RU2658833C1 - Succinic acid esters of 5-hydroxyadamantan-2-one, improving blood supply to ischemic brain - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to succinic acid esters of 5-hydroxyadamantan-2-one and to their study on local blood flow in the cerebral cortex in conditions of transient global ischemia. It has been found that compound Ia and, to a lesser extent, compound Ib, improve blood supply to the brain in rats subjected to transient global ischemia. It was also shown that compound Ia has a lower toxicity than compound Ib. GABAergic mechanism of the cerebrovascular effect of these substances was revealed via in vivo experiments. Therefore, compound Ia has significant cerebrovascular anti-ischemic activity and, unlike 5-hydroxyadamantan-2-one and nimodipine, does not decrease, and in fact within the first 30 minutes slightly increases the level of blood pressure. It is important to emphasise that the ability of drugs to lower the level of blood pressure, i.e. weaken cerebrovascular perfusion pressure, is an important factor limiting their use for the treatment of patients in the acute phase of ischemic stroke.

I a, b, where: Ia R=H; Ib R=5-adamantan-2-one.

EFFECT: disclosed are succinic acid esters of 5-hydroxyadamantan-2-one, which improve blood supply of the ischemic brain.

1 cl

Description

The invention relates to the chemistry of biologically active substances with cerebrovascular anti-ischemic activity, the General formula:

I a, b, where: I a R = H; I b R = 5-adamantan-2-one.

The inventive compounds are synthesized for the first time.

Among adamantane derivatives, glutamate receptor blockers are widely used in neurological practice. So, amantadine is used in the treatment of Parkinson's disease [M.F. Kraus, P.M. Maki, Effect of amantadine hydrochloride on symptoms of frontal lobe dysfunction in brain injury: case studies and review. J. Neuropsychiatry Clin. Neurosci., 1997, 9, 222-230; L.V. Kalia, S.K. Kalia, M.W. Salter, NMDA receptors in clinical neurology: excitatory times ahead. Lancet Neurol., 2008, 7, 8, 742-755], and memantine in cerebrovascular disorders in order to improve cognitive function [C.E. Heim, K.N. Sontag, Memantine prevents progressive functional neurodegeneration in rats. J. Neural. Transm. Suppl., 1995, 46, 117-130; C.G. Parsons, K. Gilling, Memantine as an example of a fast, voltage-dependent, open channel N-methyl-D-aspartate receptor blocker. Methods Mol. Biol., 2007, 403, 15-36]. The antiparkinsonian antimarkan developed at our Institute is at the stage of clinical research [I.S. Morozov, E.A. Waldman, T.A. Voronin et al. Anticaleptic activity of heterocyclic derivatives of 2-aminoadamantane. Chemical Pharmaceuticals Journal, 2000, 34, 4, 27-30; E.A. Waldman, Development of a pharmacological agent for the pathogenetic therapy of parkinsonism based on an analysis of the mechanisms of action of aminoadamantane derivatives: dis. Dr. med. Sciences: 14.00.25. M., 2001, 325 p.].

The 2-hexamethyleneiminoadamantane derivative in the form of a hydrochloride salt exhibits neuroprotective properties [T.S. Ganshina, E.V. Kurza, D.V. Maslennikov et al. Analysis of the mechanism of the cerebrovascular effect of hematan, Experimental and Clinical Pharmacology, 2015, 78, 10, 3-6]. These compounds contain a nitrogen atom in their structure, in contrast to the claimed compounds, in the molecule of which only oxygen-containing substituents are present.

