Pharmaceutical composition, containing indole compound

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical composition, containing compound of formula or for prevention or treatment of diseases, associated with oxidative stress, selected from group, consisting of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke episodes), MERRF syndrome (myoclonic epilepsy with ragged red fibres) or Kearns-Sayre syndrome, arrhythmia, cardioplegia or myocardium infarction. in formula (1) na stands for 1 or 2, Aa represents 5-membered heteroaryl or heterocycle, each of which has 2 heteroatoms, selected from N, O and S, Rla represents R5a-Xa-Ba-X′a-, Ba represents direct bond, Xa and X′a independently on each other represent direct bond or -OC(O)-, R5a represents hydrogen or 6-9-membered monocyclic or condensed cyclic heterocycle or heteroaryl, each of which has from 1 to 3 heteroatoms, selected from N, O and S, and is optionally substituted with oxo or C1-C6-alkyl, R2a represents -(CR8aR9a)pa-Ya-R7a, pa stands for number from 0 or 1, Ya represents direct bond or -O-, R7a represents hydrogen or phenyl, R3a, R8a, R9a, R10a represent hydrogen, R4a represents -(CH2)pa-Da-R10a-, Da represents C5-cycloalkyl or 6-membered heterocycle, which has 1 heteroatom, selected from N, S and O. Radical values for formula (2) are give in invention formula.

EFFECT: obtaining compositions for prevention or treatment of diseases, associated with oxidative stress.

19 dwg, 5 tbl, 3 ex

 

The technical field to which the invention relates

The present invention relates to pharmaceutical compositions containing the indole compound for the prevention or treatment of diseases associated with oxidative stress, mitochondrial dysfunction, hypoxic injury, necrosis and/or ischemic reperfusion injury, and to cosmetic compositions containing the indole compound having antioxidant effect.

The level of technology

Organisms, including the human body, get energy through a process of breath, and about 2% of oxygen absorbed during metabolism, is converted into reactive oxygen species (ROS), known as the “oxygen toxin”. Under the active forms of oxygen means an oxygen-containing free radical (common name of atoms or molecules with an unpaired electron), and usually this form includes lipid peroxide, lipid peroxide radical, peroxynitrite and so on. It is known that such reactive oxygen species are unstable and thus highly reactive to substances in the environment causing oxidative damage of DNA, the carrier of genetic information, and proteins or lipid molecules in the cell, resulting, ultimately, the cell is exposed to fatal damage. With �the other hand, it is concluded that, in these immune system cells like macrophages or neutrophils, production of reactive oxygen species plays a useful role in the destruction of pathogens that penetrate from the outside. Unlike the above cases and the recently confirmed cases in which reactive oxygen species play an important role in signal transduction within the cell, reactive oxygen species, in General, are seen as causing damage to cells and, thus, which is detrimental to organisms. Thus, in order to protect the cells from oxygen toxin, the cells themselves have antioxidants (e.g., vitamin C, vitamin E, small peptides such as glutathione) and antioxidant enzymes (e.g. catalase, superoxide dismutase [SOD], glutathione-dependent peroxidase [GPX], and so on).

Examples of diseases or metabolic processes associated with active forms of oxygen (ROS) or antioxidant enzymes are as follows:

1. Insulin-dependent diabetes develops as a result of damage to the beta cells of the pancreas, which is due to abnormal expression of the ROS.

2. Down's syndrome, which is currently the object of clinical attention, as it is known, is caused by a defect of chromosome 21. In this case, SOD, an antioxidant enzyme, is expressed abnormally.

3. EN�insidently enzyme (catalase, glutathione-dependent peroxidase), determined by analysis in a cell of a patient suffering from progeria, shows low enzymatic activity.

4. Even in the process of transformation of normal cells into cancer cells ROS are actively produced in the cells during the introduction of some cancer-causing substances and exposures.

5. In addition to the above, it is known that ROS are involved in atherosclerosis, Alzheimer's disease, coronary disease and so on.

Some free radicals, reactive oxygen species and peroxides produced during metabolic processes even in normal cells. However, the cells protect themselves from these harmful substances by using antioxidant enzymes such as SOD, catalase, peroxidase, and so forth, as a protective system, together with antioxidants such as vitamin E, vitamin C, glutathione, ubiquinone, uric acid, and so forth. When such protective system has a defect or production of reactive oxygen species exceeds the functional ability of the protective system due to various physical or chemical factors, is induced, however, oxidative stress. If the disease entity is associated with an imbalance between oxidative stress and the antioxidant defense system in the body, theoretically, oxidative damage can be reduced or main�ISEE the progression of the disease can be suppressed by the addition of antioxidant substances. Thus, antioxidant-functional substances, such as scavengers of free radicals, or substances that inhibit the production of peroxides are widely used at present in the polymer, food, cosmetic industries and so on. They can also be used as inhibitory or therapeutic agent in aging and various diseases caused by these oxides, which are based on a recent discovery that reactive oxygen species are involved in physiological phenomena associated with various diseases. Gradually increasing interest in the development of a therapeutic agent with their application, and the agent is in fact marketed as a dietary Supplement in the U.S. with growing popularity.

It is shown that oxidative stress is an important causative factor in induction of various diseases, including ageing. Accordingly, the prospects for antioxidant functional substances that have the ability to remove reactive oxygen species, as agent for suppressing the aging and for the treatment of diseases greatly increase. Thus, we need to develop new antioxidant-functional material that can have the function of treatment of aging caused by oxidative stress, and various Soboleva�th.

There are many antioxidants, but most of them are not delivered effectively to the mitochondria, and thus, they show weak or negligible effectiveness. So the delivery of antioxidants to mitochondria is very important in the treatment of the above diseases. In the case of Mito Q it was possible to use it as a therapeutic agent, since it is formed by the conjugation of coenzyme Q10 peptide targeted to the mitochondria.

On the other hand, it was reported that this oxidative stress is the major mechanism of ischemic reperfusion injury. Coronary heart disease suggest ectomy, organ transplants, embolization, myocardial infarction, stroke and so on.

Disclosure of the INVENTION

Technical problem

Therefore, the present invention is to provide a pharmaceutical composition comprising an indole compound suitable for the prevention or treatment of diseases associated with oxidative stress, mitochondrial dysfunction, hypoxic injury, necrosis and/or ischemic reperfusion injury, and to cosmetic compositions containing the indole compound having antioxidant effect.

Solution

The present invention provides f�rmaceuticals composition for the prevention or treatment of diseases, associated with oxidative stress, comprising a therapeutically effective amount of a compound of the following formula (1), (2) or (3), its pharmaceutically acceptable salt or isomer as an active ingredient, and a pharmaceutically acceptable carrier.

Formula 1

In the above formula (1) each of the substituents specified in the publication of international patent application no WO 2009/025477 as follows:

In the formula (1):

na denotes a number from 0 to 3,

Aarepresents a 5-membered heteroaryl or heterocycle, each of which has 1 to 3 heteroatoms selected from N, O and S,

R1ais R5a-Xa-Ba-X'a-,

Barepresents a direct bond or represents a 3-10-membered heterocycle or heteroaryl, each of which has from 1 to 4 heteroatoms selected from N, O and S,

Xaand X'aindependently from one another represent a direct bond or is selected from the group consisting of-NR6a-, -CO-, -CONR6a-, -CO2-, -OC(O)-, -S(O)ma-, -O-(CH2)ma-, -(CH2)ma-O-, -(CH2)ma-, -NR6aCO-, -(R6aO)2P(O) -, and-NHCO2- where ma denotes a number from 0 to 3 and R6arepresents hydrogen, alkyl or cycloalkyl,

R5arepresents hydrogen, nitrile, hydroxy, alkyl, alkoxy, cycloalkyl or aryl or n�ecstasy 3-10-membered monocyclic or condensed cyclic heterocycle or heteroaryl, each of which has 1 to 3 heteroatoms selected from N, O and S, and optionally substituted by oxo or alkyl, or

R5aand R6amay together form a 4-8-membered cycle,

R2ais -(CR8aR9a)pa-Ya-R7a,

pa denotes a number from 0 to 2,

R8aand R9aindependently from each other represent hydrogen or alkyl or may together form a 4-8-membered cycle,

Yarepresents a direct bond or is selected from the group consisting of-O-, -S-, -NR6a-, -NR6aC(O)-, -CO2-, -C(O)-, -C(O)NR6a-, -S(O)qa- , and-S(O)qaNR6a- where qa denotes a number from 0 to 2,

R7arepresents hydrogen, halogen, cyano, hydroxy, nitro, alkyl, cycloalkyl or aryl, or represents a 3-10-membered heterocycle or heteroaryl, each of which has 1 to 3 heteroatoms selected from N, S and O and which optionally contains oxo,

R3arepresents hydrogen, alkyl, -(CH2)qa-cycloalkyl or -(CH2)qa-heterocycl,

R4arepresents -(CH2)pa-Da-R10a-,

Darepresents a direct bond, represents cycloalkyl, optionally containing oxo, represents aryl or represents a 3-10-membered heterocycle or heteroaryl, each of which has 1 to 3 heteroatoms selected from N, S and O,

R10ais odor�d, halogen, amino, cyano, nitro, hydroxy, alkyl, alkylaryl, alkylsulfonyl or -(CH2)pa-NR8aR9a,

where alkyl, alkoxy, aryl, cycloalkyl, heterocycle and heteroaryl can be optionally substituted, and the substituents represent one or more selected from the group consisting of hydroxy, halogen, nitrile, amino, alkylamino, dialkylamino, alkyl, halogenoalkane, alkylsulfonyl, carboxyethyl, alkylcarboxylic, alkylthio, allyloxycarbonyl, alkylaminocarbonyl, Allakaket and oxo.

Formula 2

In the above formula (2) each of the substituents specified in the publication of international patent application no WO 2009/025478 as follows:

In the formula (2):

nb denotes a number from 1 to 3,

mb represents 0 or 1,

Abrepresents a direct bond, represents phenyl, or represents 6-membered heteroaryl having 1 to 2 nitrogen atoms,

Xbrepresents C or N, provided that mb is 0, when Xbrepresents N, and mb is 1, when Xbis a C,

R1brepresents hydrogen, alkyl, -(CH2)rbNR7bR8bor -(CH2)rbCO2H, where rb denotes a number from 1 to 5 and R7band R8bindependently from each other represent hydrogen, alkyl or alkylaryl, or can �place to form an optionally alkyl-substituted alkylenes chain, in which optionally one methylene is replaced by N atom,

R2brepresents hydrogen, halogen, cyano, nitro, hydroxy, alkyl, alkoxy or trialkylsilyl represents -(CH2)pbCO2R7b, -(CH2)pbOR7b, -(CH2)pbNR7bR8b, -NHR10b, -N(H)S(O)2R7b, -NHC(O)R10b, -(CH2)pbS(O)2R7bor (CH2)pb-heterocycl-R10bwhere pb denotes a number from 0 to 3, R7band R8bare as defined above, R10brepresents hydrogen, oxo, alkylsulfonyl, alkylsulphonyl, allyloxycarbonyl, alkylaminocarbonyl, alkoxy, alkyl or heterocycle,

R3brepresents hydrogen, cyano, halogen, alkyl or phenyl, or represents -(CH2)nb-heterocycle or -(CH2)nb-aryl, where nb denotes a number from 0 to 3,

R4bis-YbR11bwhere Ybrepresents a direct bond or -(CR7bR8b)pbY'b- where pb denotes a number from 0 to 3, R7band R8bare as defined above, Y'bselected from the group consisting of-O-, -S-, -NR12b-, -NR12bC(O)-, -C(O)-, -C(O)O-, -C(O)NR12b-, -S(O)qb- , and-S(O)qbNR12b-, where R12brepresents hydrogen, alkyl, aryl or heteroaryl, qb denotes a number from 0 to 2, R11bselected from the group consisting of hydrogen, cyano, haloge�and, hydroxy, thiol, carboxy, alkyl and -(CH2)tbBb-R13bwhere tb denotes a number from 0 to 3, Bbis heterocycl, heteroaryl or aryl, R13brepresents hydrogen, cyano, halogen, hydroxy, oxo, thiol, carboxy, carboxylic, alkylcarboxylic, alkyl, alkoxy, alkylthio, alkylsulphonyl or alkylsulfonyl,

R5brepresents hydrogen, alkyl, cycloalkyl, heterocycl or geterotsiklicheskie,

R6bis -(CR7bR8b)pb-Zb-Db-Wb-R14bwhere Zbrepresents a direct bond or is selected from the group consisting of-C(O)-, -C(O)O-, -C(O)NR12b- , and-S(O)yb-, yb denotes the number 1 or 2, Dbrepresents a direct bond or represents cycloalkyl, heteroaryl or heterocycle, Wbrepresents a direct bond or is-NR7b-, -C(O)-, -C(O)O-, -C(O)NR12b-, -S(O)yb-, -S(O)ybNR12b- or-NR12bS(O)yb-, where R14brepresents hydrogen, hydroxy, alkyl, alkoxy, heterocycle, heteroaryl, aryl or aralkyl,

R5band R6btogether represent alkylenes chain,

where alkyl, alkoxy, aryl, cycloalkyl, heterocycle and heteroaryl can be optionally substituted, and the substituents represent one or more selected from the group consisting of hydroxy, halogen, nitrile, amino, alkylamino, dialkylamino, carboxymethyl, �of Lila, alkoxy, carboxyethyl, alkylcarboxylic, alkylthio, allyloxycarbonyl, alkylaminocarbonyl, Allakaket and oxo.

