RU2643091C2 - 4-benzoylpyridine oxime derivatives having anticonvulsant activity as agents for treatment of epilepsy and paroxysmal states - Google Patents

Abstract

FIELD: chemistry; medicine.

SUBSTANCE: invention relates to 4-benzoylpyridine O-R-oxime derivatives of the general formula:

where R can be:

and:

where R can be:

X: oxalic acid or other organic or mineral acids, or is absent, which exhibit anticonvulsant activity, and to methods for their preparation.

EFFECT: invention provides derivatives exhibiting anticonvulsant activity and a method of preparation.

6 cl, 12 tbl, 10 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of medicine, biology, pharmacology and organic chemistry, in particular to new derivatives of O-R-oximes of 4-benzoylpyridine of the general formula:

Where

где n=2 или 3; N(R')2-морфолино или диметиламино, X=(СООН)2 или другие органические или минеральные кислоты (1а-в), или отсутствует R=O-(3,4-дихлор)бензоил; O-никотиноил; O-изоникотиноил; O-(4-хлорфеноксиацетил), X отсутствует (1г-ж),

where n = 2 or 3; N (R ') 2-morpholino or dimethylamino, X = (COOH) 2 or other organic or mineral acids (1a-b), or R = O- (3,4-dichloro) benzoyl is absent; O-nicotinoyl; O-isonicotinoyl; O- (4-chlorophenoxyacetyl), X is absent (1g-g),

which can be used to expand the arsenal of drugs used in therapy for the treatment of epilepsy and paroxysmal activity that occurs in various pathologies. The invention also relates to methods for producing new derivatives of O-R-oximes of 4-benzoylpyridine and to pharmaceutical preparations containing these compounds as active components.

The aim of the invention is the creation and detection of new derivatives of O-R-oximes of 4-benzoylpyridine with anticonvulsant activity. The goal is achieved by the synthesis of the claimed compounds by reacting 4-benzoylpyridine oxime with substituted aminoalkyl halides or acid chlorides and psychopharmacological screening of the claimed compounds.

The prevalence of epilepsy in different countries according to WHO data varies in a very wide range - from 1.5 to 50 cases per 1000 population (Sidorenko K.V., Darenskaya E.Yu. Prevalence of epilepsy in the world // Successes in modern science. 2014. No. 6 ; Hauser WA Epidemiology of epilepsy / WA Hauser // X All-Russian Congress of Neurologists with International Participation: Materials of the Congress. Nizhny Novgorod, 2012. - S. - 313-314), and in the Russian Federation it is 2.98 per 1000 population ( Geht, V.A. Hauser, L.E. Milchakova et al. // X All-Russian Congress of Neurologists 125 with international participation: materials of the congress. Nizhny Novgorod. - 2012. - S. 277).

A significant amount of antiepileptic drugs (PEPs), such as levetiracetam, topiramate, carbomazepine, finlepsin, diphenin, and others, are used to treat epilepsy, however, their active search and improvement continues, due to the fact that modern PEPs do not fully satisfy the requirements of the clinic. Despite a wide range of anticonvulsants, about 20-30% of patients remain resistant to treatment with the main anticonvulsants. In addition, there are cases of provocation of convulsive manifestations during therapy with PEP, due to the variety of mechanisms for the generation of seizures, which creates difficulties in the selection of anticonvulsant therapy. (R.J. Porter, B.S. Meldrum. Antiepileptic drugs. / In the book. B.G. Katzung. Basic and clinical pharmacology: 2 vol. T. 1. / Transl. From English. - 2nd ed. ., revised and ext. - M .; St. Petersburg: Binom Publishing House, Dialect Publishing House, 2007. S. 464-491; Karlov VA Epilepsy in children and adult women and men: a guide for Doctors / V.A. Karlov, Moscow: Medicine, 2010 .-- 717 p.).

Among the pyridine derivatives in medicine, nicardipine (methyl-2- [methyl (phenylmethyl) amino] ethyl-1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) -3,5-pyridinedicarboxylate) has been used (Jpn Kokai Tokkyo Koho JP 62.221.669 [87.221.669]) as an antihypertensive drug. Zimelidine ((Z) -3- (4-bromophenyl) -N, N-dimethyl-3- (pyradin-3-yl) prop-2-en-1-amine) has antidepressant activity (J. Med. Chem., 1981, 24, No. 2, 1499-1507). Derivatives of ([(3-pyridinylmethylene) amino] hydroxy) alkanoates are inhibitors of blood body aggregation (US Patent 794999, 1985).

Most of the original probes are produced outside of Russia, so patients are not always able to access them, which is one of the reasons for the lack of adequate care.

The objective of the invention is to obtain and identify new compounds among derivatives of O-R-oximes of 4-benzoylpyridine with anticonvulsant activity, expanding the arsenal of drugs used to treat epilepsy and other paroxysmal conditions with low toxicity.

The technical result is achieved by creating new and studying existing derivatives of O-R-oximes of 4-benzoylpyridine, which have anticonvulsant activity and are not accompanied by side effects.

The claimed compounds are synthesized according to the method consisting in the interaction of 4-benzoylpyridine oxime with substituted aminoalkyl halides or acid chlorides, followed by isolation of the products in the form of bases or salts (described in examples 1,2) and have high anticonvulsant activity in the dose range of 5-100 mg / kg with intraperitoneal administration in a number of tests presented in the examples.

