RU2572711C1 - Anticonvulsant - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method is implemented by using 9-dialkyl aminoethyl-2-(4-fluorophenyl)imidazo[1,2-a]-benzimidazole dihydrochloride of general formula I

, wherein a) NR₂ = pyrrolidino; b) NR₂ = morpholino as an agent possessing anticonvulsant activity. The compound may be used for preparing an anticonvulsant.

EFFECT: higher anticonvulsant activity.

2 cl, 8 dwg, 1 tbl

Description

The invention relates to medicines, namely, used for epilepsy. Epilepsy is a chronic brain disease characterized by repeated unprovoked attacks of impaired motor, sensory, autonomic, mental or mental functions resulting from excessive neural discharges [Rehabilitation of children with epilepsy: Methodical manual / O.V. Khaletskaya, V.A. Vorobyova, N.V. Karashtina, O.V. Konurina. N. Novgorod: Publishing House of the Nizhny Novgorod State Medical Academy, 2001. S. 3].

Currently, pharmacotherapy of epilepsy is one of the urgent medical problems. Moreover, over the past 15 years, not a single new original antiepileptic drug has been registered in Russia.

According to the International Antiepileptic League (MPEL), 6 million people suffer from epilepsy in Western and Central Europe, about 15 million are supposed to be ill over the next 20 years. The corresponding figures for Russia are approximately half a million patients with epilepsy, with an incidence of 54 thousand in year [Zenkov L.R. Treatment of Epilepsy / Ed. Corr. RAMS N.N. Yah. M., 2001. S. 8].

Alternative treatments are only rarely effective, and there are no preventative measures to prevent the development of epilepsy. Antiepileptic drugs of the first and second generations seem to be effective only in the relief of acute attacks, as well as some symptoms associated with the chronic course of the disease [Belousov Yu.B. et al. Clinical and economic evaluation of the effectiveness of treatment of patients with epilepsy // Qualitative clinical practice. - 2002. - No. 3. - S. 54-59].

Known drugs used in epilepsy are carbamazepine, phenytoin, phenobarbital, ethosuximide, diazepam [Zenkov L.R. Pharmacological treatment of epilepsy // Russian Medical Journal. - 2000. - T. 8. - No. 10. - S.411-417].

However, they cause serious side effects that are associated with effects on the nervous system and mental sphere, as well as direct toxic effects on internal organs. The most common adverse reactions include: drowsiness, decreased memory, attention, working capacity, dizziness, nausea, effect on the gastrointestinal tract and liver, hematopoietic system, as well as other manifestations characteristic of each drug individually [Glauser T., Ben-Menachem E., Bourgeois B., Cnaan A., Chadwick D., Guerreiro C, et al. ILAE treatment guidelines: evidence-based analysis of antiepileptic drug efficacy and effectiveness as initial. monotherapy for epileptic seizures and syndromes // Epilepsia. - 2006. - V. 47. - P. 1094-120]. The significant severity of these manifestations and poor tolerance directly affects the quality of life of patients and is often an occasion to search for an alternative drug.

Known funds - ethosuximide [Evason K. et al. Anticonvulsant medications extend worm life-span // science. - 2005. - T. 307. - No. 5707. - C. 258-262.], Phenytoin [Rajasekhar K. K. et al. Comparative study of conventional and microwave induced synthesis of selected heterocyclic molecules // International Journal of ChemTech Research. - 2010. - T. 2. - No. 1.], primidon [Bogdanov G.N. et al. [Anticonvulsants as bioantioxidants under stress conditions] // Biomeditsinskaia khimiia. - 2008. - T. 55. - No. 4. - C. 519-524.], Benzodiazepines [Narayana B. et al. Synthesis of some new substituted triazolo [4, 3-a] [1, 4] benzodiazepine derivatives as potent anticonvulsants // European journal of medicinal chemistry. - 2006. - T. 41. - No. 3. - C. 417-422.] And others related to heterocyclic structures.

Known means with GABA-ergic action (progabid, tiagabin) [Sato K. et al. An analysis of anticonvulsant actions of GABA agonists (progabide and baclofen) in the kindling model of epilepsy // Epilepsy research. - 1990. - T. 5. - No. 2. - P.117-124.] [Winhusen T.M. et al. A placebo-controlled screening trial of tiagabine, sertraline and donepezil as cocaine dependence treatments // Addiction. - 2005. - T. 100. - No. s1. - C. 68-77].

