The method of modulating the function of serine/threonine protein kinase with the compound based on azobenzenes, a method for identifying compounds modulating the function of serine/threonine protein kinase, the method of prevention or treatment of pathological conditions in the body, the connection-based azobenzenes, the method of its synthesis and pharmaceutical composition

 

(57) Abstract:

The invention relates to the field of medicine and organic chemistry and relates to new derivatives of azobenzenes formula I, II or III, modulating the function of serine/threonine protein kinases, methods of modulating the function of serine/threonine protein kinases, the method of identifying compounds modulating the function of serine/threonine protein kinase, the method of treatment-related serine/threonine protein kinase pathological conditions using such compounds, methods of synthesis of the above on the basis of the compounds of formula I, II or III. Connections provide effective modulation of the function of serine/threonine kinases and reduce side effects in the treatment of relevant diseases. 6 C. and 26 C.p. f-crystals, 5 PL.

The technical field to which the invention relates

The following description of the prerequisites of the invention is intended to explain the invention and cannot be considered as prior art.

Transduction of cellular signals is a fundamental mechanism by which external influences that regulate a variety of processes in the cell, is transferred into the cell. One of the key used to regulate the activity of Mature proteins by altering their structure and function.

The most characterized eukaryotic protein kinase phosphorylate proteins on the hydroxy groups of residues serine, threonine and tyrosine. These kinases are divided mainly into two groups - specific fosforiliruyusciye residues serine and threonine and specific fosforiliruyusciye tyrosine residues. Some kinases are enzymes with dual specificity, they are able to fosforilirovanii as a tyrosine residue, and residues of serine/threonine.

Protein kinase is also characterized by localization in the cell.

Some kinases are transmembrane receptor proteins that can bind ligands on the outer side of the cell membrane. Ligand binding leads to changes in the catalytic activity of the receptor protein kinase. Other kinases are preceptory proteins and do not have a transmembrane domain. These preceptory protein kinase may be present in different parts of the cells from the inner side of the cell membrane to the cell nucleus.

Many kinases are included in the regulatory cascade, where their substrates may be other kinase whose activity is regulated by phosphorylation. Ultimately, the subsequent activity of the effector modules include enzymes, which regulate many of the stages of the signaling cascade, including the cascades controlling cell growth, migration and differentiation of cells, gene expression, muscle contraction, glucose metabolism, protein biosynthesis and cell cycle regulation.

Example preceptory protein kinase, fosforiliruyusciye the target proteins at residues serine and threonine is RAF (kinase rapidly increasing fibrosarcoma). RAF modulates the catalytic activity of other protein kinases such as protein kinase, which phosphorylates and thereby activates the mitogen-activated the protein kinase (MARK). Actually the RAF is activated anchored on the membrane protein RAS, which in turn is activated in response to activation by ligands of receptor tyrosine kinases, such as receptor for epidermal growth factor (EGFR) and the receptor for platelet-derived growth factor (DERIVED). The important role of RAF in the regulation of cellular processes show evidence that modified forms of RAF can cause the development of cancer in the body. Data on the value of the RAF in the development of malignant tumors are given in the article Monia et al., Nature Medicine, 2, 668 (1996), fully incorporated in the description by reference, including all figures and the and other diseases specialists in the field of chemistry and biochemistry, created, synthesized and tested compounds that inhibit the function of protein kinases. Some low molecular weight organic compounds are a class of compounds that modulate the function of protein kinases. Examples of compounds which according to inhibit the function of protein kinases, are bis monocyclic, bicyclic or heterocyclic arylesterase connection (PCTWO 92/20642), derived vinile-azaindole (PCT WO 94/14808), 1-cyclopropyl-4-pyridylketone (U.S. patent No. 5330992), compounds of a number of sterile (U.S. patent No. 5217999), styryl-substituted pyridil (U.S. patent No. 5302606), some derivatives of hintline (application for grant of European patent No. 0566266 A1), solenoidal and selenides (PCT WO 94/03427), tricyclic polyhydroxylated compounds (PCT WO 92/21660) and connection-based benzylphosphonic acid (PCT WO 91/15495).