Synthesis scheme

The reaction of oxime (2) with dialkylaminoalkyl chlorides in the presence of sodium hydride in DMF gives bases Ia-c in the form of oils, a quantitative yield. The bases are dissolved in alcohol, dry oxalic acid is added and the oxalates of O- (2-dialkylaminoalkyl) oximes of 4-benzoylpyridine (1a-c) are isolated, and when boiled 2 with the corresponding acid chlorides in the presence of triethylamine in benzene, O- (R-benzoyl) is obtained 4-benzoylpyridine oximes (1g-g).

The experimental part of chemical synthesis

PMR spectra were recorded on a Bruker AC-250 instrument, TMS internal standard. The data of elemental analyzes of 4-benzoylpyridine oxime derivatives (1a-g) correspond to gross formulas. Physico-chemical properties are presented in table 1.

Example 1

O-2-morpholinoethyl oxime of 4-benzoylpyridine, oxalate (1a).

To 0.90 g (0.022 mol) of NaH in 7 ml of dry DMF was added dropwise a solution of 2.24 g (0.011 mol) of 4-benzoylpyridine oxime (2) (J. Pharm. Sci., 1965, V. 54, P. 393) in 20 ml of DMF under cooling with water (10-15 ° C), stirred for 1 h, add 2.56 g (0.017 mol) of chloroethylmorpholine (base obtained from 3.5 g of amine hydrochloride), stirred for 2 hours at room temperature, add 220 ml of water, extracted 3 times with ethyl acetate (40 ml each), dried over MgSO ₄ , evaporated, the residue (quantitative yield, as an oil) is dissolved in 5 ml of alcohol, 0.9 g of oxalic acid in 2 ml of alcohol are added, the resulting precipitate filtering out yielding 3 g of compound 1a.

Compounds 1b, c were prepared analogously (J. A. van Zorge, US Patent 4,297,359, 1981). Data on compounds 1A-c are given in table. one.

Example 2

O- (3,4-dichlorobenzoyl) oxime 4-benzoylpyridine (1g).

A mixture of 0.99 g (0.005 mol) of 4-benzoylpyridine oxime (2), 1.26 g (0.006 mol) of 3,4-dichlorobenzoic acid chloride and 0.60 g (0.006 mol) of dry triethylamine in 10 ml of dry benzene is boiled 2 h, left for a day, treated with water (the precipitate is dissolved), the benzene solution is washed with saturated NaHCO ₃ solution, then with water, dried over MgSO ₄ , evaporated, the oil is rubbed under petroleum ether. Yield 1.70 g (91.9%), crystallized from alcohol. Yield 1.30 g.

Compounds 1e-z were obtained in a similar manner. Data on compounds 1g-z are shown in table 1.

* Compounds 1a-c were obtained in the form of oxalates, and 1g-g in the form of bases.

Pharmacological part.

Experimental animals were obtained from the Stolbovaya nursery, GU NTsBMT (Moscow region). The keeping of animals was in accordance with the rules of laboratory practice (GLP) and normative documents “Sanitary rules for the construction, equipment and maintenance of vivariums” approved by the Chief State Sanitary Doctor on 04/06/1973 No. 1045-73 and Order of the Ministry of Health and Social Development of the Russian Federation of August 23, 2010 No. 708n “On approval of the Laboratory Practice Rules”.

Example 3

The anticonvulsant effect of dialkylaminoalkyl ester derivatives of 4-benzoylpyridine oximes on the model of primary generalized seizures caused by maximal electroshock.

The experiments were performed on white outbred male mice weighing 20-26 g. Each dose of the compound was studied in 8-10 animals. The method of maximum electroshock (MES) simulates primary-generalized convulsions - the so-called “large” (Grant mal) seizures and is a basic test in assessing the effect of substances with anticonvulsant activity (Voronina T.A., Nerobkova L.N. Methodological guidelines for the study of anticonvulsant activity of pharmacological substances. “Guidelines for preclinical studies of drugs.” Part 1. FGBU “NTsEMSP.” Moscow, Grif and K Publishing House, 2012, Chapter 14, pp. 235-250; Loscher et al., The role of technical, biological and pharmacological factors in the l aboratory evaluation of anticonvulsant drugs. II. Maximal electroshock seizure models, Epilepsy Res., 1991, v. 8, p. 79-94; Swinyard EA-Laboratory evaluation of antiepileptic drugs. Reviewof laboratory methods, Epilepsia, 1969, v. 10, pp. 107-119).

The maximum electroconvulsive seizure (MES) was created using a certified Rodent Shocker RS, type 221 (Harvard Apparatus, GmbH, Germany). Animals received electrical stimuli through special corneal electrodes (500/300 V / mA mode: 144 mA, duration 0.3 s). The following indicators were recorded: tonic extension of the hind and forelimbs, and then, clonic convulsions and the death of animals. The anticonvulsant effect of the claimed compounds was evaluated by their ability to prevent the development of tonic extension and the death of animals. Compounds were administered intraperitoneally 40 minutes before MES. To determine the ED ₅₀ (effective dose of a substance that exerts an anticonvulsant effect in 50% of animals), the probit analysis method (Finney method) was used using the StatplusV5 software package to calculate ED16, ED50, ED84 - doses at which administration an anticonvulsant effect was observed in 16%, respectively , 50% and 84% of animals.

