RU2621348C1 - Application of adamantane indole and their hydrochlorides as cholinesterase inhibitors and nmda-receptors blockers - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to application of adamantane-containing indoles and their hydrochlorides of general formula

where R₁, R₂, R₃, R₄ may be the same or different and independently represent H, F, Cl, Br, (C₁-C₆) alkyl, (C₁-C₆) alkoxy; R₅, R₆ may be the same or different and independently represent H, (C₁-C₆) alkyl, ONO₂; X represents CH₂CH(OH)CH₂ or CH₂CH₂C(O); Z=no, Cl; R₇, R₈, R₉, R₁₀ may be the same or different and independently represent H, F, Cl, Br, (C₁-C₆) alkyl, (C₁-C₆) alkoxy; R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, R₁₈ may be the same or different and independently represent H, (C₁-C₆) alkyl, as a cholinesterase inhibitors and NMDA receptors blockers, and can be used as new drugs that interact with several targets responsible for improval of memory and cognitive functions, weakening of which has various origin, including dementias of various origins, especially due to neurodegenerative diseases, such as Alzheimer's disease.

EFFECT: increased compound application effeciency.

15 cl, 1 dwg, 1 tbl, 3 ex

Description

The invention relates to the use of chemical compounds in medicine and can be used in the manufacture of drugs to improve memory and cognitive functions, which are impaired, including in dementia of various origins, especially due to neurodegenerative diseases such as Alzheimer's disease (AD).

Studies of the last decade have shown that impaired memory and cognitive functions, like some other neuropathological disorders, are closely associated with dysfunction in cholinergic and glutamatergic neurotransmitter systems [Mufson, E. J., Counts, S. E., Perez, S. E. & Ginsberg, SD Cholinergic system during the progression of Alzheimer's disease: therapeutic implications. Expert review of neurotherapeutics 2008, 8, 1703-1718; Cacabelos, R., Takeda, M. & Winblad, B. The glutamatergic system and neurodegeneration in dementia: preventive strategies in Alzheimer's disease. International journal of geriatric psychiatry 14, 3-47 (1999)].

A promising modern approach to the treatment of complex, multifactorial diseases, which include neurodegenerative disorders, including Alzheimer's disease (AD), is the creation of multi-target ligands that interact with several targets responsible for pathogenesis.

Deficiency of central acetylcholine plays an important role in the development of impaired memory and cognitive dysfunctions - the main symptom of Alzheimer's disease (AD) and dementia of a different origin. The use of acetylcholinesterase inhibitors (AChE), the main enzyme that hydrolyzes the neurotransmitter acetylcholine, increases the concentration of acetylcholine and leads to moderate improvements in memory and cognitive functions of patients with AD [Wilkinson, DG, Francis, R. T., Schwam, E. & Payne-Parrish , J. Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between pharmacological effects and clinical efficacy. Drugs & aging, 2004, 21, 453-478].

These compounds, however, cause unwanted side effects such as nausea and vomiting. Inhibition of butyrylcholinesterase (BChE) also contributes to the improvement of memory and cognitive function in dementia of various origins [Martinez A, Castro A. Novel cholinesterase inhibitors as future effective drugs for thetreatment of Alzheimer's disease. Expert Opin. Investig. Drugs, 2006, 15, 1-12], Inhibition of BChE is especially productive in the progression of diseases, when the activity of AChE is reduced and its function in the hydrolysis of acetylcholine is taken by BChE [Lane, R. M., Potkin, SG & Enz, A. Targeting acetylcholinesterase and butyrylcholinesterase in dementia. Int J Neuropsychopharmacol. 2006, 9, 101-124]. Selective BChE inhibitors, as shown by recent studies, increase the level of acetylcholine in the brain and improve cognitive function in rodents, without having the classic side effects of AChE inhibitors [Greig NH, Utsuki T, Ingram DK, Wang Y, Pepeu G, Scali C, Yu QS, Mamczarz J, Holloway HW, Giordano T, Chen D, Furukawa K,

Sambamurti K, Brossi A, Lahiri DK. Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer beta-amyloid peptide in rodent. Proc. Natl. Acad. Sci. USA, 2005, 102 (47), 17213-17218].