The closest analogues of the claimed compounds in structure is 5-acetooxyadamantan-2-one (stimantan), which has an immunostimulating effect [N.G. Artsimovich, T.A. Fadeeva, Yu.A. Shalyminova, N.V. Klimova et al. 1-Acetoxy-adamantan-4-one, which exhibits immunostimulating activity. USSR copyright certificate No. 5883607, 1977]. However, it has been experimentally established that stimantan does not exhibit cerebrovascular activity. 5-hydroxyadamantan-2-one (cemantan), which exhibits immunomodulating properties and has a positive effect in the treatment of diseases of the vascular system of the extremities of autoimmune genesis, can be considered an analogue in pharmacological action [N.G. Artsimovich, T.A. Fadeeva, T.S. Galushina. An experimental study of the immunotropic activity of a new drug, cemantan. Immunology, 1990, 6, 21-23; I.E. Kovalev, V.M. Koshkin, N.V. Shipulina, E.I. Rumyantseva, Adamantane derivative as a fundamentally new means of treatment of chronic obliterating diseases of lower limb arteries. Chemical Pharmaceuticals Journal, 2004, 38, 3, 3-5; N.I. Avdyunina, A.V. Tolkacheva, L.N., Grushevskaya and others. Development of technology for the synthesis and quality control of the immunotropic drug Kemantan. Chemical Pharmaceutical Journal, 2016, 50, 1, 39-41]. However, 5-hydroxyadamantan-2-one significantly enhances the blood supply to the brain of animals in conditions of global transient ischemia, while lowering the level of blood pressure [R.S. Mirzoyan, T.S. Ganypin, D.V. Maslennikov, A.I. Turilova, N.I., Avdyunina, B.M. Pyatin. The adamantane derivative enhances the blood supply to the ischemic brain. Experimental and Clinical Pharmacology, 2012, 75, 6, 27-30; R.S. Mirzoyan, T.S. Ganshina, A.V. Gnesdilova, D.V. Maslennikov, Cerebrovascular Pharmacology of combined and separate ischemic pathology of brain and heart. Basic and Clinical Pharmacology and Toxicology, Nordic Pharmacological Society Basic and Clinical Pharmacology and Toxicology. 2014, 115 (Suppl. 1), 12-13]. The presence of a hypotensive effect, i.e. lowering cerebral perfusion pressure is an undesirable component of the action of such a drug.

The claimed compounds Ia and Ib exhibit comparable cerebrovascular activity with 5-hydroxyadamantan-2-one at a dose of 100 mg / kg when administered intravenously. The duration of the cerebrovascular effect is 2 hours. In addition, the claimed compounds do not lower blood pressure. The cerebrovascular effect of compound 1a in its strength and duration is close to the effect of 5-hydroxyadamantan-2-one. However, he does not have the hypotensive effect characteristic of the latter. The LD _{50 of} compound IB when administered intravenously in rats is 210.0 (165.9 ÷ 254.1) mg / kg, and compound Ia is 740.0 (676.0 ÷ 804.0) mg / kg. A distinctive feature of the present invention is the absence in the spectrum of the proposed compounds of antihypertensive activity. The invention expands the arsenal of agents for the treatment of cerebrovascular disorders.

The result of the use of the proposed compounds is their manifestation of cerebrovascular selective activity and the absence of the hypotensive effect characteristic of known drugs used in the treatment of patients with cerebrovascular disorders - nimodipine, mexidol, picamilon, cinnarizine and cavinton.

Analysis of literature data showed that the claimed compounds are not described in the literature and their biological activity has not been studied.

The purpose of the invention: the search for new biologically active compounds in the series of adamantane with cerebrovascular anti-ischemic activity and not showing hypotensive effect.

This goal is achieved by the synthesis of succinic acid esters of 5-hydroxyadamantan-2-one, which showed high cerebrovascular activity in the experiment.

A method of obtaining the claimed compounds consists in the interaction of 5-hydroxyadamantan-2-one with an anhydride (compound Ia) or succinic acid chloride (compound Ib) while boiling in an aprotic solvent, for example, toluene. The reaction is monitored by TLC on Kieselgel 60 F ₂₅₄ plates (Merck) in a solvent system hexane-ethyl acetate-ethanol conc. ammonia solution in the ratio (5: 3: 1: 1) or (5: 3: 2: 1), the manifestation of iodine vapor.

The inventive compounds are white or almost white crystalline substances, soluble in alcohols, slightly soluble (Ia) and insoluble (Ib) in water.

The target compounds are obtained in high yield and high purity.