It was found that conducting MES caused tonic extension and the death of 88-100% of mice. Compound 1a in the dose range from 0.5 to 10.0 mg / kg decreased (at the level of the trend) the number of animals with tonic extension and increased the number of surviving animals compared to the control. Compound 1a in doses from 20.0 to 150.0 mg / kg statistically significantly reduced the number of animals with tonic extension and increased the number of surviving animals compared to the control. So, at a dose of 20 mg / kg, the elimination of seizures and an increase in survival were observed in 67% of animals, and in doses of 60.0; 80.0; 100.0 and 150 mg / kg in 100% of mice (table 2).

Compound 1b in doses from 25.0 to 100.0 mg / kg did not have a pronounced effect on the seizures of mice caused by MES. Only at a dose of 50.0 mg / kg, the survival of mice treated with compound 1b increased to 33.3% relative to 100% death of animals in the control group (Table 2).

Compound 1c in doses of 1.0 and 3.0 mg / kg did not significantly affect the seizures and survival rate in the MES antagonism test, and at a dose of 5.0 mg / kg under the influence of compound 1c there was a tendency to an increase in survival rate compared to with control. Compound 1c at a dose of 20.0 mg / kg significantly increased the survival rate in relation to the control by 62.5%, and at a dose of 40.0 mg / kg by 87.5% (P≤0.05). When using compound 1c at a dose of 40.0 mg / kg, the convulsive pattern is characterized by the complete absence of the tonic extensibility stage with the loss of the overturning reflex in 50% of the animals (Table 2).

Compound 1g changed the dose-dependent survival curve and these changes were dome-shaped. So, at a dose of 5.0 mg / kg, compound 1g contributed to the elimination of tonic extension and survival of 25% of mice, and at a dose of 20.0 mg / kg - 62.5%, while with an increase in dose to 40.0 mg / kg Compound 1g was reduced to 50% in terms of survival (table 2). Compound 1e, in all doses used, from 5.0 to 60.0 mg / kg, reduced the number of animals with tonic extension and increased the number of surviving animals compared to the control. The maximum anticonvulsant effect of compound 1e was recorded at the highest dose of 60.0 mg / kg and amounted to 100% animal survival relative to 12.5% of the control group (Table 2). Compound 1d in the dose range from 5.0 to 40.0 mg / kg did not have an anticonvulsant effect, which was recorded by the absence of an effect on the survival of mice with generalized seizures caused by MES. (Table 2).

Note: * - significance of differences from the control group, at p≤0.05 (Fisher's exact test).

Using the probit analysis method, it was found that the initial effective dose of compound 1a, at which the prevention of seizures and death of 16% of mice (ED ₁₆ ) is observed, is 2 (0.2 ÷ 5.7) mg / kg, the dose at which the prevention of seizures is observed and the death of 50% of mice (ED ₅₀ ) - 15 (5.2 ÷ 38.8) mg / kg and the dose at which there is a warning of seizures and death in most (84%) mice (ED ₈₄ ) is 107 (46, 5 ÷ 471.0) mg / kg. Compound 1c completely prevents the death of rats in the test of antagonism with MES at a dose of 40 mg / kg The ED _{16 of} compound 1c is 1.4 (0.4 ÷ 2.7) mg / kg, ED ₅₀ - 7 (4.0 ÷ 42.5) mg / kg and ED ₈₄ - 37 (17.3 ÷ 485.1 ) Compound 1 g prevents the death of 62.5% of animals at a dose of 20 mg / kg: ED ₁₆ is - 3.4 (1.7 ÷ 4.9) mg / kg, ED ₅₀ is - 13.9 (11.3 ÷ 48 9) mg / kg; ED ₈₄ is 56.3 (34.3-458.1) mg / kg. The initial effective dose of compound 1e, at which there is a prevention of seizures and death of 16% of mice (ED ₁₆ ), is 2.5 (0.8 ÷ 5.4) mg / kg, ED ₅₀ - 13.7 (5.8 ÷ 19 , 6) mg / kg and ED ₈₄ - 51.7 (30.0 ÷ 120.0).

Thus, derivatives of dialkylaminoethyl ether of 4-benzoyl pyridine oximes (compounds 1a, 1b, 1g and 1e) have a pronounced anticonvulsant effect in the MES test in mice, preventing the development of seizures and death of animals. The anticonvulsant effect of compound 1a develops in the dose range from 10.0 to 150.0 mg / kg, the ED ₅₀ is - 15.0 (5.2 ÷ 38.8) mg / kg, and in the dose range from 60.0 to 150 , 0 mg / kg compound 1a completely prevents the development of seizures and death of all (100%) animals. The anticonvulsant effect of compound 1c develops in the dose range from 5.0 to 40.0 mg / kg, ED ₅₀ is 17.0 (4.0 ÷ 12.5) mg / kg, and at a dose of 40.0 mg / kg, the compound 1c completely prevents the development of seizures and the death of all (100%) animals. The anticonvulsant effect of compound 1 g develops in the dose range from 5.0 to 40.0 mg / kg, the ED ₅₀ is 13.9 (11.3 ÷ 18.9) mg / kg. The anticonvulsant effect of compound 1e develops in the dose range from 5.0 to 60.0 mg / kg, the ED ₅₀ is 13.7 (5.8 ÷ 19.6) mg / kg, and at a dose of 60.0 mg / kg, the compound 1e completely prevents the development of seizures and the death of all (100%) animals.