Formula 3

In the formula (3):

Bcrepresents aryl, or represents a 4-to 8-membered heterocycle or heteroaryl, each of which has 1 to 2 heteroatoms selected from N, O and S,

R7crepresents hydrogen, halogen, hydroxy, nitrile, nitro or alkoxy,

R8crepresents C1-C6-alkyl, C3-C8-cycloalkyl, heterocyclyl, aryl, arylalkyl, cycloalkyl-alkyl or heterocyclyl-alkyl,

R9crepresents hydrogen, halogen, hydroxy, nitrile, nitro, alkoxy, allyloxy, alkylamino or arylamino,

where alkyl, alkoxy, aryl, cycloalkyl, heterocycle and heteroaryl can be optionally substituted, and the substituents represent one or more selected from the group consisting of hydroxy, halogen, nitrile, amino, C1-C6-alkylamino, di(C1-C6-alkyl)amino, carboxy, C1-C6-alkyl, halogen-(C1-C6-alkyl, C1-C6-alkoxy, aryl-C1-C6-alkoxy and oxo.

In the above definitions of the compounds of formulas (1), (2) and (3) the term “alkyl” means aliphatic hydrocarbon radical. Alkyl may be a saturated alkyl, alkenyl or al�inily fragment, or unsaturated alkyl, which includes at least one alkenyl or alkynylaryl fragment. “Alkenyl” means a group containing at least one carbon-carbon double bond, “alkenyl” means a group containing at least one carbon-carbon triple bond. Alkyl may be branched or normal chain, being used on its own or in composite form, such as alkoxy.

An alkyl group can have from 1 to 20 carbon atoms, unless otherwise defined. The alkyl group may be a medium size alkyl having 1 to 10 carbon atoms. Otherwise, the alkyl group may be a lower alkyl having from 1 to 6 carbon atoms. Its typical examples include, but are not limited to: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, ethenyl, propenyl, butenyl and so on. For example, C1-C4-alkyl has 1 to 4 carbon atoms in the alkyl chain and is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

The term “alkoxy” means alkyloxy having from 1 to 10 carbon atoms, unless otherwise defined.

The term “cycloalkyl” means substituted aliphatic 3-10-membered cycle, unless otherwise stated. Its typical examples include�up with the following, but not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and so forth.

The term “aryl” includes at least one ring having a covalent π-electronic system, for example, monocyclic or condensed polycyclic (i.e. loops which share adjacent pairs of carbon atoms) groups. In the present description, the aryl means an aromatic 4-10-membered, preferably 6-10-membered, monocyclic or polycyclic ring including phenyl, naphthyl and so on, unless otherwise stated.

The term “heteroaryl” means an aromatic 3-10-membered, preferably 4-to 8-membered, more preferably 5-6-membered cycle which has from 1 to 4 heteroatoms selected from N, O and S and which may be condensed with benzo - or C3-C8-cycloalkyl, unless otherwise stated. The monocyclic heteroaryl includes the following but is not limited to: thiazole, oxazole, thiophene, furan, pyrrole, imidazole, isoxazole, isothiazole, pyrazole, triazole, triazine, thiadiazole, tetrazole, oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine and the like. Bicyclic heteroaryl includes the following but is not limited to: indole, indolin, benzothiophen, benzofuran, benzimidazole, benzoxazole, benzizoksazola, benzthiazole, benzothiadiazole, benzotriazole, quinoline, isoquinoline, purine, properidine and the like.

The term “hetero�TC” means a 3-10-membered preferably 4-to 8-membered, more preferably 5-6-membered cycle which has from 1 to 4 heteroatoms selected from N, O and S, may be condensed with benzo - or C3-C8-cycloalkyl and is saturated or contains 1 or 2 double bonds, unless specified. Heterocycl includes the following but is not limited to: pyrrolin, pyrrolidin, imidazolin, imidazolidine, pyrazoline, pyrazolidine, PYRAN, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran and the like.

Other terms and abbreviations in the present description can be understood as having the value traditionally used in the field by a qualified technician, if not otherwise defined.

Preferred examples of compounds of formula (1) or (2) can be listed as follows:

Connection 1:

[(S)-2-(7-cyclopentylamine-5-methyl-1H-indol-2-yl)-4,5-dihydrothiazolo-4-yl]acetic acid

Connection 2:

{(S)-2-[5-methyl-7-(tetrahydropyran-4-ylamino)-1H-indol-2-yl]-4,5-dihydrothiazolo-4-yl}acetic acid

Connection 3:

[(S)-2-(7-cyclopentylamine-5-methyl-1H-indol-2-yl)-4,5-dihydrooxazolo-4-yl]acetic acid

Compound 4:

[(S)-2-(7-cyclopentylamine-1H-indol-2-yl)-4,5-dihydrothiazolo-4-yl]acetic acid

Connection 5:

[(S)-2-(7-cyclopentylamine-5-phenoxy-1H-indol-2-yl)-4,5-dihydrothiazolo-4-yl]acetic acid

Compound 6:

Compound 7:

cyclopentyl-(2-{(S)-4-[2-(3-methyl-5,6-dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazine-7-yl)ethyl]-4,5-dihydrothiazolo-4-yl}-5-phenoxy-1H-indol-7-yl)amine

Compound 8:

((S)-2-{7-[(tetrahydropyran-4-ylmethyl)amino]-1H-indol-2-yl}-4,5-dihydrothiazolo-4-yl)acetic acid

Compound 9:

(tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-diocletianopolis-4-yl)methyl-1H-indol-7-yl]amine

The connection 10:

[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indol-7-yl]-(tetrahydropyran-4-yl)methylamine

Compound 11:

[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indol-7-yl]-bis-[(tetrahydropyran-4-yl)methyl]amine

Compound 12:

{4-[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indole-7-ylamino]piperidine-1-yl}-(tetrahydropyran-3-yl)methanon

Compound 13:

[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indol-7-yl]-(1-methylsulfonylmethane-4-yl)amine

Compound 14:

((S)-2-{5-methyl-7-[(tetrahydropyran-4-ylmethyl)amino]-1H-indol-2-yl}-4,5-dihydrothiazolo-4-yl)acetic acid

Preferred compounds among the compounds of the above formula (3) are those in which

Bcrepresents aryl, or represents 5-6-membered heterocycle or heteroaryl, each of which has 1 to 2 heteroatoms selected from N, O and S,

R7crepresents hydrogen, halogen, nitrile or alkoxy,

R8crepresents C1-C6-alkyl, C3-C8/sub> -cycloalkyl, heterocyclyl, arylalkyl, cycloalkyl-alkyl or heterocyclyl-alkyl,

R9crepresents hydrogen, halogen, nitrile, alkoxy, allyloxy, alkylamino or arylamino.

More preferably, in formula (3), Bcrepresents phenyl or pyridine.

More preferably, in formula (3), R7crepresents hydrogen, halogen or alkoxy, and most preferably hydrogen

More preferably, in formula (3), R8crepresents C1-C6-alkyl, heterocyclyl, cycloalkyl-alkyl, heterocyclyl-alkyl or arylalkyl and most preferably - cyclopentyl or tetrahydropyran.

More preferably, in formula (3), R9crepresents hydrogen, halogen or alkoxy, and most preferably is hydrogen.

Illustrative compounds of formula (3) include the following:

cyclopentyl-(2-phenyl-3H-benzimidazol-4-yl)amine,

(2-phenyl-3H-benzimidazol-4-yl)-(tetrahydropyran-4-yl)amine,

cyclopentyl-(2-pyridin-2-yl-3H-benzimidazol-4-yl)amine.

Pharmaceutical composition of the present invention can be effectively used for the prevention and treatment of diseases induced by oxidative stress.

Pharmaceutical composition of the present invention can be effectively used for the prevention and treatment of diseases induced by oxidative stress, which �posedown by reactive oxygen species (ROS) or reactive nitrogen (RNS).

Pharmaceutical composition of the present invention can suppress ischemic reperfusion injury.

Pharmaceutical composition of the present invention can be effectively used for the prevention and treatment of diseases induced hypoxic damage.

Pharmaceutical composition of the present invention can be effectively used for the prevention and treatment of diseases induced necrotic cell death.

Pharmaceutical composition of the present invention can suppress mitochondrial dysfunction.

Pharmaceutical composition of the present invention can be effectively used for the prevention and treatment of MELAS syndrome caused by mitochondrial dysfunction (mitochondrial myopathy, encephalopathy, lactic acidosis and insulinopenia episodes), MERRF syndrome (myoclonic epilepsy with ragged muscle fibers) or syndrome Kearns-they, all of which are caused by mitochondrial dysfunction.

Pharmaceutical composition of the present invention can suppress the necrosis of cells secreting HMGB1 (high-motility group box 1).

Moreover, the pharmaceutical composition of the present invention can prevent and/or treat a disease mediated by HMGB1, in particular mediated by or associated with inflammation ill�tion. Such diseases include sepsis, rheumatoid arthritis, osteoarthritis, cirrhosis of the liver, hemorrhagic disease, various necrotic diseases, viral or bacterial infection, and so on.

Diseases that can be prevented and/or treated using the present invention include liver disease, heart disease, vascular disease, degenerative brain disease, disease caused by ischemic reperfusion injury, and infectious disease caused by virus or bacteria; status in liver transplantation, liver resection, embolization of the liver, liver fibrosis, cirrhosis, alcoholic/non-alcoholic fatty liver and hepatitis caused by virus or drugs (e.g., anticancer agent, acetaminophen and so forth); cardiac or cardiovascular diseases such as arrhythmia, cardioplegia, myocardial infarction and so on; degenerative brain diseases, such as disease Lou Gehrig, stroke, dementia, Parkinson's disease, Huntington's disease and so on; diabetic complex, arteriosclerosis, myocardial infarction or stroke, each of which is caused by ischemic reperfusion injury; a disease caused by infection with a virus such as influenza virus, HBV, HCV, HIV and so on, or bacteria�I.

In the present invention, the liver disease may be one or more selected from the group consisting of a state in liver transplantation, liver resection, embolization of the liver, liver fibrosis, liver cirrhosis, alcoholic/non-alcoholic fatty liver and hepatitis caused by virus or drugs (e.g., anticancer agent, acetaminophen and so forth).

In the present invention a heart or cardiovascular disease may be one or more selected from the group consisting of arrhythmia, cardioplegia, myocardial infarction, heart failure and angina.