However, these drugs exhibit significant toxicity and may have proconvulsive activity [Stahl, S. Stahl's Essential Psychopharmacology: Prescriber's Guide. Cambridge University Press: New York, NY. 2009. pp. 523-526].

Along with this, benzimidazole derivatives exhibiting agonistic interaction with GABA-A receptors were found [Larsen J. S. et al. Benzimidazole derivatives and their use for modulating the GABAA receptor complex: US Pat. 8,492,408 United States. - 2013.]. This class of substances is considered highly promising in relation to the search for new anticonvulsants [Jain P. et al. Design, synthesis and biological evaluation of some novel benzimidazole derivatives for their potential anticonvulsant activity // Archives of pharmacal research. - 2010. - T. 33. - No. 7. - C. 971-980].

The most commonly used anticonvulsant recommended by the WHO for epilepsy is sodium valproate [Zenkov L.R. Pharmacological treatment of epilepsy // Russian Medical Journal. - 2000. - T. 8. - No. 10. - S. 411-417].

Sodium valproate effectively suppresses corazole spasms.

However, its anticonvulsant activity remains not high enough.

The technical result of the invention is to increase anticonvulsant activity.

The technical result is achieved by the use of 9-dialkyl aminoethyl-2- (4-fluorophenyl) imidazo [1,2-a] benzimidazole dihydrochloride of the general formula

where a) NR ₂ = pyrrolidino;

b) NR ₂ = morpholino,

as a compound having anticonvulsant activity.

The compound can be used to make an anticonvulsant.

9- (2-Morpholinoethyl) -2- (4-fluorophenyl) imidazo [1,2-a] benzimidazole dihydrochloride and 9- (2-pyrrolidinoethyl) -2- (4-fluorophenyl) imidazo [1,2-a] dihydrochloride Benzimidazole is known as an analgesic effect (RF patent No. 2412187, IPC C07D 487/04, 2011) and kappa-opioid agonist activity (RF patent No. 2413512, IPC AK 31/4188, 2011). In the proposed case, the compounds exhibit an anticonvulsant effect, new in the series of imidazo [1,2- a ] benzimidazole.

The following are tests for the anticonvulsant activity of compounds I.

RESEARCH METHODS

Objects of study: compound RU-1205 (dihydrochloride 9- (2-morpholinoethyl) -2- (4-fluorophenyl) imidazo [1,2-a] benzimidazole) and RU-1203 dihydrochloride 9- (2-pyrrolidinoethyl) -2- ( 4-fluorophenyl) imidazo [1,2- a ] benzimidazole synthesized at the Scientific Research Institute of Physical and Organic Chemistry of the Southern Federal University (Scientific Research Institute of Physics and Chemistry of SFU). The experiments were carried out in accordance with the rules of laboratory practice (GLP), with Article 11 of the Federal Law of April 12, 2010 No. 61-ФЗ “On the Circulation of Medicines” (Collected Legislation of the Russian Federation, 2010, No. 16, Article 1815; No. 31 , Art. 4161), “Guidelines for the experimental (preclinical) study of new pharmacological substances”, ed. RU. Khabrieva, M. OJSC “Publishing house“ Medicine ”, 2005. (M., 2005).

The studies were carried out on 130 male nonlinear white mice weighing 20-22 g contained in vivarium conditions (temperature 22-24 ° C, relative humidity 40-50%) with natural light conditions on a standard diet of laboratory animals in accordance with GOST R 50258 92 [1993], in compliance with the rules of laboratory practice when conducting preclinical studies in the Russian Federation, regulated by GOST R 51000.3 96 [1996] and GOST R 51000.4 96 [1996], as well as the rules and International Recommendations of the “European Convention for the Protection of Vertebrate Animals Used for Experimentation” cops or other scientific purposes ”[European convention for the protection of vertebral animals used for experimental and other scientific purpose: Council of Europe 03/18/1986. - Strasbourg, 1986. - 52 p].

Anticonvulsant activity was evaluated using a standard model of epileptogenesis caused by subcutaneous administration of a GABA receptor antagonist with corazole (SIGMA, USA) [Swinyard, 1969].