Example 4

The anticonvulsant effect of the 4-benzoylpyridine oxime derivative of morpholinoethyl ester (1a) on the model of primary-generalized seizures caused by low-intensity electroshock.

The experiments were performed on white outbred male mice weighing 20-26 g. Each dose of the compound was studied in 8-10 animals. Animals received electric stimuli of lower intensity and duration (250/300 V / mA: 12 mA, duration 0.2 s) through corneal electrodes in comparison with the maximum electric shock. The use of electric shock of low intensity allows you to track the development of a convulsive seizure in dynamics. In the scoring system, the following severity severity indices were evaluated: 1 point — single twitching of the mouse body; 2 points - clonic convulsions; 3 points - tonic extensor cramps; 4 points - the death of the animal. The total number of points was calculated.

The test substance was administered intraperitoneally 40 minutes before the start of the experiment. It was found that in the control the number of surviving animals was 60-70%, and the severity of the convulsive seizure was 3-3.25 points (Table 3). Compound 1a in doses of 5.0 and 20.0 mg / kg increased the survival of animals and reduced the severity of a convulsive seizure statistically unreliable (Table 3). At the same time, when the dose was increased to 60.0 mg / kg, compound 1a reduced the severity of convulsive manifestations by more than 3 times (up to 1 point) relative to the control values (3.0 points) and completely prevented the death of animals. The pattern of seizures when using compound 1a at a dose of 60 mg / kg was characterized by the absence of stages of clonic seizures and tonic extension in 90% of animals (Table 3).

Thus, compound 1a at a dose of 60.0 mg / kg has a pronounced anticonvulsant effect, preventing both the development of tonic extensibility and clonic seizures caused by low-intensity electroshock and protecting 100% of the animals from death.

Example 5

Anticonvulsant action of dialkylaminoalkyl ester derivatives of 4-benzoylpyridine oximes on the model of primary generalized convulsions caused by corazole.

The experiments were performed on white outbred male mice weighing 20-26 g. Each dose of the compound was studied in 8-10 animals. The antagonism test with corazole (pentylenetetrazole, Sigma-Aldrich, USA), an antagonist of GABA-A receptors, as well as the maximum electroshock test, is the basic method for evaluating the effect of substances with anticonvulsant activity (Voronina T.A., Nerobkova L.N. Guidelines for the study of the anticonvulsant activity of pharmacological substances. "Guidelines for preclinical studies of drugs. Part. 1. FGBU" NTsEMSP ". Moscow, Publishing House Grif and K, 2012, Chapter 14, pp. 235-250; Loscher et al, The role of technical, biological and pharmacological factors in the labora tory evaluation of anticonvulsant drugs. III. Pentylenetetrazol seizure models. Epilepsy Res., 1991, v. 8, p. 171-189). In this technique, seizures are caused by chemical attack and simulate primary-generalized seizures in the so-called “small” (Petit mal) seizures.

Compound 1a, dissolved in physiological saline at doses of 1.0 and 50.0 mg / kg 40 minutes before corazole, was intraperitoneally administered to the experimental groups. Control animals were injected intraperitoneally with physiological saline in an equivalent volume. To obtain a seizure, animals were injected subcutaneously in the cervical spine with a dose of 95.0 mg / kg, causing seizures in 97% of mice. Animals were observed for 30-60 minutes after injection of corazole. The following were recorded: the latent period of the first generalized attack with the loss of the overturning reflex (LP), the time of death and the number of dead animals.

It was found that in control animals, after administration of corazole at a dose of 95.0 mg / kg, convulsive manifestations developed in the following sequence.

1. One or more myoclonic twitches of the whole body - 100% of mice. 2. Repeated clonic cramps of the front and / or hind limbs lasting more than 3 seconds without loss of the overturning reflex - 100% of mice. 3. Generalized clonic convulsions of the front and hind limbs with the loss of the overturning reflex - 90% of mice. 4. The death of animals - 80-100% of mice.

It was found that compound 1a in doses of 20.0 and 60.0 mg / kg statistically significantly increases the latent period of the first attack (LP) by 148 and 59 seconds, respectively, compared with the control group of animals, but does not prevent the development of convulsions caused by corazole and death of animals (table 4). Compound 1c at a dose of 40.0 mg / kg statistically significantly increases the latent period of the first attack (LP) by 35 seconds compared to the control group of animals, but does not prevent corazole-induced seizures and animal death (Table 4). Compound 1b in doses from 12.5 to 50.0 mg / kg did not affect the convulsive effects caused by corazole and the survival of mice (table 4). Compound 1g among the studied doses (5.0-40.0 mg / kg) exerts anticonvulsant action at a dose of 20.0 mg / kg, postponing the time of onset of animal death provoked by corazole by 986.7 seconds (an increase of more than 2 times ) (Table 4). The anticonvulsant effect of compound 1d was recorded in all studied doses from 12.5 to 50.0 mg / kg and was manifested in an increase in all recorded parameters relative to control values. At doses of 12.5 and 50.0 mg / kg, compound 1d increased the LP of the onset of seizures and the total life time of animals by 5 and 4.5 times, respectively, in doses, which increased the number of surviving mice by 44.7 and 33.5%. The total life time of animals that received compound 1d at a dose of 25.0 mg / kg increased three times, but the number of survivors remained at the control level (Table 4).