Pharmaceutical composition of the present invention can be effectively used for the prevention and treatment of diseases of the liver, heart or cardiovascular diseases, each caused by ischemic reperfusion injury, where ischemic reperfusion injury may occur as a result of mitochondrial dysfunction, hypoxic injury and/or necrotic cell death.

Pharmaceutical composition of the present invention can be effectively used for preventing or treating one or more diseases selected from the group consisting of diabetic complex, arteriosclerosis and stroke, to�zhdy of which is caused by ischemic reperfusion injury.

Specific embodiments of the synthesis of compounds of formula (1) or (2) of the present invention are disclosed in WO 2009/025477 and WO 2009/025478 respectively.

The present invention also provides a compound of formula (3) and methods for their preparation. Further methods for obtaining compounds of formula (3) will be explained on the basis of the illustrative reaction schemes in order to facilitate understanding of the present invention. However, it should be understood that a person having ordinary skills in this area, could obtain a compound of formula (3) in a variety of ways based on the structure of the formula (3), and all such methods are included in the scope of the present invention. In other words, it should be understood that the compounds of the formula (3) can be obtained by any combination of different methods of synthesis described herein or disclosed in the prior art, and this combination is included in the scope of the present invention. Methods for obtaining compounds of formula (3) is not limited to the methods described below.

First, the compound of formula (3) can be synthesized by the reduction of nitro group of compounds of formula (4), gives the amine of a compound of formula (5), and carrying out the reaction of reductive amination (RA) of the resulting amino groups with the participation of the compounds of formula (6) according to the method shown in the following reaction �fur 1.

Reaction scheme 1

where

Bc, R7c, R8cand R9care as defined in formula (3);

R10crepresents alkyl, cycloalkyl, heterocyclyl, heteroaryl or aryl;

R11crepresents hydrogen or alkyl; or

R10cand R11ccan withdraw into the cycle with the formation of cycloalkyl or heterocycle.

Compounds of the formula (5) can be obtained by reduction of compounds of formula (4). Of reduction reaction can be carried out using acidic catalysts and metals or metal catalysts in the presence of hydrogen gas.

Examples of acids which may be used in the reaction of recovery, which involves the use of acid catalysts and metals, include inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid; organic carboxylic acid such as acetic acid and trifluoroacetic acid; and amine salt and acid, such as ammonium chloride. Preferred acids are hydrochloric acid, acetic acid, ammonium chloride and so on. The amount of acids to be used is typically from 0.01 to 10 equivalents, and preferably 0.1 to 5 equivalents per 1 equivalent�alent compounds of the formula (4). Examples of metal that may be used include iron, zinc, lithium, sodium, and tin (usually the chloride of tin). The preferred metals are iron, zinc, chloride of tin, and so on. The number of metals to be used is typically from 1 to 20 equivalents and preferably 1 to 10 equivalents per 1 equivalent of the compounds of formula (4). Reaction with metals in the presence of acid catalysts can be carried out in an inert solvent. Examples of the inert solvent include alkyl alcohol, such as methanol and ethanol; ether such as tetrahydrofuran and diethyl ether; and alkyl ester, such as ethyl acetate. Preferred solvents are methanol, ethanol, tetrahydrofuran and ethyl acetate and so on. Reaction temperature is typically from -10 to 200°C and preferably from 25 to 120°C. the reaction Time is typically from 10 minutes to 60 hours and preferably from 10 minutes to 12 hours.

Examples of the metal catalyst which can be used in the reaction of recovery, which includes the use of metal catalysts in the presence of hydrogen gas include palladium, Nickel, platinum, ruthenium, rhodium and the like. Preferred metal catalysts are palladium, Nickel and so on. The amount of metal to�of telesfora, to be used, is typically from 0.001 to 2 equivalents, and preferably from 0.01 to 1 equivalent per 1 equivalent of the compounds of the formula (2). The pressure of hydrogen gas is typically from 1 to 10 ATM and preferably from 1 to 3 ATM. The reaction can be carried out in an inert solvent, for example, alkyl alcohol, such as methanol and ethanol; simple ether, such as tetrahydrofuran and diethyl ether; and allylacetate, such as methyl acetate and ethyl acetate. Preferred solvents are methanol, ethanol, ethyl acetate and so on. In the reaction of recovery, which includes the use of metal catalysts, reaction temperature is typically from -10 to 200°C and preferably from 25 to 50°C. the reaction Time is typically from 10 minutes to 60 hours and preferably from 10 minutes to 12 hours.

Compounds of the formula (6) is commercially available and can be used in the reaction, the reductive amination of the amino group of compounds of the formula (5).

The reaction of reductive amination can be carried out by reaction of the aldehyde or ketone with a reducing agent and an acid catalyst, if necessary. The amount of aldehyde or ketone to be used is typically from 1 to 10 equivalents and preferably 1 to 3 equivalents to 1 equivalent of a compound of formula(5). Examples of reducing agents that may be used include sodium borohydride, cyanoborohydride sodium (NaBH3CN) and triacetoxyborohydride sodium (NaBH(OAc)3). The amount of reducing agent to be used is typically from 1 to 10 equivalents and preferably 1 to 3 equivalents to 1 equivalent of the compounds of the formula (5). Examples of the acid catalyst which can be used include inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid; organic carboxylic acid such as acetic acid and trifluoroacetic acid; and amine salt and acid, such as ammonium chloride. Preferred acids are hydrochloric acid, acetic acid and so on. The amount of acids to be used is typically from 0.1 to 10 equivalents and preferably 1 to 5 equivalents per 1 equivalent of the compounds of the formula (5). The reaction can be carried out in an inert solvent, for example in a simple ether, such as tetrahydrofuran and diethyl ether; and chloroalkane, such as dichloro methane, chloroform and dichloroethane, preferably dichloromethane, chloroform and so on. Reaction temperature is typically from -10 to 100°C and preferably -10 to 50°C. the reaction Time is typically from 10 minutes to 60 hours, etc�doctitle from 10 minutes to 12 hours.

Compounds of the formula (4) can be obtained by the reaction of the combination and reaction of cyclization of the aldehyde compounds of formula (7) with compounds of the formula (8), as shown in the following reaction scheme 2.

Reaction scheme 2

where Bc, R7cand R9care as defined in formula (3).

First, commercially available aldehydes, such as compounds of formula (7), and commercially available diamine compounds such as compounds of the formula (8) is heated with stirring, resulting in cyclization. Then, the heating in the presence of acidic catalysts could provide the compound of formula (4). Examples of the acid catalyst which can be used include inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid; organic carboxylic acid such as acetic acid and trifluoroacetic acid; and amine salt and acid, such as ammonium chloride. Preferred acid catalysts are hydrochloric acid, acetic acid and so on. The amount of acids to be used is typically from 0.1 to 10 equivalents and preferably 1 to 5 equivalents per 1 equivalent of the compounds of the formula (5). In the case of the reaction can be carried out with use�Itanium organic acids, such as acetic acid, as solvent.

In the present description “pharmaceutically acceptable salt” encompasses non-toxic acid-additive salt containing pharmaceutically acceptable anion, for example a salt with inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, Hydrobromic acid, itestosterone acid and the like; salt with organic carboxylic acids, such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and so on; or a salt with sulfonic acids such as methanesulfonic acid, econsultancy acid, mixture of Benzenesulfonic acid, p-toluensulfonate acid, naphthalenesulfonate acid and so on. Compounds of formula (1) may also form pharmaceutically acceptable basic additive salt, for example a salt with alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and the like; salt with amino acids such as lysine, arginine, guanidine and the like; or an organic salt with dicyclohexylamine, N-methyl-D-glucamine, Tris(gidroximetil)IU�aluminum, with diethanolamine, choline, triethylamine and so on. Compounds of formula (1), (2) and (3) of the present invention can be converted into its salts according to any one of conventional methods, and the salt formation can be easily carried out by a person skilled in the art based on the structural formulas (1), (2) and (3) without further explanation in this regard.

The term “isomer” in the present description means compounds having the same chemical or molecular formula of a compound of formula (1) or their salts, but optical or sterically different from them. Compounds of formula (1), (2) and (3) of the present invention may have asymmetric(e) carbon(e) centre(s) in the structure and, thus, can exist in the form of an optical isomer (R - or S-isomer, racemate, mixture of diastereomers or of an individual diastereoisomer, and so on. If compounds have a double bond, they can exist in the form of geometric isomers (TRANS - or CIS-isomer). All isomers and mixtures thereof are also covered by the present invention.

Further compounds of formula (1), (2) and (3) include pharmaceutically acceptable salts and their isomers, unless explained otherwise. Salts and isomers should be considered as covered by the present invention. For convenience, in the present description they are briefly referred to as compounds of formula (1).

Wiseup� - mentioned pharmaceutical composition may include pharmaceutically acceptable carriers, thinner, excipient or their combinations, if necessary, together with the compounds of the present invention. Pharmaceutical composition facilitates the introduction of compounds to a living organism. There are a number of methods of introducing compounds, and they include the following, but not limited to: oral, injection, aerosol, parenteral and topical administration.

Used herein “carrier” means a substance which facilitates the entry of compounds into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a typical medium that is used to facilitate the introduction of various organic compounds in cells or tissues of living organisms.

Used herein, “diluent” is defined as a substance that is dissolved in water, which dissolves the compound, as well as stabilize the biologically active form of compounds of the compounds. Salt dissolved in a buffer solution, are used in this area as diluents. Typically used buffer solution is a phosphate buffered saline solution that mimics the salt composition of the salt solution human body. Buffer diluents rarely alter the biological activity of the compounds, since buffer salts can control the pH of the solution at low concentration.

Ispolzovaniya “pharmaceutically acceptable” means the property will not impair the biological activity and physical properties of the connection.

Of the compounds of the present invention can be composed of the formulation in the form of various pharmaceutical dosage forms in accordance with the desired objective. To obtain pharmaceutical compositions of the present invention the active ingredient, in particular compounds of formula (1), (2) or (3), their pharmaceutically acceptable salts or isomers, mixed together with various pharmaceutically acceptable carriers that can be selected in accordance with subject to obtaining the recipe. For example, pharmaceutical compositions of the present invention can be given an injectable form of the drug, a drug for oral administration and so on, according to the desired goal.

Of the compounds of the present invention can be composed of the formulation methods known in this field that use pharmaceutical carriers and excipients known in this field, and can be enclosed in a container a unit dosage form or multinational form. Form of the drug may be solutions, suspensions or emulsions in oily or aqueous media, and they typically contain dispersing agents, suspendida agents or stabilizers. In addition, for example, the drug may be in the form of dry powder, which is suitable for being transferred to its original state before the ISP�Lovanium by dissolving in sterile, apyrogenic water. Of the compounds of the present invention may be composed of the formulation in the form of a suppository forms, using a typical basis for suppositories, such as cocoa butter or other glycerides. Can be obtained in solid dosage forms for oral administration, capsules, tablets, pills, powders and granules, and capsules and tablets are especially suitable. Preferably, tablets and pellets obtained in the form of forms enteric coated. Solid dosage forms can be obtained by mixing the compounds of the present invention together with the media, such as one or more inert diluents such as sucrose, lactose, starch and so forth, lubricating agents such as magnesium stearate, disintegrators, binders and so on.

The present invention also provides a cosmetic composition having antioxidant effect comprising a compound of the above formula (1), (2) or (3), its pharmaceutically acceptable salt or isomer as an active ingredient; and an acceptable carrier.

Providing the advantage effects of the invention

Pharmaceutical composition of the present invention has excellent antioxidant activity and, thus, can effectively prevent or treat a variety of diseases, linked�s with oxidative stress.

In addition, the pharmaceutical composition of the present invention can effectively prevent or treat cardiac or cardiovascular disease suppression dysfunction of mitochondria in cardiac muscle cells, hypoxic injury, necrosis and ischemic reperfusion injury. In addition, it can be used as an agent to protect the heart at reperfusion treatment, such as operative therapy, including bypass grafts coronary artery, or percutaneous transluminal coronary angioplasty, and drug therapy with thrombolysis agents, or the like.