It is known that some indole-containing compounds (carbazoles, gamma-carbolines) have the ability to inhibit cholinesterase, while their activity and selectivity depend significantly on the structure of the substituents and the structure of the compound as a whole [Kumar NS, Mukherjee PK, Bhadra S, Saha BP, Pal Sun. Acetylcholinesterase inhibitory potential of a carbazole alkaloid, mahanimbine, from Murraya koenigii. Phytother. Res. 2010, 24 (4), 629-631; Akrami H, Mirjalili BF, Khoobi M, Moradi A, Nadri H, Emami S, Foroumadi A, Vosooghi M, Shafiee A. 9H-Carbazole Derivatives Containing the N-Benzyl-1,2,3-triazole Moiety as New Acetylcholinesterase Inhibitors. Arch Pharm (Weinheim). 2015, 348 (5), 366-374].

However, these compounds have insufficient therapeutic effect and have not found their application for therapeutic purposes.

Piperidine derivative donepezil (arisept), rivastigmine carbamate (exelon) and galantamine alkaloid [Wilkinson DG, Francis PT, Schwam, E., Payne-Parrish, currently used as compounds with the ability to inhibit cholinesterase in the treatment of AD are currently used. J. Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between pharmacological effects and clinical efficacy. Drugs & aging 2004, 21, 453-478].

At the same time, modern approaches to the treatment of memory impairment and cognitive impairment require the use of drugs with a wider spectrum of action, taking into account the multifactorial nature of such diseases. In addition, one of the drawbacks of cholinesterase inhibitors used to treat AD is their ability to inhibit carboxyl esterase (CE), which hydrolyzes numerous drugs with ester groups, which leads to undesirable drug interactions. [Tsurkan L.G., Hatfield M.J., Edwards C.S., Hyatt J.L., Potter P.M. Inhibition of human carboxylesterases hCE1 and hiCE by cholinesterase inhibitors. Chem. Biol. Interact 2013, 203, 226-230].

Known use in clinical practice as a medicine for improving memory and cognitive functions in the treatment of Alzheimer's disease is NMDA receptor blockers, which is used as memantine, belonging to the group of adamantane-containing compounds. [Lipton SA. Paradigm shift in neuroprotection by NMDA receptor blockade: memantine and beyond. Nat Rev Drug Discov. 2006, 5 (2), 160-170]

However, memantine interacts only with the MK-801 NMDA receptor binding site, which also does not meet modern requirements for the use of drugs with a wider spectrum of activity, taking into account the multifactorial nature of such diseases.

The present invention solves the problem of expanding the arsenal of multi-targeted drugs that can be used as new drugs that combine the properties of cholinesterase inhibitors and NMDA receptor blockers that interact with several targets responsible for improving memory and cognitive functions, the weakening of which has a different origin.

The present invention relates to the use, as cholinesterase inhibitors and NMDA receptor blockers, of an adamantane-containing indole or its hydrochloride, of the general formula 1,

in which R ₁ , R ₂ , R ₃ , R ₄ : may be the same or different and independently represent H, F, O, Br, (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy;

R ₅ , R ₆ may be the same or different and independently represent H, (C ₁ -C ₆ ) alkyl, ONO ₂ ;

X represents CH ₂ CH (OH) CH ₂ or CH ₂ CH ₂ C (O);

Z = no, Cl;

R ₇ , R ₈ , R ₉ , R ₁₀ may be the same or different and independently represent H, F, O, Br, (C ₁ -C ₆ ) alkyl, C ₁ -C ₆ ) alkoxy;

R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ may be the same or different and independently represent H, (C ₁ -C ₆ ) alkyl.

Among the compounds of formula 1, the following 2 groups of compounds, which can be represented by formulas 1.1 and 1.2 below, are preferred as cholinesterase inhibitors and NMDA receptor blockers.

In particular, the first group of preferred compounds of formula 1 are adamantane-containing indole or its hydrochloride of the general formula 1.1,

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, Y have the meanings defined above for formula 1.

Within the framework of adamantane-containing indoles and their hydrochlorides of group 1.1, adamantane-containing carbazole or its hydrochloride of the general formula 1.1.1 are most preferred as cholinesterase inhibitors and NMDA receptor blockers

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R, Z, R ₇ , R ₈ , R ₉ , R ₁₀ have the meanings defined above for formula 1.