* - significance of differences from the control group, at P≤0.05 (student criterion).

Thus, compound 1a at doses of 20.0 and 60.0 mg / kg and compound 1b at a dose of 40.0 mg / kg statistically significantly increase the latent period of the first convulsive attack, but do not prevent the development of convulsions caused by corazole and the death of animals. Compounds 1g in a dose of 20.0 mg / kg and 1d in doses of 12.5; 25.0 and 50.0 mg / kg increase the latent period of the onset of the first convulsive attack and the total life time of mice. However, compound 1d in doses of 12.5 and 50.0 mg / kg protects against animal death caused by a generalized attack.

Example 6

Anticonvulsant effect of the 4-benzoylpyridine oxime morpholinoethyl ester derivative (1a) on partial (focal) secondary-generalized seizures in a chronic experiment in rats with a chronic cobalt-induced epileptogenic focus.

The studies were performed on males of outbred sexually mature white rats weighing 220-250 g. The study was carried out using the technique of creating a chronic epileptogenic focus caused by the application of cobalt, which models partial (focal) and secondary-generalized seizures in a chronic experiment. The technique is widely used to study the mechanisms of action of anticonvulsants in Russia and abroad (Avakyan T.N., Nerobkova L.N., Voronina T.A., Markina N.V. Mitrofanov A.A. Effect of carbamazepine on structural-functional bonds in the development of the epileptic system // Experimental and Clinical Pharmacology, 2002, No. 2, pp. 7-10; Bregman, F. Le Saux, S. Trottier 1, P. Chauvel 1, and Y. Maurin. Chronic Cobalt-induced Epilepsy: Noradrenaline Ionophoresis and Adrenoceptor Binding Studies in the Rat Cerebral Cortex. J. Neural Transmission, 1985, v. 63, p.109-118) and is recommended by the Guidelines for Preclinical Drug Research funds, FBSI "NTsESMP" of the Ministry of Health and Social Development of Russia "(Voronina TA, Nerobkova LN Guidelines for the study of anticonvulsant activity of pharmacological substances." Guidelines for preclinical studies of drugs ", M., edition of FBSU" NTsESMP "Ministry of Health and Social Development Russia, 2012, part 1, chapter 14, pp. 235-250).

The operations of implanting long-term electrodes into rat brain structures (into the motor zone of the left and right hemisphere cortex, the dorsal hippocampus, and the lateral nuclei of the hypothalamus) were performed using a stereotactic device along the coordinates of the rat brain atlas (Bures et al. 1960). Electrode implantation operations were performed under chloral hydrated anesthesia (300 mg / kg). The indifferent electrode used in monopolar recording was placed in the nasal bone of the skull. The bioelectric activity was recorded under conditions of free movement of the animal in the experimental chamber. In order to avoid artifacts from the movement of the pins, special spring contacts were used. Brain biopotentials were recorded on a 21-channel Neurosensor neurograph operating on the basis of IBM-PC 586 with installed filters at 32 Hz, with a time constant (0.03 s) and recording digital computer EEG for subsequent data processing.

The epileptogenic focus was created by the application of metallic cobalt powder on the surface of the motor region of the cortex of the left brain of rats. For this purpose, a trepanation hole was drilled into the bones of the skull, into which a glass cannula with cobalt powder was inserted (the diameter of the cannula corresponded to the diameter of the hole and did not exceed 1 mm). The cannula descended to the surface of the cortex (the dura mater was previously opened with a thin injection needle). Application of cobalt on the cerebral cortex of the brain causes hyperactivity of neurons localized at the injection site, which is expressed in the appearance of epileptiform paroxysmal discharges on the EEG. The emerging epileptogenic focus is the beginning of the development of a dynamic, constantly becoming more complex structural and functional system. The functional organization of this system is characterized by the presence of a determinant and dependent foci; the determinant focus enhances and synchronizes the activity of other foci, combining them into a single complex. Several stages are distinguished in the development of the epileptic system caused by the application of cobalt on the sensorimotor cortex of the brain. The main ones are the stage of formation of primary and secondary epileptogenic foci 24-48 hours after surgery, the stage of generalized epileptiform activity (EPA) in various brain structures with a stable level of synchronized paroxysmal discharges 5-6 days after application of cobalt (second stage of development of EPA) .

In all rats, the epileptogenic focus was localized in the sensorimotor region of the cortex of the left hemisphere in the following coordinates: 1 mm forward from bregma and 1 mm to the side of the sagittal suture. The dynamics of EPA in rats with a cobalt epileptogenic focus was studied for 7-8 days after the application of cobalt to the sensorimotor cortical zone. EEG registration was started 48 hours after cobalt application and was performed on days 5–6 (stage of generalization of EPA).

Compound 1a was administered once (intraperitoneally) at a dose of 60.0 mg / kg after background recording. After the introduction of the substance, the EEG was recorded for 120 minutes. Registration of the background EEG of animals 48 hours after the operation (stage 1) revealed the formation of foci of epileptic activity (EPA) in all studied structures with the largest number and duration of discharges in the ipsilateral cortex and hippocampus. EPA was characterized by single sharp high-amplitude waves, peaks, as well as synchronously arising paroxysmal discharges (Table 5).