Brief description of the drawings

Fig.1 presents a photograph showing the antioxidant effect (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-diocletianopolis-4-yl)methyl-1H-indol-7-yl]amine (compound 9) in the cells, when using the probe DHR123.

Fig.2 presents a photograph showing the antioxidant effect of compound 9 in cells, when using the probe MitoSOX red.

Fig.3 shows a photograph comparing the results from the group treated with compound 9, with the results of the group not receiving treatment.

Fig.4 is a diagram showing values of ALT and AST after ischemic reperfusion injury in the treatment and without treatment soy�errors 9.

Fig.5 is a photograph showing the comparison of staining of the tissue between the group, which introduced the 13 mg/kg of compound 9, and the group in which the connection 9 is entered (IR: ischemic reperfusion injury).

Fig.6 is a diagram showing the comparison of the results in the group treated with compound 9, and in the group not receiving treatment, on the basis of microscopic observations (degeneration of hepatocytes, sinusoidal it's congestion, inflammatory cell infiltration).

Fig.7 is a diagram showing a comparison of the level of MGB1 between the group, which introduced the 13 mg/kg of compound 9, and the control group with ischemic reperfusion injury.

Fig.8 is a diagram showing a comparison of the activity of mitochondrial complex I after ischemic reperfusion injury between the group treated with compound 9, and the group not receiving treatment.

Fig.9 presents a graph showing the release of HMGB1 after treatment with compound 9.

Fig.10 is a diagram showing the change in ATP content induced by compound 9, after treatment with compound 9.

Fig.11 shows a photograph obtained on a fluorescent microscope, H9C2 cells after double staining of FDI and PI, illustrating the protective effect� compound 9 against necrotic cell death.

Fig.12 is a diagram showing a direct cell count to illustrate the protective effect of compound 9 against necrotic cell death.

Fig.13 is a diagram showing the results of FACS analysis illustrating the protective effect of compound 9 against necrotic cell death.

Fig.14 is a diagram showing the inhibitory effect of compound 9 against mitochondrial swelling.

Fig.15 shows a photograph obtained on a fluorescent microscope, showing the mitochondrial protective effect of compound 9.

Fig.16 is a diagram showing the inhibitory effect of compound 9 against swelling of the left ventricle in a model of ischemic reperfusion injury.

Fig.17 is a diagram showing the inhibitory effect of compound 9 against the shortening fraction of the left ventricle after ischemic reperfusion injury.

Fig.18 shows an image showing heart fibrosis staining method using MT (Masson-Trichrome), for comparison of compound 9 with cyclosporine A.

Fig.19 shows a photograph that shows the progress of the operation on the liver of experimental example 2.

Embodiments of the invention

Further, the present invention will be description�but in more detail with reference to the following examples, but the scope of the present invention should not be construed as limited by them in any way.

Example 1: Obtaining cyclopentyl-(2-phenyl-3H-benzoimidazol-4-yl)amine

Stage A: 4-nitro-2-phenyl-2,3-dihydro-1H-benzoimidazol

To a solution of 3-nitro-benzene-1,2-diamine (1.0 g, 6.5 mmol) in methanol (10 ml) was added benzaldehyde (0,69 g, 6.7 mmol) and stirred at 80°C for 2 days. After completion of the reaction the solvent was removed under vacuum and the residue was purified by column chromatography to obtain the titled compound in the amount of 1.1 g (yield 70%).

1H-NMR (500 MHz, DMSO): δ 8,68 (s, 1H), 7,43-7,31 (m, 5H), 7,18 (s, 1H), 6,95 (d, 1H), 6,44 (s, 1H), 6,36 (m, 1H), 6,29 (d, 1H).

Step B: 7-nitro-2-phenyl-1H-benzoimidazol

4-nitro-2-phenyl-2,3-dihydro-1H-benzoimidazol (1.1 g, 4.6 mmol) obtained in stage A, was dissolved in acetic acid (10 ml) and stirred at 80°C for 3 hours. After completion of the reaction was added ice water, extracted with ethyl acetate and washed with 1 n sodium hydroxide. After drying with anhydrous magnesium sulfate, the filtrate was distilled under vacuum and the residue was purified by column chromatography to obtain the titled compound in the amount of 0.91 g (83%).

1H-NMR (400 MHz, DMSO): δ 13,3 (ush. s, 1H), 8,37 (d, 2H), 8,15 (d, 2H), 7,65-7,56 (m, 3H), of 7.46 (t, 1H).

Step C: 2-phenyl-3H-benzoimidazol-4-ylamine

7-nitro-2-phenyl-1H-benzoimidazol (0,137 g, 0,57 mmol) rece�th B-stage, was dissolved in tetrahydrofuran (5 ml), methanol (5 ml) and water (5 ml). To this solution was added iron powder (0.40 g, 7.2 mmol) and ammonium chloride (0,39 g, 7.2 mmol) and stirred at 60°C for 1 hour. After completion of the reaction was added water and was extracted with ethyl acetate. After drying with anhydrous magnesium sulfate, the filtrate was distilled under vacuum and the residue was purified by column chromatography to obtain the titled compound in the amount of 0.12 g (yield 79%).

1H-NMR (500 MHz, DMSO): δ of 12.5 (s, 1H), 8,10 (d, 2H), 7,55-7,45 (m, 3H), 6,87 (m, 1H), to 6.67 (d, 1H), 6.32 per (d, 1H), to 5.21 (s, 1H).

Stage D: Cyclopentyl-(2-phenyl-3H-benzoimidazol-4-yl)-amine

2-phenyl-3H-benzoimidazol-4-ylamine (0,040 g, 0,19 mmol) of step C, was dissolved in dichloroethane (5 ml). To the solution was added acetic acid (0.012 g, 0,19 mmol), Cyclopentanone (0,048 g, 0,57 mmol) and triacetoxyborohydride sodium (0,049 g, 0,23 mmol) and stirred at room temperature for 18 hours. After completion of the reaction was added water, was extracted with dichloromethane and washed with saturated solution of sodium chloride. After drying with anhydrous magnesium sulfate, the filtrate was distilled under vacuum and the residue was purified by column chromatography to obtain the titled compound in the amount 0,026 g (yield 49%).

1H-NMR (40 MHz, CDCl3): δ 9,36 (ush. s, 1H), 8,02 (m, 2H), 7,56-7,45 (m, 3H), 7,17 (t, 1H), 6,83 (d, 1H), 6,47 (d, 1H), 5,06 (ush. s, 1H), was 4.02 (m, 1H), 2,13 (m, 2H), ,82 (m, 2H), 1,69 (m, 4H).

Example 2: preparation of (2-phenyl-3H-benzimidazol-4-yl)-(tetrahydrofuran-4-yl)amine

Using 2-phenyl-3H-benzoimidazol-4-ylamine (0,040 g, 0,19 mmol) obtained in stage C of example 1, and tetrahydrofuran-4-carbaldehyde (0,026 g, 0,23 mmol) of the titled compound in the amount of 0.030 g (yield 51%) was obtained following the same method described in stage D of example 1.

1H-NMR (40 MHz, CDCl3): 8,02 (m, 2H), 7,56-7,45 (m, 3H), 7,17 (t, 1H), of 6.85 (d, 1H), 6,44 (d, 1H), of 4.05 (DD, 1H), of 3.45 (dt, 2H), 3,27 (d, 2H), 2,02 (m, 1H), of 1.85 (m, 2H), 1,45 (m, 2H).

Example 3: Cyclopentyl-(2-phenyl-2-yl-3H-benzoimidazol-4-yl)amine

Using 2-carboxaldehyde-3-nitrobenzene-1,2-diamine and cyclopentanon titled compound was obtained following the same method described in example 1.

1H-NMR (40 MHz, CDCl3): 1,64 (m, 6H), 2,10 (m, 2H), 4,14 (s, 1H), 6,38-system 6.34 (d, 1H), 6,69-6,71 (d, 1H), between 7.09-7.13 (t, 1H), 7,25-7,26 (m, 1H), 7,75-7,79 (m, 1H), 8,40-to 8.41 (d, 1H), 8,54-8,55 (m, 1H).

The effect of the present invention is specifically illustrated by the experimental examples below. In experimental examples, the following compounds:

Connection 1:

[(S)-2-(7-cyclopentylamine-5-methyl-1H-indol-2-yl)-4,5-dihydrothiazolo-4-yl]acetic acid

Connection 2:

{(S)-2-[5-methyl-7-(tetrahydropyran-4-ylamino)-1H-indol-2-yl]-4,5-dihydrothiazolo-4-yl}acetic acid

Connection 3:

[(S)-2-(7-cyclopentylamine-5-methyl-1H-indol-2-yl)-4,5-dihydro�xazal-4-yl]acetic acid

Compound 4:

[(S)-2-(7-cyclopentylamine-1H-indol-2-yl)-4,5-dihydrothiazolo-4-yl]acetic acid

Connection 5:

[(S)-2-(7-cyclopentylamine-5-phenoxy-1H-indol-2-yl)-4,5-dihydrothiazolo-4-yl]acetic acid

Compound 6:

4-{2-[(S)-2-(7-cyclopentylamine-5-phenoxy-1H-indol-2-yl)-4,5-dihydrothiazolo-4-yl]ethyl}piperazine-2-he

Compound 7:

cyclopentyl-(2-{(S)-4-[2-(3-methyl-5,6-dihydro-8H-[1,2,4]triazolo[4,3-a]pyrazine-7-yl)ethyl]-4,5-dihydrothiazolo-4-yl}-5-phenoxy-1H-indol-7-yl)amine

Compound 8:

((S)-2-{7-[(tetrahydropyran-4-ylmethyl)amino]-1H-indol-2-yl}-4,5-dihydrothiazolo-4-yl)acetic acid

Compound 9:

(tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-diocletianopolis-4-yl)methyl-1H-indol-7-yl]amine

The connection 10:

[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indol-7-yl]-(tetrahydropyran-4-yl)methylamine

Compound 11:

[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indol-7-yl]-bis-[(tetrahydropyran-4-yl)methyl]amine

Compound 12:

{4-[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indole-7-ylamino]piperidine-1-yl}-(tetrahydropyran-3-yl)methanon

Compound 13:

[5-(1,1-diocletianopolis-4-yl)methyl-2-phenyl-1H-indol-7-yl]-(1-methylsulfonylmethane-4-yl)amine

Compound 14:

((S)-2-{5-methyl-7-[(tetrahydropyran-4-ylmethyl)amino]-1H-indol-2-yl}-4,5-dihydrothiazolo-4-yl)acetic acid

Example 1: cyclopentyl-(2-phenyl-3H-benzimidazol-4-yl)amine

Example 2: (2-Hairdryer�l-3H-benzimidazol-4-yl)-(tetrahydropyran-4-yl)amine

Experimental example 1: the Antioxidant effects of inhibitors of necrosis

To measure the antioxidant effect of each compound were carried out the following experiments: measurement of the total antioxidant effect of DPPH free radical method, the measurement of antioxidant effect method using dihydrorhodamine 123, the measurement of inhibition of necrosis by a method using tBOOH, which causes oxidative stress of cells, the measurement method that determines the ability to remove ONOO-(peroxynitrite), and the measurement method for determining direct antioxidant effects against H2O2(hydrogen peroxide) and tBOOH.

Experimental example 1-1: Measurement of antioxidant using DPPH method

DPPH free radical (1,1-diphenyl-2-picrylhydrazyl) is a stable free radical and has a purple color. Antioxidant effect of the compounds was measured using the principle that the color of the DPPH free radical is changed to yellow when DPPH method the connection is restored. DPPH method shows a strong absorption at 517 nm, and when restored DPPH method, the color changes to yellow and the absorbance at 517 nm decreases. Here the term “IC50” refers to the concentration of a compound that reduces the absorbance at 517 nm up to 50% of the initial value.