Within subgroup 1.1.1, the most preferred are

1- (adamantan-1-ylamino) -3-carbazol-9-yl-propan-2-ol - compound 1;

1- (adamantan-1-ylamino) -3- (3,6-dibromo-carbazol-9-yl) -propan-2-ol - compound 2;

1- (adamantan-1-ylamino) -3- (3,6-dichloro-carbazol-9-yl) -propan-2-ol compound 3;

3- (3,5-dimethyladamantan-1-ylamino) -1-carbazol-9-yl) propan-2-ol compound 4;

1- (3,6-dibromocarbazol-9-yl) -3- (3,5-dimethyladamantan-1-ylamino) propan-2-ol - compound 5

3- (3,5-dimethyladamantan-1-ylamino) -1- (3,6-dichlorocarbazol-9-yl) propan-2-ol - compound 6;

1- (adamantan-1-ylamino) -3-carbazol-9-yl) propan-2-ol hydrochloride - compound 7;

1- (adamantan-1-ylamino) -3- (3,6-dibromo-carbazol-9-yl) -propan-2-ol hydrochloride - compound 8;

1- (adamantan-1-ylamino) -3- (3,6-dichloro-carbazol-9-yl) -propan-2-ol hydrochloride - compound 9;

3- (3,5-dimethyladamantan-1-ylamino) -1-carbazol-9-yl) propan-2-ol hydrochloride — compound 10;

1- (3,6-dibromocarbazol-9-yl) -3- (3,5-dimethyladamantan-1-ylamino) propan-2-ol hydrochloride - compound 11;

3- (3,5-Dimethyladamantan-1-ylamino) -1- (3,6-dichlorocarbazol-9-yl) -propan-2-ol hydrochloride - compound 12.

The second subgroup of the most preferred as cholinesterase inhibitors and NMDA receptor blockers are adamantane-containing tetrahydrocarbazole or its hydrochloride of the general formula 1.1.2,

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ have the meanings given above for formula 1

Preferred compounds of this subgroup are:

1- (adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 13;

1- (3,5-dimethyl-adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 14;

1- (adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 15;

1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 16;

1- (adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 17;

1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 18;

1- (adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 19;

1- (3,6-dimethyl-adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol - compound 20;

1- (adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) -propan-2-ol hydrochloride - compound 21;

1- (3,5-dimethyl-adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride - compound 22;

1- (adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride - compound 23;

1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride — compound 24;

1- (adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride — compound 25;

1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride - compound 26;

1- (adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride - compound 27;

1- (3,6-dimethyl-adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride - compound 28;

A third subgroup of compounds of formula 1.1, the most preferred as cholinesterase inhibitors and NMDA receptor blockers are adamantane-containing gamma-carboline or its hydrochloride of the general formula 1.1.3,

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ have the meanings defined above for formula 1.

Preferred compounds of this subgroup are:

1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) propan-2-ol - compound 29 ;

1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) -propan-2-ol hydrochloride - compound thirty.

A second group of preferred compounds of formula 1 are adamantane-containing indole or its hydrochloride salt of the general formula 1.2,

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, Y have the meanings defined above for formula 1.

Among the compounds of formula 1.2, the following 3 subgroups of compounds, which can be represented by formulas 1.2.1, 1.2.2, and 1.2.3 below, are preferred as cholinesterase inhibitors and NMDA receptor blockers.

The first subgroup of preferred adamantane-containing indoles of group 1.2 are adamantane-containing carbazole of the general formula 1.2.1,

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ have the meanings defined above for formula 1.

Preferred compounds of this subgroup are:

N- (adamantan-1-yl) -3-carbazol-9-yl-propionamide - compound 31;

N- (3,5-Dimethyl-adamantan-1-yl-3-carbazol-9-yl) -propionamide - compound 32.

The second subgroup of adamantane-containing indoles of group 1.2, preferred as cholinesterase inhibitors and NMDA receptor blockers, is adamantane-containing carbazole of the general formula 1.2.2,

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ have the meanings defined above for formula 1.