Compounds that can pass through cell membranes and are resistant to acid hydrolysis, have a significant advantage as medicines since become bioavailable after oral administration. However, many of these inhibitors have a weak inhib and therefore, when used as medicines will cause a lot of side effects.

The invention

The present invention partially relates to methods of modulating the function of serine/threonine protein kinases by the compounds based on azobenzenes. Methods include cells expressing the serine/threonine a protein kinase, such as RAF. In addition, the application describes methods of prevention and treatment of pathological conditions associated with serine/threonine protein kinase using compounds according to the invention. Moreover, the invention relates to pharmaceutical compositions comprising compounds identified by the methods according to the invention.

1. Methods of testing compounds for modulation of the function of serine/threonine protein kinase

The methods of the present invention provide a means for modulating functions as a receptor and cytoplasmic serine/threonine protein kinases. These methods provide a means of modulating enzymes both in vitro and in vivo. For the application of in vitro methods according to the invention partially relate to a method of identifying compounds modulating the function of serine/threonine protein kinases.

The term "function" means the role played by the serine/threonine protein kinase in the cell. Family of serine/threonine protein kinases includes enzymes that regulate many of the stages of signaling cascades, including the cascades controlling cell growth, migration and differentiation of cells, gene expression, muscle contraction, glucose metabolism, protein biosynthesis and cell cycle regulation.

The term "modulates" refers to the connection property to change the function of protein kinases. The modulator preferably activates the catalytic activity of protein kinases, more preferably activates or inhibits the catalytic activity of protein kinases, depending on the concentration of compounds influencing the protein kinase, or most preferably inhibits the catalytic activity of protein kinases.

The term "catalytic activity" used in this text means the rate at which the protein kinase phosphorylates the substrate. The catalytic activity can be determined, for example, the PTA is in the active centre of the protein kinase. Usually the active center is a cavity (protein globule), in which the substrate binds to the protein kinase and fosfauriliruetsa.

The term "substrate" used in this text, refers to a molecule, fosforilirovanii serine/threonine protein kinase. Preferably the substrate is a peptide, more preferably a protein. Regarding the protein kinase RAF preferred substrate is MEK (mitogen and extracellular regulatory kinase), and substrate MEK is MARK.

The term "activate" means the activation of the cellular functions of protein kinases. The preferred function of protein kinases is the interaction with a natural binding partner, most preferably catalytic activity.

The term "inhibits" means a reduction in the cellular function of the protein kinase. The preferred function of protein kinases is the interaction with a natural binding partner, most preferably catalytic activity.

The term "modulate" also means changing the function of the protein kinase by increasing or reducing the probability of complex formation between the protein kinase and a natural binding partner. Preferably, module, what more preferably increases or decreases the probability of formation of a complex between the protein kinase and the natural binding partner depending on the concentration of compounds affecting protein kinase, most preferably reduces the probability of formation of a complex between the protein kinase and a natural binding partner.

The term "complex" means an Assembly of at least two molecules. related to each other. Transducers signal complexes often contain at least two related to each other protein molecules. For example, subunit of the receptor protein kinase, GRB2, 80S, RAF and RAS are going in transducible the complex signal in response to mitogenic ligand.

The term "natural binding partner" means polypeptides that are associated with protein kinase in cells. Natural binding partner can perform the function of amplifying the signal in the process of signal transduction by protein kinase. The change of interaction between the protein kinase with a natural binding partner can manifest itself as an increased or decreased probability that the interaction will occur, or increase or decrease the concentration of the complex protein kinase/prirodno with intracellular fragment of a protein kinase. High affinity means the equilibrium constant of the order of 10-6or less. In addition, natural binding partner may also briefly interact with the intracellular portion of the protein kinase and modify it due to chemical reactions. Natural binding partners of the protein kinase is chosen from the group which includes (but is not limited to) the SRC homology domain 2 (SH2) or domain 3 (SH3), other phosphorilation-binding domains (PTB), guanidoacetic metabolic factors, proteinopathy and other protein kinases. Means for determining changes in the interaction of protein kinase C natural binding partners are well known in the art.