A study of the effect of a derivative of 60.0 mg / kg morpholinoethyl ester derivative of 4-benzoylpyridine oxime (compound 1a) on rat brain epileptiform activity showed that at the 1st stage of the formation of the epileptic system (48 hours after surgery) 30 minutes after the substance was administered the number of EPA discharges (on average per minute) statistically significantly decreased in the electrocorticograms of the ipsi and contralateral cortex, hippocampus and hippo-thalamus. Moreover, the greatest severity of the effect (by the number of discharges) was determined in the ipsilateral cortex and hypothalamus, and a shortening of the average duration of discharges in these structures was also observed (Table 5).

At the 2nd stage of the development of EPA (5-6 days), in the control (background) rats, attenuation of the primary focus was observed, recorded by a decrease in the number of discharges and their duration in electrograms of the ipsilateral cortex and the formation of secondary foci recorded by an increase in the number of discharges and their duration in electrograms of the contralateral cortex, hippocampus and hypothalamus (Table 6).

30 minutes after the administration of compound 1a at a dose of 60.0 mg / kg, a significant and statistically significant decrease in the number of discharges and their duration was observed in all studied structures. The most pronounced effect was revealed in the contralateral cortex, where a decrease in the number of discharges by 5 times and their duration by 7.5 times was observed (Tables 6, 7).

The data obtained indicate that, in the rat cobalt-induced epilepsy model, the dialkylaminoethyl ester derivative of 4-benzoylpyridine oxime, compound 1a, has a pronounced anticonvulsant effect, significantly reducing the number of convulsive discharges and their duration in all brain structures, both in the first and second stages of the formation of the epileptic system. At the first stage of the development of the epileptic system, the most noticeable decrease in the number of convulsive discharges and their duration is observed in electrograms of the ipsilateral cortex and hypothalamus - by 1.6 times. In the second stage of the development of the epileptic system, the effect of compound 1a was most pronounced in the contralateral cortex and hypothalamus, which was recorded by a 5-fold and 3.1-fold decrease in discharge activity, respectively.

Thus, under the conditions of the partial (focal) epilepsy technique, which simulates secondary-generalized convulsions in a chronic experiment in rats with a chronic cobalt-induced epileptogenic focus, the 4-benzoylpyridine derivative of dialkylaminoethyl ester - compound 1a at a dose of 60.0 mg / kg anticonvulsant effect on both primary and secondary epileptic foci in various brain structures, statistically significantly reducing both the number of convulsive discharges and their duration. At the first stage of the development of the epileptic system, the greatest effect of compound 1a is detected in the ipsilateral cortex and hypothalamus, and in the second stage of generalization of EpA, in the contralateral cortex and hypothalamus.

Example 7

The effect of the 4-benzoylpyridine oxime morpholinoethyl ester derivative (1a) on the epileptic status caused by homocestein thialactone in cobalt-induced rats.

The experiments were carried out on white outbred male rats weighing 220-260 g. According to the method described in Example 4, a cobalt-induced epileptic focus was created. On the 7-8th day after the application of cobalt to rats, with persistent changes in the bioelectrical activity in the brain structures, homocysteine thialactone (DL-homocysteinethiolactone, HCT) was administered at a dose of 5.5 mmol / kg, diluted in 3.5 ml / kg of saline immediately before using. The provocation of epileptic status was carried out according to the method described previously (Voronina T.A., Nerobkova L.N. Guidelines for the study of the anticonvulsant activity of pharmacological substances. A guide to the experimental (preclinical) study of new pharmacological substances. - M., 2005. - P. 277-294; Walton NY et al., Lamotrigine vs. Phenytoin for treatment of status epilepticus: comparison in an experimental model. EpilepsyRes., 1996, v. 24, pp. 19-28).

The background bioelectric activity of rat brain was recorded within 15 minutes before and within 2 hours after administration of neurotoxin. To assess the ability of substances to eliminate the developed epileptic status, compound 1a at a dose of 60.0 mg / kg (intraperitoneally) was administered after the development of the electrographic status and the effect of the compound was recorded 30 minutes after its administration. The ability of a substance to eliminate epileptic status was evaluated by reducing the manifestations of a developed epileptic seizure in electrograms of various brain structures (ipsi and contralateral cortex, hippocampus and hypothalamus) and by motor-behavioral indicators.

It was found that neurotoxin caused the development of epileptic status in 70% of rats, which was characterized by persistent and prolonged clinical and behavioral motor manifestations: twitching of the muscles of the back and both forepaws, tilting of the head.

After administration of neurotoxin in rats, an increase in paroxysmal activity on the EEG is observed, which increases until a high-amplitude long-lasting (from 10 to 15 sec) discharge appears on the EEG and secondary generalized tonic-clonic seizures (VGTKS) appear: 4.1 VGTKS on average per rat. VGTKS is characterized by EEG paroxysmal activity (in 100% of rats) and motor manifestations (in 70% of rats). VGTKS ends with twitching of the muscles of the back and both front paws, tipping the head, drumming and the death of the animal. Separate focal twitches were observed in 80% of rats, complex motor complex movements - in 60% of rats, drumming in 40% of rats. At the end of the attacks, 50% of the animals had a lateral position, ending in the death of the animals (Table 9). Thus, clinical and behavioral motor manifestations of epileptic status were accompanied by the appearance of characteristic patterns of convulsive activity in the electrograms of the cortex, hippocampus, and hypothalamus.