A preferred compound as cholinesterase inhibitors and NMDA receptor blockers of formula 1.2.2 is

N-adamantan-1-yl-3- (3,4-dihydro-1H-carbazol-9-yl-propionamide - compound 33.

The third subgroup of adamantane-containing indoles of group 1.2, which are preferred as cholinesterase inhibitors and NMDA receptor blockers, is adamantane-containing carbazole of the general formula 1.2.3.

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ have the meanings defined above for formula 1.

Preferably one of the following compounds is used as cholinesterase inhibitors and NMDA receptor blockers of formula 1.2.3

1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) propionamide - compound 34;

1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) propionamide hydrochloride - compound 35.

Thus, the technical effect of the proposed group of compounds is the discovery of the properties of multi-target drugs in the known compounds, combining the properties of butyrylcholinesterase inhibitors with an IC _{50 of the} order of 10 ^-6 M (IC ₅₀ is the concentration of the compound, which is required to reduce the enzyme activity by 50%) with a weak inhibition of acetylcholinesterase, the same order blockers of NMDA receptors with IC ₅₀ values (IC ₅₀ - the concentration of compound required to reduce the binding of labeled ligand to the receptor by 50%), that n It allows one to use them as drugs, significantly enhancing memory and cognitive function.

It was previously shown that the compounds to which the compounds of formula 1 belong have both the property of stabilizing microtubules and can be used to treat and prevent diseases associated with disorders of the microtubule system (patent application No. 2015146079, priority from 10.27.15), and cytoprotection property and can be intended for the treatment and prevention of diseases associated with an increase in the cytosolic concentration of calcium (application for invention No. 2015146080, priority from 10.27.15)

As a result of biological tests, a new, previously unknown property of compounds of the general formula 1 was discovered: the ability to inhibit cholinesterase (AChE and BChE) and block NMDA receptors (both MK-801 and the site of binding of ifenprodil), which does not follow from the structure of the compound itself.

The rational design strategy of multi-target drugs is based on linking two pharmacophores that are active against two different molecular targets via a linker [Youdim M.V., Buccafusco J.J. Multi-functional drugs for various CNS targets in the treatment of neurodegenerative disorders. Trends Pharmacol. Sci. 2005, 26, 27-35; Zhang H.Y. One-compound-multiple-targets strategy to combat Alzheimer's disease. FEBS Lett. 2005, 579, 5260-5264].

However, the selection of pairs of pharmacophores is not predictable and depends on the structure of the new molecule as a whole. Preservation of the properties of both constituent parts in a new molecule depends both on the structure of each of the parts and on the structure of the linker.

Thus, the discovery of new unexpected properties of the compounds of formula 1 gives the entire invention compliance with the criteria of "novelty" and "inventive step". The synthesis of compounds 1 is carried out by known methods described in applications for the invention No. 2015146079 and No. 2015146080, which gives the entire invention the criterion of "industrial applicability".

Table 1 shows the data characterizing the inhibitory activity of conjugates of the general formula 1 with respect to AChE, BChE, and CE and their effect on the binding of labeled [3H] MK-801 and [3H] ifenprodil ligands to different sites of the NMDA receptor.

In FIG. Figure 1 shows the dependences of the inhibitory activity of compound 10 with respect to BChE on the substrate concentration obtained for 3 increasing concentrations of the inhibitor.

Derivatives of adamantane-substituted substituted indoles of the general formula 1 possessing the claimed properties can be obtained (application for invention No. 2015146079, priority of 10.27.15) by combining indole and aminoadamantane moieties corresponding to the claimed composition through the claimed molecular spacer (conjugation). The process of obtaining compounds of general formula 1.1 is carried out by heating an equimole mixture of the corresponding substituted indoles and aminoadamantanes in a solvent (ethanol, propanol, iso-propanol) at a temperature of 79-90 ° C for 2-6 hours. To obtain adamantane-containing indole hydrochlorides, the corresponding adamantane-containing indoles are dissolved in a 10% solution of HCl in isopropanol.

The process of obtaining compounds of general formula 1.2 is carried out by heating an equimole mixture of the corresponding substituted indoles and aminoadamantanes in dimethyl sulfoxide or dimethyl foramide in the presence of a catalyst selected from the series: sodium methylate, potassium tert-butylate, potassium hydroxide or cesium fluoride cesium, at a temperature of 100-140 ° C for 2 -12 hours.