The term "serine/threonine protein kinase" refers to the enzyme amino acid sequence which is at least 10% identical to other enzymes, fosforiliruyusciye proteins at residues serine and threonine. Serine/threonine protein kinase catalyzes attach phosphate groups to proteins, the residues of serine and threonine. Serine/threonine protein kinases can be in the form of membrane-associated proteins, as well as in the form of plasma proteins.

The term "contacting" as used in danaidae environment for culturing cells according to the method. The solution containing the compound according to the invention may also comprise another component, such as dimethylsulfoxide (DMSO), which promotes the uptake by cells of the compound or compounds on the basis of azobenzenes. The solution containing the compound based on azobenzenes, can be added to the medium for culturing cells using any of the dosing device, such as a pipette or syringe.

The term "connection-based azobenzenes" refers to organic substituted usabasketball.com connection.

Connection-based azobenzenes represented by the General formula:

The term "substituted" used in this text refers to connection-based azobenzenes, which is derived from any number of alternates.

The preferred embodiment of the invention is a method of modulating the function of serine/threonine protein kinases, in which the protein kinase RAF means.

RAF protein kinase phosphorylates proteins target the residues of serine or threonine. One such protein is the protein kinase (MEK), which phosphorylates and activates the mitogen-activated the protein kinase (M is ImageName receptor tyrosine kinases, such as the receptor for epidermal growth factor (EGFR) and the receptor for platelet-derived growth factor (DERIVED).

The methods of the invention allow the detection of compounds that modulate the function of protein kinase RAF in cells. RAF phosphorylates the protein kinase MEK, which in turn phosphorylates mitogen-activated the protein kinase (MARK). The methods of analysis that allow us to determine the levels of phosphorylation of the protein kinase MEK enzyme RAF, are not sensitive enough due to the very low level of phosphorylation of MEK. To increase the sensitivity of the method according to method according to the invention control the phosphorylation of two enzymes MEK and MARK. The phosphorylation signal MARK amplifies the signal of the phosphorylation of MEK and allows to determine the level of RAF-dependent phosphorylation using an analytical method such as enzyme-linked immunosorbent assay. Furthermore, the method of analysis according to the invention is performed with high productivity, which allows for a short period of time to analyze many compounds.

Another aspect of the invention is a method for identifying compounds modulating the function of serine/threonine protein kinase, comprising the following stages to the entrances.

The term "monitoring" means monitoring the effect obtained after adding the test compound to the cells according to the invention. The result can manifest itself in the form of a change in cell phenotype, cell proliferation, catalytic activity of protein kinases or interaction between a protein kinase and a natural binding partner.

The term "effect" means a change or absence of change in cell phenotype or cell proliferation. "Effect" means a change or absence of change of the catalytic activity of protein kinases. "Effect" means a change or absence of change of interaction between the protein kinase and a natural binding partner.

The preferred embodiment of the invention is a method for identifying compounds modulating the function of serine/threonine protein kinases, where the effect is a change or absence of change in cell phenotype.

The term "cellular phenotype" refers to the appearance of cells or tissue or to the function of cells or tissues. Examples of cell phenotype are the size of cells (decrease or increase), cell proliferation (increase or decrease in the number cleto is spruce cells) or adoption metabolicheskikh nutrients (for example, the consumption of glucose). Change or no change in cell phenotype is easily determined by methods known in the art.

Another preferred embodiment of the invention is a method for identifying compounds modulating the function of serine/threonine protein kinases, where the effect is a change or absence of change of cell proliferation.

The term "cell proliferation" refers to the speed with which the division of groups of cells. The number of cells grown in a vessel for culturing, can be calculated by a person skilled in the art when visually count the number of cells in a certain volume, using an ordinary light microscope. In alternative rate of cell proliferation is calculated using devices in which the density of the cell suspension in an appropriate nutrient medium is measured optical and conductometric methods.