180 µl of the DPPH free radical solution (200 µm in DMSO) was distributed into each well of 96-lonodn�th tablet and added in 20 μl of compounds which serially diluted twofold, on the basis of 2000 μm. Then they are well mixed and kept at room temperature for 30 minutes. After 30 minutes measure the optical density (O. D.) at 517 nm using an ELISA reader, SpectraMAX. The results presented in the following table 1.

td align="center"> 12,5
Table 1
Number of connectionsAntioxidant effect against DPPH free radical (IC50, micron)
Connection 124
Compound 224
Union 317,5
Connect 415
Connection 520
Compound 623
Connection 725
Compound 820,5
Connection 916
Connection 1022
Connection 11
Connection 1219
The connection 1317
The connection 1428

Experimental example 1-2: Measurement of the antioxidant effects using dihydrorhodamine 123

Dihydrorhodamine 123 itself is not fluorescent, but is transformed into fluorescent rhodamine 123, when it is oxidized ROS (reactive oxygen species) or RNS (reactive nitrogen). Antioxidant effects in cells or in vitro was measured using such property. H9C2 cells were distributed into each well of 96-hole tablet (1,5×104cells/100 μl/well). The next day cells were pre-treated with each compound serially diluted three-fold, based on the highest concentration, for 1 hour and then treated with 10 μl of a basic solution DHR123 with a concentration of 12.5 μm (final concentration of 1.25 µm) in the incubator at 37°C for 30 minutes. Then the cells were immediately treated with 400 μm tBOOH and incubated for 2 hours. Then fluorescence was measured, which was excited at 480 nm and opuscules at 538 nm using a reader for microplates SpectraMAX Gemini. Here the term “IC25” refers to the concentration of a compound that cause th�no inhibition at 25% value where fluorescence after 2 h after treatment with tBOOH, which was espousals due to the oxidation of DHR123 due to increased oxidative stress was taken as 100%. The results are presented in table 2. In addition, to test whether the compounds have an antioxidant effect on the cells, after the above analysis, cells in a 96-well plate were fixed with 4% formaldehyde and washed three times with PBS. Then 100 ál of PBS was added to each well of 96-well plates and measured the fluorescence of each cell by immunofluorescence microscopy (fluorescence microscope Olympus). (DHR123 as is found in the cells, the fluorescence emitted is proportional to the oxidation). The results are presented in Fig.1. In addition, inhibition of generation of superoxide was measured with a probe MitoSOX red (final concentration 5 μm) by the same method.

Table 2
The results of the determination of the antioxidant effect using DHR 123
Number of connectionsIC25(μm)
Connection 90,10
Connection 120,24
The connection 13 <0,1

Experimental example 1-3: the Effect of protecting cells from oxidative stress induced tBOOH

It is known that tert-butylhydroperoxide (tBOOH) induces the necrosis of cells, causing oxidative stress in various cells. Thus, inhibition of cell death due to the antioxidant effect was measured by tBOOH treatment of H9C2 cells. H9C2 cells were distributed per well of 96-hole tablet (1,5×104cells/100 μl/well). The next day cells were pre-treated with each compound and then treated with tBOOH (final concentration 400 μm) for 2 hours. To each well was added 50 µl of formaldehyde to fix the cells. Cells were fixed for 30 minutes and washed with distilled water three times. Then the tablet was fully dried in a drying Cabinet at 50°C and added a solution of SRB in the wells to stain the remainder of the protein. The tablet was washed with 1% acetic acid and dried. Then 100 μl of Tris solution (10 mm) was added to plate wells for re-dissolving the dye and counted IC50by measuring the optical density (O. D.) at 590 nm and 650 nm using an ELISA reader SpectraMAX. Here the term "IC50"refers to the concentration of compound that protects 50% of the cells from necrosis induced by tBOOH treatment. The results are presented in the following table�e 3.

Experimental example 1-4: Measurement of ability to remove ONOO-(peroxynitrite) using dihydrorhodamine 123

ONOO-(peroxynitrite), which is known as a kind RNS, plays a very important role in the oxidative stress. Thus, it is highly important to the evaluation of the ability of inhibitors of necrosis delete ONOO-. It is well known that the probe DHR123 is oxidized ONOO-. Thus, using this property, we measured the ability of each connection.

10 µl of each compound which was sequentially diluted 3-fold on the basis of 50 μm, were distributed into each well of 96-hole tablet and then to each well was added 170 μl of the mixture for analysis (10 μm DHR123, 1×PBS, 100 μm DPTA without ONOO-). At the end was added 20 μl of a solution of ONOO-(final concentration 1 μm) to initiate the reaction. Here the term “IC50” refers to the concentration of compound that causes 50% inhibition of oxidation of DHR123 under the action of ONOO-.

The results of the study of dependence “structure-activity (SAR) regarding the ability of compounds to remove ONOO-presented in table 4.

tr>
Table 4
The ability of compounds to remove ONOO-
Number of connectionsThe ability to remove ONOO-(IC50, micron)
Connection 53,4
Connection 90,5
Connection 120,21
The connection 130,9

Experimental example 1-5: Direct antioxidant effect against H2O2and tBOOH

To determine the antioxidant effects of inhibitors of necrosis having an indole structure, has been evaluated by direct antioxidant effects against specific ROS, H2O2(hydrogen peroxide) and tBOOH.

Added 10 µl of the probe DHR123 (final concentration of 1.25 µm) to 10 ál each compound, which was diluted 100 ál of complete DMEM. Added H2O2and tBOOH in the concentration shown in table 5, and after 2 h, fluorescence was measured at wavelengths (480 nm (excitation)/538 nm (emission)) using a SpectraMAX Gemini. Here the term “IC25” refers to the concentration of a compound that causes a reduction by 25% of the fluorescence in the assumption that the number of emitted due to oxidation of DHR123-induced H2O2or tBOOH was 100%.

Table 5
Direct antioxidant effect against H2O2and tBOOHCompound No.IC25(μm)H2O2(0.6 mm)H2O2(0.3 mm)tBOOH (0.4 mm)tBOOH (0.2 mm)Union 32,152,22,42Connect 43,62,35,83Connection 920,852,91,45Connection 122The 1.6531,95

Experimental example 2: Protective effect of compound 9 against liver damage due to ischemia/reperfusion in dogs Beagle

Study design: the study took 27 healthy dogs breed Beagle (weight t�La 8,5-11,5 kg). The study was conducted after obtaining permission from the ethics Commission Asan Hospital Ethics Commission. To minimize the accumulation of glycogen in the liver, the dogs were fasted for 24 hours before surgery; and they hair was removed. Dogs were randomly divided into control group (n=9), group B (n=6), group C (n=6) and group D (n=6). Underwent endotracheal intubation and were anestesiologi dogs solutiom and 2.5% of enflurane. Cefazolin and Ketorolac were used, respectively, as an antibiotic and analgesic in accordance with the procedure approved by the Commission.

Surgery: the Dogs were laid on the operating table with warm blankets and bottles of warm water on both sides. The left external jugular vein and right long saphenous vein of the leg we carried out cannulation IV catheter through a 21 gauge open approach. The right internal carotid artery was cut off, and we carried out cannulation, so that during the operation, it was possible to observe the hemodynamics. After receiving the full intravenous access anesthesia was maintained by inhalation of 2.5% enflurane. Crystalloid solution was administered with an infusion rate of 15 ml/kg/h.

20 minutes before the vascular occlusion of the left lobe of the liver administered 13 mg/kg, 4.5 mg/kg and 1.5 mg/kg of the drug, respectively, groups B, C and D through IV. The combined dose and infusion were conducted in such a way that Cmax12.5 mg/�l (at 20 min) was consistent with steady concentration (C ss5 mg/ml. during the operation of the blood pressure, the frequency of heart beats, respiratory rate and saturation of arterial blood with oxygen was monitored every 15 minutes for all dogs. The surgery was performed by a reverse T-shaped incision. Hepato-duodenal ligament was isolated after the division of the lesser omentum. The gallbladder and the 4th lobe of the liver was fixed and the overlying structure carefully examined along with root structures. Fig.19a shows the left and right lobe of the liver, which were divided along the line of CANTLIE In Fig.19b illustrates a typical root structure. People have a right leg forms 60-70% of the total liver volume. Occlusion of the hepatic vessels was performed for 90 minutes to induce ischemic injury, and the clamp is separated to cause reperfusion injury within 60 minutes (Fig.19c). Therefore, this model represents a system that uses 90-minute ischemic injury and 60-minute reperfusion injury. After the operation the abdominal cavity and the skin was closed.

Blood sampling and biopsy: blood sampling and biopsy was performed in intervals, including a complete count of blood corpuscles (FBE; hemoglobin Hb, leukocyte formula WCC, hematocrit HCT, Plt platelets, MCR, the average content of hemoglobin MCH and the average concentration of hemoglobin in the erythrocyte CHC), liver function tests (LFT; AST, ALT, LDH and total bilirubin), determination of urea and electrolytes (U&E; BUN and creatine), liver biopsy (histopathology and H&E-staining) and measurement of HMGB1. Light micrograph and electron micrograph was used to study the shape of liver cells. Dogs breed Beagle contained after surgery in a warm and clean environment with the introduction of analgesics and continuous provision of soft and high-protein diet. 48 hours later they were sacrificed according to the procedure. The results are shown in Fig.3, 4 and 5.

Histological analysis after IR damage: Biopsy samples of liver were fixed and legalrational and then embedded in paraffin. Paraffin blocks were cut with a thickness of 4 µm were stained using Mayer's hematoxylin-eosin and examined under a light microscope. Each sample was randomly split and analyzed blind. Researched degeneration and necrosis hepatocyte cells, sinusoidal and portal venous congestion and inflammatory cell infiltration. The results of histological analysis quantitatively ranked according to the published method (Hafez T at al., Journal of Surgical Research 2007; 138: 88-99): 0 (0%, no), 1 (1-25%, weak), 2 (26-50%, moderate) or 3 (51-100%, patently). The results are shown in Fig.6.

Measurement of HMGB1: HMGB1 was measured according to the Protocol produces�La, using a ELISA analysis for HMGB1. The results are shown in Fig.7.

Measuring the activity of mitochondrial complex I: the liver Tissue was washed 2 times with chilled ice with Buffer A (320 mm sucrose, 1 mm EDTA, 10 mm Tris (pH 7,5)). They were cut into small pieces and homogenized using 4 ml AT-buffer (75 mm sucrose, 225 mm mannitol, 1 mm EGT and 0.01% BSA, pH 7,4) at 1 g pieces, in a glass-Teflon homogenizer. The homogenate was centrifuged at 4°C for 5 minutes at 1000 g and the resulting supernatant was centrifuged twice at 13,000 g. Enriched with mitochondria mass was used to measure the activity of mitochondrial complex I. the Activity of complex I (NADH: CoQ-oxidoreductase) was measured by monitoring the rotenone-sensitive recovery of NADH in the presence of desilusionado at 340 nm. The results are shown in Fig.8.

Experimental example 3: Preventive effect against the release of HMGB1 connection 9 after treatment with tBOOH

It is well known that HMGB1 is released when there is necrosis of the cells. Thus, after treatment with 400 μm tBOOH, which causes oxidative stress, tested the preventive effect of compound 9 against HMGB1 release.

H9C2 cells were distributed in an amount of 1.5×104cells/100 μl/well. The next day there was the processing of compound 9 in con�entrale 10 μm for 30 minutes. After treatment with tBOOH (final concentration 400 μm) measured the levels of HMGB1 from 50 µl of the supernatant within an hour of ELISA analysis for HMGB1. The results are shown in Fig.9.

Experimental example 4: Monitoring changes in total ATP content after treatment with tBOOH

Cells show a decrease in the content of ATP in the process of necrosis. Maintaining the amount of ATP is very important for the survival of cells. Thus, after treatment with tBOOH observed the change of ATP content.