The following are the results of biological tests of compounds of general formula 1, which illustrate but do not exhaust the present invention.

Example 1. Determination of the inhibitory activity of the compounds of General formula 1 in relation to acetylcholinesterase, butyrylcholinesterase and carboxyl esterase.

Enzyme preparations: For kinetic studies, commercial preparations (Sigma, USA) of human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), horse serum butyrylcholinesterase (BChE, EC 3.1.1.8) and porcine liver carboxyl esterase (EC, EC 3.1.1.1) are used ), which has high homology, a similar structure of the active center, and close inhibitory specificity with human liver carboxyl esterase hCE-1 [Satoh T., Hosokawa M. The mammalian carboxylesterases: from molecules to functions. Annu. Rev. Pharmacol Toxicol. 1998, 38, 257-288].

Determination of enzyme activity: The activity of AChE and BChE is determined by the Ellman method (λ 412 nm) [Ellman GL et al. A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol 1961, 7, 88-95] using acetylthiocholine (1 mM) and butyrylthiocholine (1 mM) as a substrate, respectively; determination conditions: 0.1 M phosphate buffer pH 7.5, 25 ° C. CE activity is determined spectrophotometrically by the release of 4-nitrophenol (λ 405 nm), the substrate is 4-nitrophenyl acetate (1 mM); determination conditions: 0.1 M phosphate buffer pH 8.0, 25 ° C [Sterri SH et al. A radiochemical assay method for carboxylesterase, and comparison of enzyme activity towards the substrates methyl [1- ¹⁴ C] butyrate and 4-nitrophenyl butyrate, Biochem. Pharmacol 1985, 34 (15), 2779-2785]. Measurements are carried out on a FLUOStar Optima microplate spectrophotometer (Labtech, Germany).

Determination of IC ₅₀ values for inhibition of AChE, BChE and CE.

An initial assessment of the inhibitory activity of compounds is carried out by determining the degree of inhibition of enzymes at a concentration of 20 μM, then for the most active compounds, IC ₅₀ values are determined - the concentration of the compound, which is required to reduce enzyme activity by 50%. To determine the IC ₅₀ inhibition of AChE, BChE, and CE, a sample of the corresponding enzyme is incubated with the test compound (DMSO concentration 2 vol%) for 10 minutes, then the residual enzyme activity is determined. The concentration range of the test compound is 1 × 10 ^-12 - 1 × 10 ^-4 M. Each experiment is carried out in triplicate. The measurements are carried out in 96-well plates on a FLUOStar Optima microplate spectrophotometer (BMG Labtech, Germany). The calculation of the IC _{50 is} carried out using the program Origin 6.1. (see Table 1).

As can be seen from Table 1, both the compounds of group 1.1 and the compounds of group 1.2 demonstrate high selectivity for BChE compared to AChE. Data on the weak inhibitory activity of the compounds against carboxyl esterase, which hydrolyzes many drugs containing ester groups [Imai T. Human carboxylesterase isozymes: catalytic properties and rational drug design. Drug Metab Pharmacokinet. 2006, 21 (3), 173-185], indicate the absence of undesirable drug interactions in the therapeutic use of the claimed compounds.

Example 2. The determination of the mechanism of inhibition of BChE by the compound of General formula 1 and the calculation of the kinetic constants of inhibition.

To determine the inhibition mechanism, the dependence of the residual activity of BChE on the concentration of the inhibitor at various substrate concentrations is determined. Data is analyzed in the double inverse coordinates of Lineuiver-Burke. The inhibition mechanism is determined from the analysis of the dependences 1 / V = f (1 / S) obtained for three increasing concentrations of the inhibitor. The calculation of the kinetic constants of inhibition K _i (competitive component) and αK _i (non-competitive component) is carried out using the Origin 6.1 program.

The intersection of the direct dependences 1 / V = f (1 / S) for compound 10 (Fig. 1) obtained for different concentrations of the inhibitor in the upper left quadrant indicates a mixed type of inhibition of BChE by the test compound. The inhibition constants of BChE by compound 10 were determined, which are K _i = 2.11 ± 0.17 μM (competitive component) and αK _i = 8.48 ± 0.38 μM (non-competitive component).