The structure and personality of the claimed compounds is proved by IR and ¹ H NMR spectra and elemental analysis data. In the IR spectra of the claimed compounds, taken in KBr, there are characteristic absorption bands of carbonyl and carboxyl groups (ester, ketone and acid) in the range of 1729-1715 and 1685 cm ^-1 , stretching vibrations of the hydrogen atom of the hydroxyl group at 3430 cm ^-1 . Absorption bands characteristic of the adamantane cycle (C-H) are observed in the region of 2930-2840 cm ^-1 . In the ¹ H NMR spectrum of compound Ia, a singlet signal (~ 15 ppm) belonging to the proton of the hydroxyl acid group is observed.

The invention is illustrated by the following examples.

Example 1. A mixture of 4.15 g (0.025 mol) of 5-hydroxyadamantan-2-one, 3.03 g (0.030 mol) of succinic anhydride and 0.15 ml of concentrated sulfuric acid in 40 ml of dry toluene is boiled for 10 hours. reaction cooled reaction mass is filtered. The precipitate is washed with 10 ml of dry toluene. 25 ml of hexane was added to the obtained filtrate and left under periodic stirring at 0 + 5 ° С for 10-12 hours. The precipitated precipitate Ia was filtered off, washed with 2 × 15 ml of hexane, then dried at 80 ° С for 2-3 hours. White crystalline powder with so pl. 113-115 ° C (from ethyl acetate). The yield of 5-hydroxyadamantan-2-one (Ia) succinic acid monoester is 4.5 g (70%); Rf 0.05.

Found,%: C 63.17; H 6.96; C ₁₄ H ₁₈ O ₅ .

Calculated,%: C 63.14; H, 6.81.

¹ H NMR Spectrum (DMSOd ₆ , δ, ppm): 1.75-2.02 (4H, m, H-Ad); 2.17-2.55 (13H, m, 9H-Ad and 4H (CH ₂ ) ₂ ); ~ 15 (1H, OH acid).

Example 2. A mixture of 10.00 g (0.06 mol) of 5-hydroxyadamantan-2-one, 5.20 g (3.7 ml, 0.033 mol) of succinic acid dichloride in 35 ml of dry toluene is boiled for 10.5 hours At the end of the reaction, toluene is distilled off, 30 ml of water are added to the residue. The precipitate formed is filtered off, washed repeatedly with water. Ib is dried to constant weight at 80-85 ° C. White crystalline powder with so pl. 154-156 ° C (from ethyl acetate). The yield of 5-hydroxyadamantan-2-one (IB) succinate was 11.00 g (90%). Rf 0.45.

Found,%: C 69.32; H, 7.07. C ₂₄ H ₃₀ O ₆ .

Calculated,%: C 69.54; H, 7.30;

¹ H NMR (DMSO d ₆ ; δ, ppm): 1.76-2.03 (8H, m, H-Ad), 2.18-2.37 (14H, m, 10H-Ad and 4H - (CH ₂ ) ₂ ; 2.38-2.54 (8H, m, H-Ad).

Pharmacological study of the claimed compounds Ia and Ib.

To assess the pharmacological effects of the claimed compounds, we used basic certified methods recommended by the Pharmacological Committee of the Ministry of Health of the Russian Federation for the study of substances with cerebrovascular and anti-ischemic effects (Guide to the experimental (preclinical) study of substances with pharmacological activity. M., Grif and K, 2012, p. 255, p. 485).

The pharmacological activity of the claimed compounds was studied in comparison with analogues in structure (5-hydroxyadamantan-2-one and succinic acid) and the action of nimodipine. The effect of the claimed compounds on the blood supply to the brain of rats under the conditions of its experimental ischemic lesion, which was modeled using global transient cerebral ischemia, was studied.

The experiments were performed on 90 anesthetized (chloral hydrate 350-400 mg / kg, intraperitoneally) non-linear male rats weighing 250-300 g.

The state of cerebral circulation was assessed using laser Doppler flowmetry in the parietal region of the rat cerebral cortex using an ALF-21 flowmeter from Transonic System Inc. (USA). At the same time, changes in blood pressure were recorded through a polyethylene catheter previously inserted into the femoral artery. Recording of blood flow and blood pressure was performed on a polygraph company "BIOPAK" USA, connected to a personal computer. Global ischemia in rats was caused by a 10-minute occlusion of both common carotid arteries with a simultaneous decrease in blood pressure to 40-50 mm Hg. bloodletting and subsequent reinfusion. The test substances were injected through a polyethylene catheter into the femoral vein of animals.