H9C2 cells were distributed in an amount of 1.5×104cells/100 μl/well. The next day there was the treatment with compound 9 at a concentration of 10 μm for 30 minutes. After treatment with tBOOH (final concentration 400 μm) measured the amount of ATP remaining in the cells using a kit for monitoring ATP (Perkin Elmer Life Science). The results are presented in Fig.10.

Experimental example 5: the Protective effect of compound 9 against cell death after in vitro ischemic reperfusion injury

To measure the protective effect of compound 9 against necrosis of H9C2 cells after ischemic reperfusion injury following experiments are: double staining with FDA and PI, counting the number of cells, FACS analysis and measurement of mitochondrial swelling.

Cell culture and hypoxic conditions damage: H9C2 Cells were incubated in DMEM, �tereasa FBS and antibiotic. When 80-90% of the cells were confluent, the experiment was carried out. 24 hours before the experiment, H9C2 cells were sown in 35-mm vessel and 150-mm vessel. When about 80% of the cells were confluent, the medium was changed to DMEM containing 0.5% FBS and antibiotic. Cells were incubated in a hypoxic chamber for 24 h. At the point of 23.5 h in a hypoxic chamber cells were treated with different chemicals (with 0.01% DMSO, compound 9 (20 μm), vitamin C (10 μm) and z-VAD-fmk (20 μm)). Cells were transferred to an incubator (37°C) and then treated with H2O2(400 μm) directly by reincubate. The reoxygenation was performed for 1.5 hours.

Experimental example 5-1: Measurement of the protective effect by double staining with FDA and PI after ischemic reperfusion injury in H9C2

H9C2 cells incubated in 35-mm vessel for measuring fluorescence, double-stained with FDA (fluorescein diacetate, Sigma) and PI (iodide of propidium). Preparing the mother liquors FDA and PI. Cells were washed with PBS and 1.5 ml of cell cultural medium was added to 4.5 μl of a solution of FDA (5 mg/ml) and 4 µl of PI solution (2 mg/ml). Using a fluorescent microscope the received image, painted FRA/PI. The results are shown in Fig.11.

Experimental example 5-2: Measurement of the protective effect by counting cells subjected to double staining with FDA and PI after ischemic reperfusion�about damage in H9C2

After receiving the image, painted FRA/PI, using a fluorescence microscope in experimental example 5-1 counted the number of living cells and dead cells using Image Pro. The results are presented in Fig.12.

Experimental example 5-3: Measuring the protective effect of FACS analysis of cells subjected to double staining with annexin V and PI after ischemic reperfusion injury in H9C2

Apoptotic or necrotic cells were stained using the kit to detect, including FITC and annexin V, and then analyzed using flow cytometer. Cells incubated in 35-mm vessel, divided into three (3) groups according to PI staining and annexin V - FITC: not processed, single staining and double staining. After reoxygenation, the collected cells are centrifuged at 2000 rpm at 4°C for 10 minutes. After removal supernatants environment the cells are re-suspended with buffer, annexin V binding, stained with 5 μl PI and 5 μl of annexin V - FITC for 15 minutes in the dark and then analyzed by FACS (cell sorter fluorescence-activated cells). The results are shown in Fig.13.

Experimental example 5-4: Measurement of inhibitory effect against mitochondrial swelling after ischemic reperfusion injury in H9C2

Experimental example 5-5: Measurement of mitochon�territorial opacities and necrosis of nuclei using double staining of MitoTracker and PI after ischemic reperfusion injury in H9C2

Visualization of mitochondria and a three-dimensional image: to visualize the mitochondria of H9C2 cells incubated in 35 mm SOUD, prepared 1 mm MitoTracker Green FM, dissolved in DMSO. Cells were washed with medium for cell cultures and then subjected to dual-color 0.5 mm MitoTracker Green FM and 4 µl of PI solution. After 1 h cells were analyzed in the fluorescence microscope and received three-dimensional image using the Imaris program.

Electron microscope: Used H9C2 cells, which were incubated in 24-well plate, having at the bottom coverslip. Cells were fixed in 0.1 M sodium phosphate buffer (pH 7.4) containing 2% glutaraldehyde. Cells were washed with PBS buffer and then postvaccinial 1% OsO4and 1.5% potassium hydroxide for 1 h. the Cells were sequentially washed with PBS buffer and twice with distilled water and then treated with filtered 0.5% uranylacetate at 4°C over night. Cells were washed twice with distilled water, legalrational ethanol and twice embedded in Epon for 1 h each time. The cells in the resin Epon was polymerized at 60°C for 2 days and then analyzed by electron microscopy.

Fluorescent immunoablative: the tissue Sections, filled OCT, washed with ice-a 0.05% CORRESPONDENT for 10 minutes three times. The tissue sections were treated with blocking solution containing 1% BSA at room temperature�reux for 1 h. To conduct immunofluorescent staining for cardiac myosin heavy chain, the tissue sections were treated with antibody to heavy chain cardiac myosin (1:100, Abcam) as primary antibodies. The tissue sections were washed twice with PBS-Tween 20 and then stained with a secondary antibody, donkey artemisinin 633 IgM (Invitrogen) and washed twice with PBS-Tween 20. For staining agglutinin from wheat germ tissue sections were processed by the agglutinin from wheat germ Alexa Fluor 555 (Invitrogen) at 37°C for 10 minutes. To conduct immunofluorescent staining for alpha-sarcomeric actin tissue sections were treated with primary antibody, an antibody against alpha-sarcomere actin at 4°C for 24 hours. The tissue sections were washed twice in 0.05% CORRESPONDENT and then stained with a secondary antibody, donkey antimachine IgG Alexa Fluor 555 (Invitrogen). The tissue sections were stained with hematoxylin for 5 seconds to stain the nucleus. After mount preparations of tissue sections on a fluorescent microscope received image. The results are shown in Fig.15.

Experimental example 6: the Protective effect of compound 9 against heart damage by ischemia/reperfusion in SD rats

Pets: Male SD rats (270-360 g) were purchased from Seoul National University Animal Center. Rats were kept in polyspecific free from pathogens when the light turns�and I was off on a 12-hour cycle temperature and humidity were maintained at 22°C and 55%, respectively. Provided standard solid food for rodents and drinking water ab libitum. Before the experiment, which was conducted with the permission of the IACUC at Seoul National University, each animal was given an adaptation period of 1 week.

Preparation for surgery: after an adaptation period of 1 week, rats were anestesiologi a mixture of ketamine (100 mg/kg, Yuhan Corp., Seoul, Korea) and xylazine (10 mg/kg, Bayer, Shawnee Mission, Kansas, USA) by intraperitoneal infusion. After appropriate anesthesia performed basic echocardiography and then all rats were submitted 100% oxygen.

Ischemic reperfusion was induced by temporary ligation of the anterior descending branch of the left artery (LAD) for 45 minutes. LAD was ligated using 8-0 Ethilon (Ethicon) using a polyethylene tube 10 within 45 minutes. Ischemia was determined by the naked eye discoloration of the myocardium and ventricular tachyarrhythmias.

Rats were divided into three groups: control group (5 ml 0.9% saline solution), group treated with cyclosporine (25 mg/kg in 5 ml of 0.9% saline solution), and the group treated with compound 9 (30 mg/kg in 5 ml of 5% dextrose). The drug was administered by infusion over 20 minutes via the tail vein using an infusion pump for the animals.

Reperfusion was started UDA�Yaya ligature and a plastic tube 10 and the characteristics caused by typical hyperemic changes in the damaged myocardium during the first few minutes, was defined as the reperfusion. Loose pieces of seams kept to determine the ischemic area to the end.

After the operation, in the case of the group treated with compound 9, compound 9 was administered orally once daily at the same dose for 3 days. The remaining groups were the introduction of the same dose.

The study of the function of infarcted rat heart by echocardiography Under anesthesia hair was removed the left half of the chest. Transthoracic echocardiography was performed immediately before coronary ligation and data was collected using Doppler echocardiography system with a linear sensor. The image of the heart obtained in a two-dimensional view mode okoloplodnoj long axis and short axis. View along the short axis, including papillary muscle, was used to define the location of the cursor M-mode vertically relative to the interventricular septum and the posterior wall of the left ventricle. Images obtained during the training period were not registered. End-diastolic (LVEDD) and end-systolic (LVESD) dimensions of the left ventricle was measured according to the method advanced technology American echocardiographic society. Percentage (%) shortening fraction Le�wow ventricle (LV) was calculated by the equation 100×(LVEDD-LVESD)/LVEDD.

Analysis of tissue infarcted rat heart: Later, 3 days after the events of myocardial ischemia, rats were anestesiologi. To estimate the size of the breach, filled with paraffin representative slices LV cut into sections with a thickness of 4 μm. Neutrophils were stained with hematoxylin and eosin and then examined under a light microscope. On an area of 1 mm2each slice was measured, the number of inflammatory cells. The area of fibrosis, painted trichromat by Mason, was investigated using a system of image analysis (Image Pro, version 4.5; MediaCybernetics, Bethesda, MD, USA). Measuring the area of fibrosis was performed for 2 individual slices, and the average value was used for numerical analysis.

For staining histological method and study of rat hearts were placed in OCT compound, having the 8 µm sections (Tissue-Teck, Sakura, TORRANCE, CA, USA), quickly frozen and preserved. Staining was quantitatively measured by the number of positive cells to the staining in 10 fields of view under high magnification (HPF) for each animal (n=3). For assessment of apoptosis was performed TUNEL analysis (set Chemicon S7100, Chemicon, Temecula, CA, USA) and staining of histological method for caspase-3. Positive cells by TUNEL analysis or by staining for caspase-3 was investigated in 10 fields under a microscope for at least 3 different sections for each animal salt licking�.

Immunofluorescent staining was performed using protivootechny troponin I (Santa Cruz Biothechnology). For detection used anywhereman with FITC goat entirelife antibody to IgG (Molecular Probes). The red staining PI was performed to show the nucleus, and staining Alexa HMGB1 using Fluro 350 conducted to show the core. Colocalization red Dil-labeled MSC and green connexin-43 or expression cardiotrophin I examined in the fluorescence microscope (LSM 510 META, Carl Zeiss, Peabody mA, USA).

Measurement of infarct size: infarct Size was determined after 72 hours after I/R-injury (ischemic reperfusion injury) of the myocardium was evaluated by the percentage of AAR (area at risk). The ratio of AAR to LV (left ventricle) is the level of I/R-damaged myocardial tissue. The ratio of IS/AAR was an accurate index to determine IS in the damaged myocardium and is the primary endpoint of assessing the effectiveness of drug therapy. To determine the AAR, the left coronary artery was again bandaged and 4% Evans Blue dye was administered by infusion through the thoracic aorta retrograde method. The heart was rapidly removed, washed in 0.9% saline solution and then kept in a freezer (-20°C). The heart was cut into five 1-mm transverse sections. Sections of the hearts were kept in 1% solution of chloride triphenyltin�Azalia for 15 min at 37°C and then placed in formaldehyde solution. Normal tissue were stained in red and infarct tissue was stained in white. Image slices of the heart were obtained in digital mode (Canon EOS 400D) under the microscope and using the software ImagePro (version 1.34), IS measured, AAR and the total area of LV.

Numerical analysis: All data were represented by a mean (SE). Sequential variables were compared using t-test, and multiple comparisons were performed variance analysis with a posteriori correction of Bonferroni. If p-value less than 0.05, the value was considered as statistically significant, and all analyses were performed using SPSS, version 17.0 (SPSS, Chicago, Il, USA).

The effect of suppressing compound 9 increased mass of the left ventricle (LV) is shown in Fig.16, and the effect of suppressing the connection 9 of the LV shortening fraction after I/R-damage shown in Fig.17, and the result of comparative analysis for compounds 9 and CsA (cyclosporine A) in relation to fibrosis of the myocardium is shown in Fig.18.