The studied substances were administered intraperitoneally in a dose range of 0.1-20.0 mg / kg. The positive control group received sodium valproate (Convulex, Austria) in the dose range of 50-300 mg / kg. The negative control group was injected with an equivalent volume of solvent (distilled water). The anticonvulsant activity of the substances was evaluated by their ability to prevent the development of clonic seizures lasting more than 3 seconds.

For the test compounds and the reference preparation, the values of ED _{50 were} determined by the least squares method.

Acute toxicity was studied in 60 white nonlinear male mice weighing 18-22 g with intraperitoneal administration. Calculation of LD _{50 was} carried out according to the classical Lichfield-Wilcoxon method using regression statistics (Microsoft Excel), which allows calculating this indicator based on the results of pharmacological tests of the studied substances according to tests with an alternative reaction form.

The conditional therapeutic index of compounds, which was calculated as the ratio of LD ₅₀ to ED _{50, was} used as an integral indicator of the conditional breadth of therapeutic action (TI).

Electrophysiological studies were conducted on white non-linear rats of both sexes, weighing 200-250 g. In the Research Institute of Neurocybernetics named after A.B. Kogan SFU. The experiments were performed in the field of somatosensory cortex of rats according to the method [Sukhov, 1992].

To study the effect of new compounds on the formation of normal and pathological rhythms of the brain of rats, a microapplication technique of 30.5 μM solution of RU-1205 and 23.7 μM solution of RU-1203 in a volume of 1 μl was used. Microapplication was performed by local injection at a distance of up to 100 μm from the recording microelectrodes at a depth of 1000 μm. The volume of introduced substances was a fraction of a microliter, the time of microapplication took ≈30 seconds.

To study the antiepileptic properties of compounds RU-1205 and RU-1203, the method of artificial genesis of epiactivity was used using electrical stimulation of the brain surface (rhythmic current stimulation with parameters: voltage 120 V, duration 10 s, stimulation frequency 10 Hz).

To enhance bioelectric activity, a 10-channel amplifier UBS 1/10 (Russia) with a passband from 0.1 Hz to 2000 Hz was used. The background focal and evoked bioelectric activity was recorded on a computer hard magnetic disk using a 16-channel L-761 ADC (L-Card, Russia) with a signal sampling frequency of 1 kHz. For stimulation, ESL-2 electric stimulators (Russia), GEFI-3-BU (Russia) were used. Stimulation was controlled through the TTL outputs of the L-761 board (L-Card, Russia) and was set programmatically.

For processing and analysis of electrical activity, we used methods of statistical analysis of time series: power spectra, coherence spectra, phase cross-spectra using the Spectrum [Strokun], DataViewRus [Gusach] and Statistica 5.0 programs. The significance of differences between the samples was evaluated by the criterion of signs (p <0.05) [V.Yu. Urbach, 1963]. When building charts and graphs used the MS Exel software package.

TEST RESULTS

In the test of seizures induced by corazole, the compounds RU-1205 (2- (4-fluorophenyl) -9-morpholinoethylimidazo [1,2- a ] benzimidazole) and RU-1203 (2- (4-fluorophenyl) -9-pyrrolidinoethylimidazo [1 , 2- a ] benzimidazole) showed a dose-dependent anticonvulsant effect and statistically significantly prevented the development of clonic seizures lasting more than 3 seconds (p <0.05).

Figure 1 shows the dose-dependent effect of the compound RU-1205, RU-1203 and sodium valproate on the development of clonic seizures caused by subcutaneous administration of corazole (95% of the CI are indicated by a dashed line). As a result of the experiment, it was shown that the median anticonvulsant activity of the compounds RU-1205 (ED ₅₀ = 8.4 mg / kg) and RU-1203 (ED ₅₀ = 10.5 mg / kg) exceeds the activity of sodium valproate (ED ₅₀ = 107, 0 (mg / kg) 12 and 10 times, respectively.

Table 1 shows the value of anticonvulsant activity (ED ₅₀ ), acute toxicity index (LD ₅₀ ) and therapeutic index (TI = LD ₅₀ / ED ₅₀ ) of the test substance RU-1205 and sodium valproate in a model of corazole seizures. Calculations showed that the therapeutic index of compound RU-1205 is 36.6, that is, 8.5 times higher compared to the reference drug. The therapeutic index of compound RU-1203 is 29.8, i.e. 7 times higher than that of sodium valproate. The data obtained indicate a relatively high therapeutic safety of these compounds.