Compound 1a at a dose of 60.0 mg / kg completely eliminated EEG and behavioral motor manifestations (twitching of the muscles of the back and both forepaws, tilting of the head and secondary generalized tonic-clonic seizures (VHTC) (Table 8). The disappearance of VHTC was accompanied by the restoration of bioelectric activity in electrograms of the cortex, hippocampus and hypothalamus.

Compound 1a at a dose of 60.0 mg / kg caused a decrease in the number of animals with focal motor seizures up to 30% (in the control 80%) and completely (up to 0%) eliminated complex motor complex movements, drumming and lateral position, and also protected against death of 100% of animals (table 9).

Thus, the derivative of dialkylaminoethyl ester of 4-benzoylpyridine oxime - compound 1a at a dose of 60.0 mg / kg in 100% of the animals completely eliminates the electrographic and behavioral manifestations of the status epilepticus caused by the neurotoxin homocysteine thiolactone in cobalt-induced rats.

Example 8

Antihypoxic effect of derivatives of dialkylaminoalkyl ethers of 4-benzoylpyridine oxime on a model of normobaric hypoxia with hypercapnia in a hermetic volume (“canned” hypoxia)

Outbred mice, males weighing 22-24 g, were used in the experiment. The study of antihypoxic properties was carried out under conditions of normobaric hypoxia with hypercapnia - a model of "canned" hypoxia. The experiments were carried out according to the guidelines for the study of drugs with a nootropic type of action (T.A. Voronina, R.U. Ostrovskaya, T.L. Garibova. Guidelines for preclinical studies of drugs. Edition: FSBI "NTsEMP" Ministry of Health and Social Development of Russia // Moscow 2012. Part 1. Chapter 17. S. - 285).

Animals of the same weight (a spread of not more than 2 g per group) were placed individually in hermetically sealed jars of 200 cm ³ volume and the life time to an agonal sigh was recorded.

Compounds 1a and 1c were administered once, intraperitoneally, and effects were recorded 40 minutes after administration.

When studying compound 1a in doses of 20.0 and 60.0 mg / kg on a hypoxia model with hypercapnia, a statistically significant increase in life expectancy was found to be 172 and 449 seconds, respectively, relative to control values (Table 10).

Compound 1c at a dose of 40.0 mg / kg causes an increase in the average life expectancy of mice - by 212 seconds compared with the control in normobaric hypoxia with hypercapnia in a hermetic volume (Table 10).

Thus, the derivatives of 4-benzoylpyridine oxime dialkylaminoethyl ester (compounds 1a and 1c) have a distinct antihypoxic effect on the hypoxia model with hypercapnia in the hermetic volume, which is expressed in a statistically significant increase in the life span of animals under hypoxia.

Example 9

Study of possible side effects (neurological deficit) of dialkylaminoalkyl ester derivatives of 4-benzoylpyridine oximes.

The experiments were performed on white outbred rats males weighing 220-260 g and outbred mice-males weighing 20-22 g. The study was carried out according to the "Guidelines for preclinical studies of drugs. Edition: FSBI "NTsEMSP" Ministry of Health and Social Development of Russia. Moscow 2012. Part 1. Chapter 13. C - 224). The study of the ability of substances to cause neurological deficit in tests of impaired coordination of movements, in tests of a rotating rod and pull-ups on a horizontal bar. To assess impaired coordination of movements and muscle relaxation in animals, we used a certified installation of a rotating rod (Rota Rod, Ugo Basile, Italy). Mice were placed on a horizontal rod 3 cm in diameter, rotating at a speed of 10 rpm. Counted the number of drops from the rod. In the test, pull-ups on the horizontal bar of the mice were suspended with their forelimbs by a wire (diameter 0.4 cm), stretched at a height of 20-30 cm from the table surface. The inability of animals to pull the hind limbs onto the wire was recorded as a manifestation of a neurological deficit.

Compounds 1a and 1c were administered once, intraperitoneally, and effects were recorded 40 minutes after administration.

It was found that compound 1a in the dose range from 0.5 to 150.0 mg / kg does not cause an increase in excitability, tremor, convulsive manifestations, hyperkinesis, twitching, discoloration of the skin and mucous membranes, ruffling of hair, catalepsy, ptosis, stereotypy, aggression and grooming. The pain, corneal and pinneal reflexes do not change.

Compound 1a (0.5-100.0 mg / kg) does not adversely affect the coordination of movements in mice, recorded by the ability of animals to stay on a rotating rod, and does not cause a violation of muscle tone in the pull-up test on the bar in rats. However, with an increase in the dose of compound 1a to 150.0 mg / kg, drops from the rod were observed in 70% of rats and an inability to catch on the bar in 30% of animals (Table 11).

Compound 1c in the therapeutic dose range from 1.0 to 40.0 mg / kg does not cause a neurological deficit in the tests of the rotating rod and pulling to the crossbar (Table 11).

Thus, the derivatives of dialkylaminoethyl ether of 4-benzoylpyridine oximes (compounds 1a and 1c) in a wide range of doses (0.5-150.0 mg / kg) do not cause excitability, tremors, convulsive manifestations, discoloration of the skin and mucous membranes, piloerection , catalepsy, ptosis, stereotyping and grooming; the thresholds of pain sensitivity and aggression do not change. Compound 1a (0.5-100.0 mg / kg) and compound 1c (1.0-40.0 mg / kg) do not cause impaired coordination of movements and muscle tone (neurological deficit) in the tests of the rotating rod and pulling to the bar.