Pharmaceutical composition for prevention or treatment of diseases associated with oxidative stress is selected from the group consisting of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and insulinopenia episodes), MERRF syndrome (myoclonic epilepsy with ragged muscle fibers) or syndrome Kearns-they, arrhythmia, cardioplegia �whether myocardial infarction, containing a therapeutically effective amount of a compound of the following formula (1) or (2), its pharmaceutically acceptable salt or isomer as an active ingredient, and a pharmaceutically acceptable carrier:

in the formula (1):
na denotes the number 1 or 2,
Andandrepresents a 5-membered heteroaryl or heterocycle, each of which has 2 heteroatom selected from N, O and S,
Rlais R5a-Xa-Ba-X'and-,
Inandrepresents a direct link,
Xandand X'andindependently from one another represent a direct bond or-OC(O)-,
R5arepresents hydrogen or 6-9-membered monocyclic or condensed cyclic heterocycle or heteroaryl, each of which has 1 to 3 heteroatoms selected from N, O and S,
and optionally substituted with oxo or C1-C6-alkyl,
R2ais -(CR8aR9a)pa-Ya-R7a,
pa denotes the number 0 or 1,
R8aand R9aindependently from each other represent hydrogen,
Yarepresents a direct bond or-O-,
R7arepresents hydrogen or phenyl,
R3arepresents hydrogen,
R4arepresents -(CH2)pa-Da-R10a-,
Darepresents C5-cycloalkyl or 6-membered heterocycle they�et 1 heteroatom, selected from N, S and O
R10arepresents hydrogen,

in the formula (2):
nb denotes the number 1,
mb stands for 1,
Abrepresents phenyl,
Xbpresents With,
R1brepresents hydrogen,
R2brepresents hydrogen,
R3brepresents hydrogen,
R4bis-YbR11bwhere Ybrepresents a direct bond, R11brepresents -(CH2)tbBb-R13bwhere tb denotes the number 1, Bbrepresents a 6-membered heterocycle that has 2 heteroatom selected from N, S and O, R13brepresents oxo,
R5brepresents hydrogen or 6-membered heterocyclic1-C6-alkyl, where heterocyclyl has 1 heteroatom selected from N, S and O
R6bis -(CR7bR8b)pb-Zb-Db-Wb-R14bwhere R7bR8bindependently from each other represent hydrogen, pb denotes the number 0 or 1, Zbrepresents a direct bond, Dbrepresents a direct bond or a 6-membered heterocycle, which has 1 heteroatom selected from N, S and O, Wbrepresents a direct link, -C(O)- or-S(O)yb-, yb denotes the number 2, where R14brepresents C1-C6-alkyl or 6-membered heterocycle, which has 1 heteroatom selected from N, S and O.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention deals with method of obtaining 5(6)-amino-2-(4-aminophenyl)benzimidazole by catalytic hydration of 2',4,4'-trinitrobenzanilide in medium of amide solvent from the series: dimethylformamide, dimetylacetamide, N-methylpyrrolidone, with separation of formed 2',4,4'-triaminobenzanilide from hydrolysate in form of monosulphate salt with further cyclodehydration of monosulphate salt of 2',4,4'-triaminobenzanilide with heating in aqueous sulphuric acid, separation of sulphate salt of 5(6)-amino-2-(4-aminophenyl)benzimidazole by its crystallisation and filtration and neutralisation with base, characterised by the fact that separation of monosulphate salt of 2',4,4'-triaminobenzanilide is carried out in presence of lower aliphatic alcohol or acetone and cyclodehydration is carried out in presence of activated coal.

EFFECT: elaborated is novel method of obtaining compound, characterised by higher productivity and low expenditures on reagents.

2 cl, 1 dwg, 4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of organic chemistry and deals with method of obtaining 5(6)-amino-2-(4-aminophenyl)benximidazole, including catalytic hydration of 2',4,4'-trinitrobenzanylide in medium of amide solvent, separation of formed 2',4,4'-triaminobenzanylide from hydrogenisate in form of salt of mineral acid, cyclodehydration of 2',4,4'-triaminobenzanylide salt in mineral acid medium, separation of formed 5(6)-amino-2-(4-aminophenyl)benximidazole in form of mineral acid salt with further purification of 5(6)-amino-2-(4-aminophenyl)benximidazole salt in water solution with activated coal and neutralization with base, characterised by the fact that 2',4,4'-triaminobenzanylide is separated from hydrogenisate in form of trihydrochloride in presence of lower aliphatic alcohol or acetone.

EFFECT: elaborated is novel method of obtaining 5(6)-amino-2-(4-aminophenyl)benximidazole, characterised by high productivity and reduction of energy consumption for amide solvent regeneration.

2 cl, 4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of general formula (I) , where is a substituted 5-member heteroaryl ring selected from thienyl, thiazolyl, oxazolyl, pyrrolyl, imidazolyl or pyrazolyl, W is selected from a group comprising N and -C=; M is selected from a group comprising -C(O)N(R1)OR2, -CXCONR1R2 and -C(O)OR1, or M is -C1-C2alkyl-C(O)N(R1)OR2, wherein is , R1 and R2 are independently selected from a group comprising -H, C1-C3-alkyl, C6-aryl, and C1-C3-alkyl-C6-aryl; R is selected from a group comprising H, C1-C3alkyl, halogen, NR1R2, -OR1 and C6aryl; n is an integer from 0 to 1; L and Y are as indicated in the claim; and to compounds of formula (II) , where L2 is selected from a group comprising H, - C0-C3alkyl- C6aryl, -C0-C3alkyl-heteroaryl, where the heteroaryl is pyridyl; -C1-C6alkyl, Y and M are the same as for compounds of formula (I). The invention also relates to a pharmaceutical composition based on compounds (I) and (II), having inhibiting action on histone deacetylase (HDAC), a method of inhibiting and a method of treating a disease which is sensitive to the HDAC inhibitor.

EFFECT: compounds of formula I and II as histone deacetylase inhibitors.

18 cl, 18 dwg, 10 tbl, 19 ex

FIELD: chemistry.

SUBSTANCE: invention relates to agriculture. 2-(1N-benzimidazol-2-yl)-5'-nitrobenzoic acid of formula 1 is antidote for protection of sunflower seedlings from negative action of herbicide of 2,4-dichlorophenoxyacetic acid: Interaction of o-phenylenediamine with 2-formyl-5-nitrobenzoic acid in ratio 1:1 in acetic acid at room temperature for 1.5 hour is carried out.

EFFECT: invention makes it possible to reduce temperature mode and reduce reaction time.

2 cl, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to an agent for activation of lipoprotein lipase containing a benzene derivative of general formula (1) which is used for preventing and treating hyperlipidemia and obesity. The invention also refers to the benzene derivatives of general formula (1a).

EFFECT: composition improvement.

8 cl, 6 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new bicyclic heterocyclic derivatives of general formula wherein radicals and symbols are specified in the patent claim. Said compounds are FGFR receptor (fibroblast growth factor receptor) inhibitors. The invention also refers to a method for preparing a preferential group of compounds of formula (I), to a pharmaceutical composition containing said compounds, and to the use of said compounds for treating diseases, e.g. cancer.

EFFECT: preparing the new bicyclic heterocyclic derivatives.

22 cl, 16 tbl, 422 ex

FIELD: chemistry.

SUBSTANCE: antifungal agents are 5(6)-alkoxycarboxamine-derivatives of 2-aryl-1-hydroxybenzimidazole-3-oxide of formulae I , II, where R-Me, Ar=Ph (a); R=Et, Ar=Ph (b); R=PhCH2, Ar=Ph (c); R=Me, Ar=4-BrC6H4 (d); R=Me, Ar=3-NO2C6H4 (e), R=Me, Ar=4-MeOC6H4 (f). The invention also relates to a method for synthesis of said antifungal agents.

EFFECT: novel antifungal agents based on compounds of formulae I, II, have high fungicidal and fungistatic activity towards Microsporum canis, Trichophyton rubrum, Candida albicans.

3 cl, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel benzimidazole derivatives of formula

and pharmaceutically acceptable salts and esters thereof, where R1 denotes C1-10alkyl, lower alkoxy group-lower alkyl, lower alkoxy group-carbonyl-lower alkyl, C3-6cycloalkyl, C3-6cycloalkyl-lower alkyl, phenyl, phenyl-lower alkyl, di(phenyl)-lower alkyl, heterocyclyl, such as piperidinyl, tetrahydropyranyl, 2-oxo-pyrrolidinyl-lower alkyl, where the cycloalkyl, phenyl or heterocyclyl group is optionally substituted with 1-2 substitutes independently selected from a group comprising lower alkyl, lower alkoxy group, lower alkoxy group-carbonyl, morpholinyl, formylamino group and halogen; R2 denotes hydrogen or lower alkyl; R3 denotes lower alkyl, C3-6cycloalkyl, partially unsaturated cyclohexyl, phenyl, phenyl-lower alkyl, pyridinyl, benzodioxolyl, tetrahydropyranyl, where the phenyl group is optionally substituted with 1-2 substitutes independently selected from a group comprising a halogen, lower alkyl, lower alkoxy group, fluoro-lower alkyl, fluoro-lower alkoxy group, N(lower alkyl)2; R4 denotes: a) heteroaryl which is an aromatic 5-6-member monocyclic ring or a 9-10-member bicyclic ring containing 1 or 2 heteroatoms selected from nitrogen, oxygen and/or sulphur, which is optionally substituted with 1-2 substitutes independently selected from a group comprising lower alkyl, phenyl, lower alkoxy group, -N(lower alkyl)2, oxo group, NH2, halogen, cyano group and morpholinyl; b) unsubstituted naphthyl, naphthyl or phenyl, which are substituted with 1-3 substitutes independently selected from a group comprising halogen, hydroxy group, NH2, CN, hydroxy-lower alkyl, lower alkoxy group, lower alkyl-carbonyl, lower alkoxy group-carbonyl, sulphamoyl, di-lower alkyl-sulphamoyl, lower alkyl-sulphonyl, thiophenyl, pyrazolyl, thiadiazolyl, imidazolyl, triazolyl, tetrazolyl, 2-oxopyrrolidinyl, lower alkyl, fluoro-lower alkyl, fluoro-lower alkoxy group, N(lower alkyl)2, carbamoyl, lower alkenyl, benzoyl, phenoxy group and phenyl which is optionally substituted with 1-2 substitutes independently selected from halogen and fluoro-lower alkyl; or c) if R3 denotes cycloalkyl and R1 denotes cycloalkyl, then R4 can also denote phenyl; R5, R6, R7 and R8 independently denote H, halogen, lower alkoxy group or lower alkyl, or R6 and R7, which are bonded to each other, form a 6-member aromatic carbocyclic ring together with carbon atoms to which they are bonded; provided that the compound of formula (I) is not selected from a group comprising butylamide 2-[2-(2-chlorophenyl)benzoimidazol-1-yl]-4-methylpentanoic acid and 2-(2-benzo[1,3]dioxol-5-ylbenzoimidazol-1-yl)-N-benzyl-butyric acid amide. The invention also relates to a pharmaceutical composition based on the formula I compound.

EFFECT: novel benzimidazole derivatives which are useful as farnesoid X receptor antagonists are obtained.

30 cl, 379 ex

FIELD: chemistry.

SUBSTANCE: invention concerns novel compounds of the formula I , where: n equals 0 or 1; Z denotes N, CR8, where R8 denotes H, C1-C6alkyl; R1 R2, R4 and R5 independently denote H, halogen, OH, -XNR9R10, where X denotes a chemical bond, and R9 and R10 denote H; R3 denotes - NR11S(O)2R12, -NR11C(O)R12, -C(O)OR11 -NR11R12, S(O)2NR11R12 and - C(O)NR11R12, where R11 and R12 independently denote H, C1-C6alkyl; R7 denotes hydrogen, C1-C6alkyl; R7 denotes phenyl substituted with 1 or 2 groups independently selected from fluorine and C1-C6alkyl, and pharmaceutically acceptable salts thereof.

EFFECT: compounds exhibit TRO mimetics activity, which enables preparation of medicinal agents from them for increasing level of thrombocytes in the blood of an animal.