Example 3. Determination of the binding of compounds of General formula 1 to NMDA receptors.

Radioactive ligands: Two radioactive ligands were used: [ ³ H] MK-801 (disocilpin) with a specific activity of 210 Ci / mol, binding to all isolated NMDA receptors, and [ ³ H] ifenprodil with a specific activity of 79 Ci / mol, binding only to NMDA receptors containing NR2B subunits (labeled ligands obtained from the Institute of Molecular Genetics, Russian Academy of Sciences).

The method of preparation of the preparation of the membrane of the brain for radioligand analysis.

A membrane preparation for radioligand analysis was prepared according to the known method [Nowak G., Trullas R., Layer RT, Skolnick P., Paul IA Adaptive changes in the N-methyl-D-aspartate receptor complex after chronic treatment with imipramine and 1-aminocyclopropanecarboxylic acid . J. Pharmacol. Exp. Ther. 1993, 265, 1380-1386]. The hippocampal tissue was pulverized in a Potter homogenizer (Teflon-glass) in buffer No. 1 (5 mM HEPES / 4.5 mM Tris buffer, pH 7.6) containing 0.32 M sucrose in a ratio of 1 g of tissue: 10 ml of buffer. The homogenate was diluted with assay buffer No. 2 (5 mM HEPES / 4.5 mM Tris buffer, pH 7.6) at a ratio of 1:50 and centrifuged for 10 min at 1000 g. The supernatant was then taken and centrifuged again for 20 min at 25,000 g. The pellet was homogenized in buffer No. 2 at a ratio of 1:50 and centrifuged for 20 min at 8000 g. The supernatant and its soft, unsteady supernatant were collected and centrifuged for 20 min at 25,000 g. The resulting pellet was suspended in buffer No. 3 (5 mM HEPES / 4.5 mM Tris buffer, pH 7.61, 1 mM Na ₄ EDTA), and the suspension was again centrifuged. This washing procedure is carried out four times, and with the last washing of Na ₄ EDTA is excluded from the composition. The final pellet was resuspended in buffer No. 2 in a ratio of 1: 5 and stored in liquid nitrogen. The reaction mixture (final volume 0.5 ml) contains 200 μl of buffer No. 2, 50 μl of 50 nM solution of labeled ligand and 250 μl of protein suspension. Nonspecific binding is determined in the presence of 50 μl of 1 M unlabeled ligand.

Researching

Radioligand binding of labeled compounds with preparations of brain membranes was carried out according to the methods of [Zhou L.M., Gu Z.Q., Costa A.M., Yamada K.A., Mansson P.E., Giordano T., Skolnick P., Jones K.A. (2S, 4R) -4-methylglutamic acid (SYM 2081): a selective, high-affinity ligand for kainate receptors. J. Pharmacol. Exp. Ther. 1997, 280, 422-427; Coughenour L.L., Barr B.M. Use of trifluoroperazine isolates a [(3) H] Ifenprodil binding site in rat brain membranes with the pharmacology of the voltage-independent ifenprodil site on N-methyl-D-aspartate receptors containing NR2B subunits. J. Pharmacol. Exp. Ther. 2001, 296, 150-159].

The reaction mixture is incubated at room temperature for 2 hours. At the end of the incubation, the samples are filtered through GF / B glass fibers (Whatman), pre-soaked in 0.3% polyethyleneamine for 2 hours at 4 ° C. Each tube is washed once with cold buffer No. 2, then the filters are washed three times with the same volume of buffer. The filters are dried in air until completely dry and transferred to scintillation vials, to which 5 ml of scintillation liquid containing 4 g of diphenyloxazole (PPO), 0.2 g of diphenyloxazoylbenzene (POPOP) and 1 l of toluene are added. Radioactivity was determined on a TriCarb2800 TR scintillation counter (PerkinElmer, Packard, USA) with a counting efficiency of about 65%.