Statistical data processing was performed using the Statistika 8.0 software package (Statistika Inc., USA). The normality of the distribution was determined using the Shapiro-Wilk test. For further data processing, a non-parametric two-sample sign-ranking Wilcoxon test was used. The average data in the text is presented as a median. The results were considered significant at p≤0.05.

The effect of succinic acid esters of 5-hydroxyadamantan-2-one and their analogues on the cerebral circulation of rats that underwent global transient ischemia.

Compound Ib at a dose of 100 mg / kg when administered intravenously to anesthetized rats under conditions of global transient cerebral ischemia causes a slowly increasing increase in cerebral blood flow. Changes in blood flow after administration of the compound are observed from 10 minutes, then gradually increase, reaching a maximum in the 70th minute - 47.6%. The level of blood pressure under the influence of IB in these experiments did not statistically significantly change. It should be noted that IB has a selective vasodilating effect on the tone of the vessels of the ischemic brain. A study of the acute toxicity of Ib, which was carried out on awake rats after intravenous administration, showed that the LD _{50 of} this compound is 210.0 (165.9 ÷ 254.1) mg / kg.

Compound Ia at a dose of 100 mg / kg in anesthetized rats under conditions of global transient cerebral ischemia 10 minutes after administration causes an increase in local blood flow in the cortex by an average of 25%. The vasodilating effect of this derivative increases with time and reaches a maximum by 80 minutes, amounting to 59.3%. The level of blood pressure under the influence of Ia in these experiments immediately after administration increases statistically significantly on average by 16.5%, after 10 minutes - by 10.6% and after 30 minutes by 5.3%, then decreases to the initial level and remains to end of experiment. Therefore, a prolonged and gradually developing increase in cerebral blood flow is not caused by a hypertensive reaction to the administration of an adamantane derivative, because an increase in pressure is observed at the beginning of the experiment, and the cerebrovascular effect increases by 80 minutes or more, and blood pressure corresponds to the control level.

A study of acute toxicity Ia, which was carried out on awake rats after intravenous administration, showed that the LD _{50 of} this compound is 740.0 (676.0 ÷ 804.0) mg / kg.

Next, comparison preparations of 5-hydroxyadamantan-2-one, succinic acid and nimodipine were studied.

Experiments have shown that 5-hydroxyadamantan-2-one at a dose of 100 mg / kg when administered intravenously causes a slowly developing increase in local cerebral blood flow, which reaches a maximum value by 60 minutes (76.5%). The blood flow remains above the baseline until the end of the experiment (90 minutes). It should be emphasized here that the level of blood pressure under the influence of this adamantane derivative in rats under conditions of ischemic brain damage decreases by an average of 11%.

Succinic acid at a dose of 100 mg / kg when administered intravenously under conditions of global transient ischemia immediately after administration causes a short-term increase in local cerebral blood flow, which averages 18% during the first 10 minutes. Further, restoration of the initial level of cerebral blood flow is noted, which then continues to decrease gradually, amounting to 15% of the initial level by 50-60 minutes. In the first minutes after administration of succinic acid, there is an increase in blood pressure by an average of 11%, by 10 minutes after administration of the drug, blood pressure is restored, which then gradually decreases. Consequently, parallel changes in local cerebral blood flow and blood pressure are observed, which indicates the absence of a direct effect of succinic acid on the tone of the vessels of the brain.

Nimodipine (0.03 mg / kg) in animals after global transient cerebral ischemia immediately after administration briefly increases local cerebral blood flow by an average of 44.3%. Then, blood flow gradually decreases, but remains elevated until the end of the experiment (14%). The drug in animals under these conditions causes a decrease in blood pressure by an average of 39.6%.

Thus, compound Ia significantly and continuously enhances the blood supply to the ischemic brain, without lowering the level of blood pressure, which compares favorably with its analogues both in structure and effect. It should also be noted that compound Ia is less toxic than compound IB.

Study of the mechanism of cerebrovascular effect of compounds Ia and IB

Further, an analysis of the cerebrovascular mechanism of 5-hydroxyadamantan-2-one succinic acid esters was performed. Since GABAergic mechanisms play an important role in the regulation of vascular tone of the brain vessels, it can be assumed that these mechanisms also take part in the implementation of the cerebrovascular anti-ischemic activity of the claimed compounds.