INFLUENCE OF RU-1203 AND RU-1205 COMPOUNDS ON BACKGROUND RHYTHMOGENESIS

In fig. Figure 2 shows the effect of compound RU-1203 on the formation of background focal rhythms in individual somatosensory cortical columns of the rat brain. A - before local microapplication. B - 2 minutes after administration. B - 5 minutes after administration. Designations: K1 and K2 upper (500 μm) and lower (1400 μm) layers, respectively, of the control (adjacent) cortical column; KZ and K4 are the upper (400 μm) and lower (1500 μm) layers of the neural column into which microapplication of a 23.7 μM solution of compound RU-1203 was performed.

In the course of electrophysiological studies, an alpha-like (8-12 Hz) rhythmic activity of the rat cerebral cortex was recorded, which corresponds to a calm, drowsy state of the animal. In Figure 2A, one can see the steady development of alpha rhythm in individual columns of the rat somatosensory cortex, obtained under conditions prior to microapplication of the studied substances.

The RU-1203 compound (23.7 μM solution in a volume of 1 μl) in the immediate vicinity of the recording channels only slightly changes the frequency characteristics of the background rhythm for a short time (1-2 min) (Fig. 2B). However, after 5 min of observations, the dominance of alpha rhythm is restored (Fig. 2B).

In fig. Figure 3 shows the power spectra of the focal background activity of cortical neural columns before and after local microapplication of RU-1203 compound. A - before local microapplication. B - 2 minutes after administration. B - 5 minutes after administration.

Designations: K1 and K2 upper (500 μm) and lower (1400 μm) layers, respectively, of the control (adjacent) cortical column; K3 and K4 are the upper (400 μm) and lower (1500 μm) layers of the neural column into which microapplication of a 23.7 μM solution of compound RU-1203 was performed.

In Fig. 3A presents the corresponding histogram of the power spectrum of the obtained electrocorticogram (ECoG). This figure shows that the prevailing background rhythm has a frequency of 9 Hz.

The calculated power spectra for the one shown in Fig. 2B and 2B ECOG are shown in figures 3B and 3B. It can be noted that within 1-2 minutes after the introduction of the RU-1203 compound, the dominant frequencies are 1, 6, 9, and 12 Hz (Fig. 3B). After 5 min of observations, the rhythmic activity almost recovered to the initial level, where the leading frequencies are 1 and 9 Hz (Fig. 3B).

In fig. Figure 4 shows the background focal activity immediately after administration of compound RU-1205 (3.5 μM solution). Microapplication of the solution was carried out in the immediate vicinity of K1 and K2. The designations are the same.

The microapplication of the RU-1205 substance caused a statistically significant suppression of the amplitude characteristics of the focal background rhythm and a change in the frequency parameters with a leading frequency of 5 Hz.

3 minutes after the introduction of compound RU-1205 in accordance with Fig. 5, the appearance of slow (about 1 Hz) oscillations at K1 and K2 (experimental neural column) and higher-frequency rhythms at K3 (control neural column located at a distance of 1000 μm from the experimental one) can be noted. After 5 min of observations, one can note a tendency to restore focal background rhythm to the initial level (Fig. 6).

ANTI-EPILEPTIC ACTIVITY OF THE RU-1203 AND RU-1205 COMPOUNDS ON THE BRAIN SURFACE ELECTRIC STIMULATION MODEL

In fig. 7 shows the antiepileptic effect of the compound RU-1203, detected under conditions of preliminary rhythmic (10 Hz) stimulation of the surface of the brain (for 10 s) by electric current (12 V). A - Electrocorticogram of a separate neural column before and after electrical stimulation before microapplication of compound RU-1203. B - before and after preliminary electrical stimulation one minute after the introduction of the compound RU-1203. B - before and after preliminary electrical stimulation 5 minutes after the introduction of RU-1203. C * - Electrocorticogram of the selected area and calculated power spectra. Designations: K1 and K2 upper (400 μm) and lower (1500 μm) layers, respectively, of a neural column into which microapplication of a 23.7 μM solution of compound RU-1203 was performed. Arrows indicate the time of stimulation.

In fig. Figure 7 shows that prior to electrical stimulation, the formation of alpha-like background rhythms is observed, and after electrical stimulation in the upper and lower layers of the cortical column, high-amplitude epi-discharges develop. The duration of the induced epiactivity is 16 s. Then a gradual restoration of focal activity occurs, but with the presence of a theta-like rhythm corresponding to the stress state of the rat brain.