The effect of the studied compounds on the binding of [ ³ H] MK-801 or [ ³ H] ifenprodil to rat hippocampal membranes was characterized by IC ₅₀ values. The determination of IC ₅₀ (the concentration of the compound, which is required to reduce the binding of the labeled ligand to the receptor by 50%) was carried out by adding 50 μl of the test compounds to the incubation medium in a concentration range of 10 ^-7 -10 ^-3 M. IC _{50 was} calculated for the studied connections using GraphPadPrism 4 Demo (see Table 1).

In cases where the inhibition of the investigated substances at a concentration of 100 μM did not exceed 50%, the value of the IC ₅₀ value was not determined (n / a).

Table 1 presents data on the blockade of the compounds of the ifenprodyl site and the MK-801 binding site of NMDA receptors.

As can be seen from Table 1, both the compounds of group 1.1 and the compounds of group 1.2 possess, in addition to the blockade ability of the MK-801 binding site, known from the memantine compound belonging to the adamantane group, an additional ability to more effectively block the binding site of ifenprodil NMDA receptors, which enhances cognitively stimulating the properties of these compounds compared to compounds that only block the MK-801 binding site [Higgins GA, Ballard T.M., Enderlin M., Haman M., Kemp JA Evidence for improved performance in cognitive tasks following selective NR2B NMDA receptor antagonist pre-treatment in the rat. Psychopharmacology (Berl). 2005, 179 (1), 85-98].

Thus, the above examples confirm the achievement of the claimed effect — the discovery in adamantane-containing indoles and their hydrochlorides, of general formula 1, of advantages over the known means that improve memory and cognitive functions due to the simultaneous exposure to several targets responsible for this purpose. When using these compounds, the effect is achieved by two mechanisms - by inhibiting cholinesterase and additional blockade of NMDA receptors, by binding them both to the MK-801 site and to the ifenprodil site. Moreover, they mainly inhibit BChE, i.e. should not cause side effects characteristic of AChE inhibitors and, weakly inhibiting carboxyl esterase, exclude undesirable drug interactions in the therapeutic use of the claimed compounds, which should lead to an increase in the therapeutic effect and increase their safety.

Claims (75)

1. The use of adamantane-containing indoles or their hydrochlorides of the general formula 1 as cholinesterase inhibitors and NMDA receptor blockers

wherein R ₁ , R ₂ , R ₃ , R ₄ may be the same or different and independently represent H, F, Cl, Br, (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy; R ₅ , R ₆ may be the same or different and independently represent H, (C ₁ -C ₆ ) alkyl, ONO ₂ ; X represents CH ₂ CH (OH) CH ₂ or CH ₂ CH ₂ C (O); Z = no, Cl;

where R ₇ , R ₈ , R ₉ , R ₁₀ may be the same or different and independently represent H, F, Cl, Br, (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy; R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ may be the same or different and independently represent H, (C ₁ -C ₆ ) alkyl. 2. The use of compounds according to claim 1, in which adamantane-containing indoles and their hydrochlorides of the general formula 1.1 are taken as cholinesterase inhibitors and NMDA receptor blockers of formula 1

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, Y have the meanings defined above for formula 1. 3. The use of compounds according to claim 2, in which adamantane-containing carbazoles and their hydrochlorides of the general formula 1.1.1 are taken as cholinesterase inhibitors and NMDA receptor blockers of formula 1.1

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, R ₇ , R ₈ , R ₉ , R ₁₀ have the meanings defined above for formula 1. 4. The use of compounds according to claim 3, in which one of the following compounds is used as cholinesterase inhibitors and NMDA receptor blockers of formula 1.1.1: 1- (adamantan-1-ylamino) -3-carbazol-9-yl-propan-2-ol; 1- (adamantan-1-ylamino) -3- (3,6-dibromo-carbazol-9-yl) -propan-2-ol; 1- (adamantan-1-ylamino) -3- (3,6-dichloro-carbazol-9-yl) -propan-2-ol; 3- (3,5-dimethyladamantan-1-ylamino) -1-carbazol-9-yl-propan-2-ol; 1- (3,6-dibromocarbazol-9-yl) -3- (3,5-dimethyladamantan-1-ylamino) propan-2-ol; 3- (3,5-dimethyladamantan-1-ylamino) -1- (3,6-dichlorocarbazol-9-yl) propan-2-ol; 1- (adamantan-1-ylamino) -3-carbazol-9-yl-propan-2-ol hydrochloride; 1- (adamantan-1-ylamino) -3- (3,6-dibromo-carbazol-9-yl) -propan-2-ol hydrochloride; 1- (adamantan-1-ylamino) -3- (3,6-dichloro-carbazol-9-yl) -propan-2-ol hydrochloride; 3- (3,5-dimethyladamantan-1-ylamino) -1-carbazol-9-yl-propan-2-ol hydrochloride; 1- (3,6-dibromocarbazol-9-yl) -3- (3,5-dimethyladamantan-1-ylamino) propan-2-ol hydrochloride; 3- (3,5-dimethyladamantan-1-ylamino) -1- (3,6-dichlorocarbazol-9-yl) propan-2-ol hydrochloride. 5. The use of compounds according to claim 2, in which adamantane-containing tetrahydrocarbazoles and their hydrochlorides of the general formula 1.1.2 are taken as cholinesterase inhibitors and NMDA receptor blockers of formula 1.1

