyl) glycine (3-hydroxy-hippuric acid) is mixed with sodium ethylate (molar ratio 1: 2) and boiled in benzene for 60 minutes. After cooling, the product is separated by filtration, washed with a small amount of an alcoholic solution of sodium hydroxide and dried. Get a white crystalline substance (yield 91%) with a melting point of 320 ° C with decomp. IR spectrum, cm -1: 1559.65 (-COO ^- ); 1267.55, 1254.70, 1242.65 (CO); 3382.91 (-CONH-); 1555-1424 (C ₆ H ₄ ). Calculated,%: C 45.20; H 2.95; Na 19.23; Ν 5.86; O 26.76. Found,%: C 45.56; H 2.89; Na 19.20; Ν 5.99; O 26.36.

Example 2. The study of antihypoxic activity of disodium 3-hydroxygippurate

The study was performed on male mice 18-20 g contained in standard vivarium conditions in compliance with all the rules of laboratory practice. The antihypoxic effect of the claimed compounds and comparison drugs was studied on the models of "normobaric hypoxia with hypercapnia" and "hemic hypoxia caused by nitrite intoxication."

To create "normobaric hypoxia with hypercapnia", animals were placed in hermetically closed chambers with a volume of 80 cc and the latent period of the onset of apnea was recorded. An increase in the life span of animals under hypoxia (compared with the control) under the influence of the studied compound indicates the presence of antihypoxic effect

"Hemic hypoxia" was caused by a single subcutaneous injection of sodium nitrite at a dose of 250 mg / kg. The antihypoxic effect of the test compound was judged by the increase in the time of onset of apnea in mice of the experimental groups compared with the control group of animals. In both series, the claimed compound and comparison preparations were administered intraperitoneally (in the left iliac region) 40 minutes before modeling experimental hypoxia in the following doses: disodium 3-hydroxygippurate - 8 mg / kg, glycine - 10 mg / kg, sodium oxybutyrate - 100 mg / kg, Mexidol - 100 mg / kg. The control group of animals was injected with physiological saline in an equivalent volume.

Table 1 presents the results of a study of the antihypoxic activity of disodium 3-hydroxyhippurate.

When reproducing normobaric hypoxia with hypercapnia and hemic hypoxia, the administration of all compounds to varying degrees contributed to an increase in the onset of apnea, while disodium 3-hydroxygippurate had an antihypoxic effect comparable to mexidol, slightly inferior in effectiveness to the reference drug sodium hydroxybutyrate (table 1).

EXAMPLE 3. The effect of disodium 3-hydroxyhippurate on the survival of animals and the degree of their neurological deficit in incomplete brain ischemia reproduced by irreversible simultaneous bilateral occlusion of the common carotid arteries

The study was performed on outbred male rats of 180-200 g, 3.5-4 months old, kept under standard vivarium conditions in compliance with all laboratory practice rules (animals were obtained from the nursery of FSUE NII GTP FMBA of Russia). As an experimental model of incomplete cerebral ischemia, simultaneous bilateral ligation of the common carotid arteries (OSA) was chosen [Mirzoyan P.C. 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.].

To assess the effect of disodium 3-hydroxyhippurate on the survival of animals and the degree of their neurological deficit in incomplete brain ischemia, the following groups were formed: 1) LO (pseudo-operated) rats - all manipulations were performed with this group of animals as well as with the control-ischemia group, but after bringing ligatures under the common carotid arteries they did not drag out; 2) control-ischemia - rats who underwent simultaneous bilateral occlusion of the common carotid arteries (OSA) and received physiological saline; 3) ischemia + disodium 3-hydroxy hippurate - animals with bilateral OCA, treated with disodium 3-hydroxy hippurate at a dose of 8 mg / kg; 4) ischemia + piracetam - animals with bilateral OCA treated with piracetam at a dose of 800 mg / kg; 5) ischemia + glycine — animals with bilateral OCA treated with glycine compound at a dose of 10 mg / kg. Each group consisted of 20 rats. The animals were injected with disodium 3-hydroxygippurate / piracetam / glycine / physiological saline 30 minutes before ligatures were applied to the carotid arteries and 2 hours after the end of surgical procedures.