Compound RU-1203 statistically significantly eliminates epileptiform discharges caused by electrical stimulation (Figure 7B). This figure shows that within 1-2 minutes of observation after local injection of the substance in the same model of the formation of the epileptiform focus, the development of epi-activity is not observed.

Further observations (5-6 minutes after administration of compound RU-1203) show that immediately after electrical stimulation, not only epileptiform discharges do not appear in a separate neural column of the somatosensory cortex, but alpha-like rhythmic activity corresponding to a calm or drowsy state of the animal is steadily developing ( Figure 7B). For a more significant reliability of this fact, a fragment (C *) of the alpha spindle is highlighted in Figure 8B, and a detailed version of this segment of ECoG with amplitude and time marks and its histogram of the power spectrum are shown in Figure 8C *. It is important to note that in the histogram of the power spectrum, the leading frequency is 9 Hz.

In fig. Figure 8 shows the effect of compound RU-1205 on the development of epileptiform activity caused by electrical stimulation of the surface of the brain. A - before microapplication of the test substance. B - immediately after microapplication of the RU-1205 compound (0.457 mM solution) into the neural columns of the rat somatosensory cortex. B - 6 minutes after intracortical microapplication of the substance. G - 12 minutes after local administration of compound RU-1205. Designations: K1 - upper (500 μm) and K2 - lower (1300 μm) layers of rat somatosensory cortex.

Data are presented that testify to the antiepileptiform properties of the RU-1205 compound, which were identified in a model of epileptiform activity caused by electrical stimulation of the animal’s brain surface.

The figure shows that electrical stimulation leads to a prolonged (over 40 s) epileptiform activity, both in the upper and lower cortical layers. It is important to note that at the final stage of epileptiform activity, one can observe more high-amplitude, but with a lower frequency of oscillations of the focal potential.

Microapplication of the solution of compound RU-1205 completely blocks the development of epileptiform activity after electrical stimulation of the brain surface, which indicates the antiepileptic properties of the compound (Figure 8B). Similar results were obtained in 10 animals, while the average values statistically significantly differ from the control parameters (p <0.05).

The duration of the connection RU-1205 is 5 minutes, followed by the extinction of the effect (Figure 8B and 8G). With an increase in time after local microapplication of the test substance, the duration of epileptiform activity caused by electrical stimulation increases (about 10 s at 6 minutes after the substance is injected (Figure 8B) and about 20 s at 12 minutes (Figure 8G). It is important to note that epileptiform activity developing in response to electrical stimulation a long time after the introduction of RU-1205, has amplitude-frequency parameters of the oscillation of the focal potential, similar to the final stage of epipodic activity Anna before application of the substance.

CONCLUSION

In the model of corazole seizures, the RU-1203 compound exceeded sodium valproate in activity by 10 times and in value of the therapeutic index by 7 times. The RU-1205 compound exceeded sodium valproate in activity and relative safety index by 12 and 8.5 times, respectively.

In the course of studying the effect of substances on focal background rhythm, microapplication of a solution of compound RU-1203 (23.7 μM) only for a short time (1-2 minutes) slightly changed the frequency characteristics of the background rhythm with its full recovery after 5 minutes of observation. Compound RU-1205 showed a pronounced inhibitory effect at a low solution concentration (3.5 μM). The effect of the RU-1205 compound was reversible: after carrying the solution of the RU-1205 compound to the surface of the brain, the restoration of normal background activity was recorded after 5 minutes of observation.

Compounds RU-1203 and RU-1205 also effectively suppressed electro-induced epileptic activity and restored normal neuron rhythmogenesis.

Thus, RU-1203 and RU-1205 are superior to sodium valproate in anticonvulsant activity and in value of therapeutic index, and also suppress electro-induced epileptic activity and restore normal rhythmogenesis of neurons.

Claims (2)

1. The use of 9-dialkylaminoethyl-2- (4-fluorophenyl) imidazo [1,2-a] benzimidazole dihydrochloride of the general formula

where a) NR ₂ = pyrrolidino;
b) NR ₂ = morpholino,
as a compound having anticonvulsant activity. 2. The use of claim 1 for the manufacture of an anticonvulsant.

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