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ have the meanings defined above for formula 1. 6. The use of compounds according to claim 5, in which one of the following compounds is taken as inhibitors of cholinesterases and blockers of NMDA receptors of formula 1.1.2: 1- (adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (3,5-dimethyl-adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (3,6-dimethyl-adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol; 1- (adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride; 1- (3,5-dimethyl-adamantan-1-ylamino) -3- (1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride; 1- (adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride; 1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-methyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride; 1- (adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride; 1- (3,6-dimethyl-adamantan-1-ylamino) -3- (6-fluoro-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride; 1- (adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride; 1- (3,6-dimethyl-adamantan-1-ylamino) -3- (3,6-dimethyl-1,2,3,4-tetrahydrocarbazol-9-yl) propan-2-ol hydrochloride. 7. The use of compounds according to claim 2, in which adamantane-containing gamma-carbolines and their hydrochlorides of the general formula 1.1.3 are taken as cholinesterase inhibitors and blockers of NMDA receptors of formula 1.1

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ have the meanings defined above for formula 1. 8. The use of compounds according to claim 7, in which one of the following compounds is used as inhibitors of cholinesterases and blockers of NMDA receptors of formula 1.1.3: 1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) propan-2-ol; 1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) propan-2-ol hydrochloride. 9. The use of compounds according to claim 1, in which adamantane-containing indoles and their hydrochlorides of formula 1.2 are taken as inhibitors of cholinesterases and blockers of NMDA receptors of formula 1

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , Z, Y have the meanings defined above for formula 1. 10. The use of compounds according to claim 9, in which adamantane-containing carbazoles of the general formula 1.2.1 are taken as cholinesterase inhibitors and NMDA receptor blockers of formula 1.2

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ have the meanings defined above for formula 1. 11. The use of the compounds of claim 10, wherein one of the following compounds is used as cholinesterase inhibitors and NMDA receptor blockers of formula 1.2.1: N-adamantan-1-yl-3-carbazol-9-yl-propionamide; N- (3,5-Dimethyl-adamantan-1-yl-3-carbazol-9-yl) propionamide. 12. The use of compounds according to claim 9, in which adamantane-containing tetrahydrocarbazoles of the general formula 1.2.2 are taken as cholinesterase inhibitors and NMDA receptor blockers of formula 1.2

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ have the meanings defined above for formula 1. 13. The use of compounds according to claim 12, in which, as inhibitors of cholinesterases and blockers of NMDA receptors of the formula 1.2.2 take N-adamantan-1-yl-3- (3,4-dihydro-1H-carbazol-9-yl) propionamide. 14. The use of compounds according to claim 9, in which adamantane-containing gamma-carbolines and their hydrochlorides of the general formula 1.2.3 are taken as cholinesterase inhibitors and NMDA receptor blockers of formula 1.2

in which R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ have the meanings defined above for formula 1. 15. The use of compounds according to claim 14, in which one of the following compounds is used as cholinesterase inhibitors and NMDA receptor blockers of formula 1.2.3: 1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) propionamide; 1- (adamantan-1-yl) -3- (2,8-dimethyl-3,4-dihydro-1H-pyrido [4,5-b] indol-5-yl) propionamide hydrochloride.

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