The number of surviving animals was recorded 6, 12, 24, 48, and 72 hours after surgery to model incomplete ischemia. The severity of neurological deficit was determined on a McGrow scale in the modification of Gannushkina I.V. Assessment of the behavioral activity and cognitive functions of animals was carried out in standard neuropsychiatric tests: “Conditional passive avoidance reaction” (passive avoidance reaction), “extrapolation deliverance test” (TEI), and “open field”.

The test "open field" (OP) (Khaunina R.A., Lapin I.P. 1976, Voronina T.A. 1982) is designed to study the behavior of rodents in new conditions. During the test, the animal is placed in the center of a round white arena with its tail to the experimenter. Visual control of the animal’s behavior was carried out for 3 minutes, the following indicators were recorded: the number of sectors crossed, the number of racks (both closed and open racks were taken into account), and the number of examined holes-minks. Orientation and research activity is judged by the sum of the racks and peering into the holes, and by the number of sectors crossed - spontaneous motor activity.

The technique for assessing the conditioned reflex of passive avoidance ”(passive avoidance reaction) is the basic model for identifying specific nootropic activity of substances. It is used to assess the effect of substances on the formation, preservation and reproduction of a memorable trace in the norm and in conditions of its violation. This technique was performed as follows:

1) the formation of a passive avoidance reflex - the rat was placed in the illuminated compartment (100 W lamp) in the central zone, tail to hole in the dark compartment, examining the bright compartment, the rat turned into dark, through the electric floor of which a current of 40 V was supplied, 3 pulses of 1 s, with an interval of 0.5 s, thereby the animal received electric pain irritation. The animal was observed for 3 minutes, if it again entered the dark compartment, electric pain irritation was repeated, rats that did not enter 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 - all animals must not re-enter the dark compartment of the unit, animals that entered 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 with training, plus the total time spent in the dark compartment.

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 test, passive avoidance reaction method includes the stage of learning the skill (development of 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.

Statistical data processing was carried out using the software package Microsoft Excel and BioStat 2008 5.2.5.0. using the Kruskal-Wallace test followed by a Dunn test, Fisher’s exact test.

It was found that when modeling incomplete cerebral ischemia in experimental animals by the 72nd hour after OSA ligation, the mortality rate was significantly lower in those animals that received disodium 3-hydroxyhippurate in relation to the group of animals that received saline (table 2). The cerebroprotective effect exerted by him was comparable to that of piracetam and superior to glycine.

When assessing neurological deficit in animals with experimentally induced cerebral ischemia, it was found that disodium 3-hydroxyhippurate significantly prevented neurological symptoms from increasing in rats, which was manifested in a decrease in the McGrow neurological deficit score (Table 3). The smallest neurological abnormalities 72 hours after modeling the pathology were observed in animals treated with disodium 3-hydroxygippurate at a dose of 8 mg / kg.

The patented compound at a dose of 8 mg / kg prevented the occurrence of severe violations of locomotor and orientational research activity of animals, reduced memory impairment caused by incomplete and transient ischemia (table 4).

Thus, given that disodium 3-hydroxygippurate increases the survival of animals and reduces the severity of neurological disorders compared with the control ischemia group in conditions of incomplete brain ischemia caused by simultaneous ligation of both carotid arteries, the claimed compound can be used to create new medicinal drugs for the treatment of diseases of the nervous system of the vascular, traumatic, toxic, hypoxic genesis.

Example 4. 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 LD50 was carried out by the method of samples and analysis.

Studies of acute toxicity showed that according to the classification of toxicity of substances when they are introduced under the skin and in the abdominal cavity of animals (K.K. Sidorova, 1973), the claimed substance belongs to the class of low toxicity (table 5).

Claims (1)

Water-soluble disodium salt of 3-hydroxyhippuric acid (disodium salt of N- (3-hydroxybenzoyl) glycine) of the formula

possessing antihypoxic and cerebroprotective action.