9 cl, 2 dwg, 15 ex

FIELD: medicine.

SUBSTANCE: in new compounds of formula (I): R1 is specified from the group including -OR7 and -NR8R9; where R7 means hydrogen; R8 and R9 are independently chosen from the group including hydrogen, aryl, substituted aryl containing one to three substitutes, preferentially one to two substitutes independently chosen from the group including alkenyl, substituted alkenyl containing one to three, preferentially one to two substitutes independently chosen from the group including carboxyl and carboxyl esters; R2 and R12 are independently chosen from the group including hydrogen, alkyl and alkyl substituted with 5-hydroxy-indolyl group; or R2 and R12 together with carbon atom whereto attached, form cycloalkyl group; R3 means hydrogen; each R4 independently means halogen; Q means oxygen; X means oxygen; R5 means alkylene; R6 is specified from the group consisting of substituted aryl containing one to three, preferentially one to two substitutes independently chosen from group consisting of acylamino, aryl, substituted aryl, containing one to three, preferentially from one to two substitutes chosen from halogen; and n is equal 0 to 3; or to its pharmaceutically acceptable salts. Besides the invention concerns a pharmaceutical composition, to the method of treatment or prevention of virus infections in mammals.

EFFECT: production of the new biologically active compound active with respect to treatment of virus infections in mammals, partially mediated with a member of flaviviridae virus family.

12 cl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula 1c

, where A, B, R1, R2 and n have values given in the description, and pharmaceutically acceptable salts thereof. The invention also relates to pharmaceutical compositions based on compounds of formula 1c, which are used as modulators of ATP-binding cassette ("ABC") transporters or fragments thereof, including cystic fibrosis transmembrane conductance regulator ("CFTR"). The present invention also relates to a method of modulating ABC-transporter activity and methods of treating ABC-transporter mediated diseases using compounds of formula 1c.

EFFECT: improved method.

32 cl, 3 tbl, 118 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing 3-substituted 2-amino-1-hydroxy-5,6-dicyanoindoles of general formula: where R denotes H(a); CH3(b); OCH3(c); Cl(d); F(e), comprising a first step where 4-bromo-5-nitrophthalonitrile reacts with a sodium salt of 2-substituted oxobutenitrile in molar ratio 1:2, respectively, at temperature T=19…25°C for 1-2 hours in a dimethyl formamide (DMF), after which the reaction mass is diluted with tenfold excess water with T=0…25°C. The released resinous residue is extracted with dichloromethane, thoroughly washed with water and chromatographed on silica gel. The eluent (solvent) is evaporated. The residue of the intermediate product is filtered and re-crystallised from alcohol. At the second step of the method, the tin dichloride solution in concentrated hydrochloric acid is mixed with the solution of the obtained intermediate product in ethyl alcohol in molar ratio 3.5-4.5:1, respectively, at temperature T=30…50°C and reaction time of 1-2 hours, after which the reaction mixture is diluted with tenfold excess water with T=0…25°C, and the precipitate is filtered and re-crystallised from alcohol.

EFFECT: obtaining desired products with output of 69-81% which can be used in synthesis of biologically active substances, fluorescent materials and phthalocyanines.

1 dwg, 1 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to hair and/or scalp treatment compositions. There is described a hair and/or scalp care composition containing 0.05 to 20 wt % of amino-oxo-indole-ylidene compound of general formula (I):

where: R1 and R4 - hydrogen, R2 - naphthyl, R3 - C1-3-alkylene aryl.

EFFECT: said amino-oxo-indole-ylidene compounds have an effect of CBR-activators, and the offered composition on the basis enable treatment and prevention of dandruff symptoms such, as scalp itching and desquamation.

8 cl, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to novel substituted 4(6)-bromine-5-hydroxy-1H-indol-3-yl acetic acids and their esters, their pharmaceutically acceptable salts and/or hydrates which have antiviral activity, particularly against influenza virus having general formula or : where: R1 is an amino group substitute selected from a hydrogen atom, C1-C5alkyl optionally substituted with a hydroxy group, mono- or di(C1-C5 alkyl)amino group; acyl which is benzoyl optionally substituted with a halogen; or sulphonyl which is (C1-C5alkyl)sulphonyl, phenylsulphonyl optionally substituted with a C1-C5alkyl group, phenyl-C1-C3 alkylsulphonyl, 6-member nitrogen-containing heteroarylsulphonyl; R2 is an alkyl substitute selected from a hydrogen atom, a halogen atom, hydroxyl, amino, mono-(C1-C5 alkyl)- or di(C1-C5 alkyl)amino group; R3 is hydrogen optionally substituted with C1-C5alkyl; R4 is a hydroxyl group substitute selected from a hydrogen atom, C1-C5alkyl, C1-C5acyl; R5 is cyclic system substitute selected from hydrogen or mono(C1-C5 alkyl)- or di(C1-C5 alkyl)aminomethylene group. The invention also relates to a focused library, a medicinal agent and a pharmaceutical composition.

EFFECT: obtaining of compounds with antiviral activity.

26 cl, 1 tbl, 18 ex

The invention relates to new derivatives of indole-2,3-dione-3-oxime of the formula I

< / BR>
where R1means hydrogen, C1-C6alkyl; R3means Het or a group of the formula

< / BR>
where Het means tetrahydrofuryl, which can be substituted one or more times with substituents selected from the group consisting of halogen, C1-C6of alkyl, C1-C6alkoxy and oxo; at least one of R31, R32and R33independently represents hydrogen, C1-C6alkyl or hydroxy WITH1-C6alkyl and at least one of R31, R32and R33independently represents (CH2)nR34where R34represents hydroxyl, carboxyl,1-C6alkoxycarbonyl,3-C7-cycloalkyl-C1-C6-alkoxycarbonyl, isoxazolidine ring which may be substituted one or more times with substituents selected from the group consisting of halogen, C1-C6of alkyl, C1-C6alkoxy, oxo, or СОNR35R36where R35and R36is hydrogen, C1-C6alkyl, гUB>6alkoxycarbonyl,3-C7cycloalkyl-C1-C6-alkoxycarbonyl, or R35and R36together with the N atom to which they are attached, form a saturated 6-membered heterocyclic ring may contain one additional atom On; n represents 0, 1, 2 or 3; R5means phenyl which may be substituted by phenyl or SO2NR51R52where R51and R52each independently mean C1-C6alkyl, or R51and R52together with the N atom to which they are attached, form a saturated 6-membered monocyclic heterocyclic ring; a is a ring condensed with the benzene ring at the positions marked "a" and "b", and formed the following bivalent radicals: a-CH2-NR6-CH2-CH2-b, where R6means1-C6alkyl, or pharmaceutically acceptable salt

Crystals // 2556206

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention describes crystals of 2-{4-[N-(5,6-diphenylpyrazin-2-yl)-N-isopropylamino]butyloxy}-N-(methylsulphonyl)acetamide ("compound A"), as a form I of the compound A crystal, which shows diffraction peaks at 9.4 degrees, 9.8 degrees, 17.2 degrees and 19.4 degrees in its power X-ray diffraction spectrum, as a form II of the compound A crystal, which shows diffraction peaks at 9.0 degrees, 12.9 degrees, 20.7 degrees and 22.6 degrees in its power X-ray diffraction spectrum, as a form III of the compound A crystal, which shows diffraction peaks at 9.3 degrees, 9.7 degrees, 16.8 degrees, 20.6 degrees and 23.5 degrees in its power X-ray diffraction spectrum. There are also described methods for producing the forms I, II and III of the compound A crystal, based pharmaceutical composition and PGI2 receptor agonist agent, an accelerating agent for angiogenic therapy, gene engineering or autoimmune bone marrow transplantation, and an accelerating agent for angiogenesis for peripheral artery recovery or angiogenic therapy on the basis thereof; there are also described a preventive or therapeutic agent for a wide range of diseases and conditions.

EFFECT: preparing the new therapeutic agent for the wide range of diseases and conditions.

11 cl, 6 dwg, 6 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention represents a mixture of two structural isomers: 2,6-di(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)-4-methylphenol and its diastereomers, and 2-(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)-6-(2,2,1-trimethylbicyclo[2.2.1]hept-5-yl)-4-methylphenol, and their diastereomers with the ratio of the first and second structural isomer isomers from 60:40 wt % to 95:5 wt %.

EFFECT: extension of the arsenal of means, possessing simultaneously haemorheological, anti-aggregate, anti-thrombogenic, retinoprotecting, endothelium-protecting, neuroprotecting, anti-arrhythmia and anti-ischemic activity, enhancing the cerebral blood flow.

4 dwg, 20 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to synthetic biologically active heterocyclic substances having antagonist activity on purinoreceptors and which are products of modifying pyridoxin, particularly n-(1,5-dihydro-3-R-8-methyl-9-hydroxy-[1,3]dioxepino[5,6-c]pyridinyl-6-azo)phenyl sulphonic acid and salt forms thereof of general formula I , where is selected from: a hydrogen atom, ethyl, heptyl or octyl.

EFFECT: compounds of the given formula have high antagonist activity on purinoreceptors and can be used in medicine and veterinary.

2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to use of synthetic biologically active heterocyclic substances as purinoreceptor antagonists, said substances having antagonist activity on purinoreceptors and being a product of modifying pyridoxin, particularly n-(1,5-dihydro-3-R1-3-R2-8-methyl-9-hydroxy-[1,3]dioxepino[5,6-c]pyridinyl-6-azo)phenyl sulphonic acid and salt forms thereof of general formula I , where R1 is a hydrogen atom or methyl, R2 is methyl, isopropyl.

EFFECT: compounds of the given formula have high antagonist activity on purinoreceptors and can be used in medicine and veterinary.

2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula

,

possessing properties of binding with delta opioid receptors. In formula I R1 is selected from the group, consisting of phenyl, pyridinyl and thiazolyl, with R1 being optionally substituted with one or two substituents, independently selected from the group, consisting of C1-4alkoxy, fluorine atom, chlorine atom, bromine atom and cyanogroup; in addition, R1 is optionally substituted with di(C1-4alkyl)aminocarbonyl; Y represents O, S, H3, vinyl, ethinyl or S(O); R2 represents a substituent, selected from the group, consisting of hydrogen, C1-4alkyl, C1-4alkoxy, C1-4alkylthio, fluorine atom, chlorine atom, bromine atom and hydroxy; Ra represents hydrogen or methyl; R3 is selected from the group, consisting of pyrrolidin-2-ylmethyl; pyrrolidin-3-ylmethyl; piperidin-2-ylmethyl, piperidin-3-ylmethyl, piperidin-4-ylmethyl, piperidin-2-ylethyl, piperidin-3-ylethyl, piperidin-4-ylethyl, pyridine-4-yl-(C1-2)alkyl, azetidin-3-ylmethyl; morpholin-2-ylmethyl, morpholin-3-ylmethyl, imidazolylmethyl, thiazolylmethyl, (amino)-C3-6cycloalkyl, 3-hydroxy-2-aminopropyl, 8-azabicyclo[3.2.1]octanyl, 1-azabicyclo[2.2.2]octanyl, guanidinylethyl, 4-(imidazol-1-yl)phenylmethyl, 2-(methylamino)ethyl, 2-diethylaminoethyl, 4-diethylaminobut-2-yl, piperidin-3-yl, piperidin-4-yl and pyrrolidin-3-yl; with piperidin-3-yl being optionally substituted on a carbon atom with phenyl; with pyrrolidin-2-yl in pyrrolidin-2-yl-methyl, pyrrolidin-3-yl, piperidin-3-yl and piperidin-4-yl being optionally substituted on a nitrogen atom with methyl, phenylmethyl, phenethyl or methylcarbonyl.

EFFECT: compounds can be used in the treatment of pain, induced by diseases or conditions, such as osteoarthritis, rheumatoid arthritis, migraine, burn, fibromyalgia, cystitis, rhinitis, neuropathic pain, idiopathic neuralgia, toothache, etc.

24 cl, 3 tbl, 19 ex

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