Example 10

The study of acute toxicity of derivatives of dialkylaminoalkyl ethers of 4-benzoylpyridine oxime.

The experiments were carried out on white outbred male mice weighing 22-26 g, which were kept in a vivarium under the same living and feeding conditions (standard briquetted food). Compounds were administered once, intraperitoneally. Registration of the death of animals was carried out 24 hours and 14 days after administration. Using the probit analysis method (Finney method) with the help of the Statplus V5 software package, LD16, LD50, LD84 - doses were calculated, with the introduction of which 16%, 50% and 84% of animals died, respectively.

It was found that compound 1a, when administered at a dose of 220 mg / kg after 24 hours, caused the death of one animal out of ten (10%). Over the next 14 days after a single administration of compound 1a, no animal deaths were recorded. With the introduction of compound 1A at a dose of 300.0 mg / kg, the death of 40 animals (4 out of 10) was observed 24 hours after administration; no change in survival over the next two weeks was observed. After administration of compound 1a at a dose of 350.0 mg / kg, 24% of animals died in 24 hours (5 out of 10), and at a dose of 400.0 mg / kg, 90% of animals (9 out of 10) died; 14 days after the administration of compound 1a, no additional death of the rats was observed (Table 12).

Compound 1c 24 hours after a single injection at a dose of 100.0 mg / kg caused the death of 10% of animals, at a dose of 150.0 mg / kg - 30% of animals, at a dose of 200.0 mg / kg - 80% and at a dose of 400 mg / kg - 90% of mice. 14 days after a single injection of compound 1c, the death rates increased: from a dose of 100.0 mg / kg, death of 40% of animals was observed, from a dose of 150.0 mg / kg - 80% of animals, from a dose of 200.0 mg / kg - 90% and from a dose of 400.0 mg / kg, 100% of mice (Table 12).

Calculation of lethal doses (LD) by the probit analysis method (Finney method) showed that the initial dose of compound 1a, with the introduction of which 16% of mice (LD ₁₆ ) dies, is 254.0 (178.6 ÷ 285.0) mg / kg , the dose at the administration of which 50% of mice (LD ₅₀ ) dies is 316.0 (275.5 ÷ 355.7) mg / kg and the dose of LD ₈₄ is 397.0 (353.7 ÷ 533.0) mg / kg For compound 1c, the dose of LD ₁₆ is 81.0 (51.2 ÷ 98.0) mg / kg, LD ₅₀ - 116.0 (95.0 ÷ 140.0) mg / kg and LD ₈₄ - 166.0 ( 138.0 ÷ 255.6) mg / kg.

To determine the therapeutic breadth of compounds 1a and 1c, therapeutic indices (TI) were calculated by the formula: TI = LD ₅₀ : ED ₅₀ .

The calculated LD ₅₀ for compound 1a and compound 1c are 316.0 mg / kg and 116.0 mg / kg, respectively, while the ED ₅₀ for compound 1a is 15.0 mg / kg and for compound 1c 7.0 mg / kg. The therapeutic indices calculated for these indicators are for compound 1a - 21, and for compound 1c - 16. Thus, the derivatives of dialkylaminoethyl ether of 4-benzoylpyridine oximes (compound 1a and 1c) belong to the class of non-toxic substances and have a large therapeutic breadth, since they therapeutic indices are greater than 10.

Claims (27)

1. Derivatives of O-R-oximes of 4-benzoylpyridine of the General formula:

where R may be:

as well as:

where R may be:

X: oxalic acid or other organic or mineral acids, or absent. 2. Compounds 1a, d, e according to claim 1, having anticonvulsant activity. 3. Derivatives of 4-benzoylpyridine oximes: O- (2-dimethylaminoethyl or propyl) oxalates of 4-benzoylpyridine oximes (1b, c) and O- (3,4-dichlorobenzoyl) 4-benzoylpyridine oxime (1g):

possessing anticonvulsant activity. 4. The method of obtaining compound 1A, which consists in the interaction of the oxime (2)

with 1-morpholinyl-2-chloroethane in the presence of NaH in DMF, the base obtained is added to the target product 1a by adding dry oxalic acid, and the known bases 1b and 1c are converted to the corresponding oxalates. 5. The method of obtaining derivatives of O-R-oximes of 4-benzoylpyridine, indicated in paragraph 1, in which the reaction of the oxime (2)

with dialkylaminoethyl chlorides (5)

where R have the above meanings, in the presence of sodium hydride in DMF, bases are obtained (6)

where R have the above meanings, in the form of oils, quantitative yield; the bases (6) are dissolved in alcohol, dry oxalic acid is added and the oxalates of O-2-dialkylaminoalkyl oximes of 4-benzoylpyridine (1) are isolated, and when boiling the oxime (2) with the corresponding acid chlorides (7)

in the presence of triethylamine in benzene, O- (R-benzoyl) oximes of 4-benzoylpyridine (1g-g) are obtained. 6. The use of derivatives of O-R-oximes of 4-benzoylpyridine, indicated in paragraphs 1 and 3, as anticonvulsants for the prevention and treatment of paroxysmal conditions, including epilepsy.