We studied the effect of compound Ia (100 mg /, intravenously) on the local cerebral blood flow in the cerebral cortex of rats after global transient ischemia and on the background of the action of the GABA _A blocker of bicucullin receptors (50 μg / kg, intravenously, Sigma). The experiments showed that under these conditions, compound Ia causes a slight and short-term (3.7% at 30 minutes) increase in blood flow in the cortex. It should be noted that in control experiments, this adamantane derivative causes a prolonged increase in blood flow, which by the 90th minute is 53.9% of the initial level. The blood pressure level in these experiments immediately after the administration of Ia begins to decrease and by 60 minutes is 10.9% of the initial level. In control experiments, blood pressure under the influence of compound Ia first slightly (by 5.6%) rises, and then decreases to the initial level.

A study of the effect of IB on the local blood flow in the cerebral cortex of rats after global transient ischemia and bicucullin showed that, under these conditions, compound IB, in contrast to control experiments, does not cause an increase in cerebral blood flow, i.e. the cerebrovascular effect of the compound against the background of the action of bicucullin is completely blocked. The level of blood pressure in these experiments immediately after the administration of Ib slightly decreases and by 60 minutes is 9.7% of the initial state. The data obtained indicate the participation of the GABAergic mechanism in the implementation of the cerebrovascular effect of the claimed compounds.

Further, in order to clarify the mechanism of the GABAergic component in the implementation of the cerebrovascular effect of the claimed compounds by in vitro radioligand analysis, the effect of Ia and Ib and 5-hydroxyadamantan-2-one on the interaction with GABA _A receptors of rat brain membranes using specific ligand binding was studied [ ³ H] -SR 95531 (gabazina). Radioreceptor analysis of the effect of substances on specific binding was carried out according to modified methods published previously [R.S. Mirzoyan, T.S. Ganshina, A.V. Gnezdilova, G.I. Kovalev, Yu.Yu. Firstova, V.V. Bezuglov, N.M. Greek. GABAergic component in the mechanism of cerebrovascular anti-ischemic effect of docosahexaenoic acid. Experimental and Clinical Pharmacology, 2015, 78, 1, 16-20]. For the preparation of membrane preparations containing GABA _A receptors in the frontal cortex of rats, 15 non-linear male rats were used. The radioactivity of each sample was measured on a Tri-Carb 2900 TR scintillation counter (PerkinElmer) with a counting efficiency of 42-45%. The results were processed using GraphPad 5.0 Prizm software.

The experiments showed that compounds Ia and Ib, as well as 5-hydroxyadamantan-2-one, do not interact with the GABA _A receptors of the cerebral cortex. However, without data on the identity of GABA _A receptors located in the nerve tissue and blood vessels of the brain, we cannot argue that the cerebrovascular anti-ischemic effect of the studied substances, detected in experiments in vivo, is not due to a direct effect on cerebral receptors of the same name. It can also be assumed that the observed effect in in vivo experiments is mediated either through other receptor systems or is due to the direct effect of substances on cell membranes. Additional research is needed to resolve this issue.

The results of a pharmacological study of the claimed compounds showed that compound Ia and, to a lesser extent, compound Ib improve blood supply to the brain in rats subjected to global transient ischemia. It was also shown that compound Ia has less toxicity than compound Ib. In in vivo experiments, the GABAergic mechanism of the cerebrovascular effect of these substances was revealed.

Thus, compound Ia has significant cerebrovascular anti-ischemic activity and, unlike 5-hydroxyadamantan-2-one and nimodipine, does not lower, and even slightly increases the level of blood pressure during the first 30 minutes. It should be emphasized that the ability of drugs to lower blood pressure, i.e. to weaken cerebrovascular perfusion pressure is an essential factor limiting their appointment for the treatment of patients in the acute phase of ischemic stroke.

Claims (3)

5-hydroxyadamantan-2-one succinic acid esters that improve blood supply to the ischemic brain, general formula

Ia, b, where: Ia R = H; Ib R = 5-adamantan-2-one.