Another preferred embodiment of the invention is a method for identifying compounds modulating the function of serine/threonine protein kinases, where the effect is a change or absence of treason is"interaction", used in this text, refers to the complex formed between the intracellular fragment of a protein kinase and a natural binding partner or compound. The term "interaction" can also be extended to the complex formed between the compound according to the invention and the intracellular and extracellular fragments of the studied protein kinases. Although plasma protein kinase has no extracellular fragment, receptor protein kinase has both fragments.

The term "intracellular fragment" used in this text, means that part of a molecule protein kinase, which is located inside the cell, the Term "extracellular fragment" used in this text, means that part of a molecule protein kinase, which is exposed on the outer side of the membrane.

The preferred embodiment of the invention is a method for identifying compounds modulating the function of serine/threonine protein kinase, which, in addition, includes the following stages: (a) the lizirovania cells with obtaining a lysate containing serine/threonine the protein kinase; (b) the adsorption of serine/threonine protein kinases on the antibody; (C) incubating the adsorbed serine/threonine protein kinase with a substrate of the ring effect on the cells includes measuring the concentration of phosphate in the substrate or in the substrate.

The term "lizirovania" used in this text, refers to the manner of destruction of the cells with the release of the inner content. The lizirovania cells carried out in various ways known to specialists in this field of technology. Preferably cells destroy by sonication or homogenization, more preferably by treatment with a detergent.

The term "antibody", as used herein, refers to a protein that specifically binds a protein kinase. Preferably the antibody binds to a single class of protein kinases, more preferably specifically binds the protein kinase RAF.

The term "specifically binds", as used in this text. refers to an antibody that binds a protein kinase with a higher affinity than another protein kinase or a cellular protein. Specifically binds the protein kinase antibody adsorbs a larger amount of specific protein kinases compared with another protein kinase or cell protein.

The term "adsorption is carried" used in this text, refers to the binding of molecules on the surface of the antibody or solid media. Examples of solid carriers are chemically modificar the AMI, well-known specialists in this field of technology. See, for example, the guide Harlo & Lane. Antibodies, A Laboratory Manual, 1989, Cold Spring Harbor Laboratories.

The term "measuring the phosphate concentration" used in this text refers to techniques well-known to specialists in this field of technology. These techniques may include quantitative determination of the phosphate content in the substrate or determining the relative amount of phosphate in the substrate. These techniques may include the adsorption of the substrate on the membrane and the determination of the phosphate content in the substrate for the measurement of radioactivity.

Another preferred embodiment of the invention is a method for identifying compounds modulating the function of serine/threonine protein kinase, which, in addition, includes the following stages: (a) the lizirovania cells with obtaining a lysate containing RAF; (b) the adsorption of the RAF on the antibody; (C) incubating the adsorbed RAF to MEK and MARK; (g) the adsorption of MEK and MARK on a solid medium or antibodies. Stage monitoring of the effect on the cells includes determining the concentration of phosphate in the above MEK and MARK.

The preferred embodiment of the invention is a method of identification with what has the structure represented by formulas I, II or III, as defined in the description, or any formula of the subgroups presented in the description.

The term "compound" means a compound or its pharmaceutically acceptable salt, ester, amide, prodrug, isomer or metabolite.

The term "pharmaceutically acceptable salt" refers to a form of connection, which does not alter the biological activity and other properties of the connection.

Pharmaceutical salts can be obtained by the interaction of the compounds according to the invention with inorganic or organic acids, such as hydrochloric acid, Hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonate acid, econsultancy acid, para-toluensulfonate acid, salicylic acid, etc.

The term "prodrug" means the agent, which becomes a source of medicinal compound in vivo. In some situations, prodrugs, you can enter more simple way than the original drug connection. For example, the prodrug may be bioavailable by oral administration, and the source connection does not possess such a property, or a prodrug may have a good solution is retene is a method for identifying compounds modulating the function of serine/threonine protein kinases, in which the connection-based azobenzenes has a structure represented by formula I, II or III, and the connection of azobenzenes chosen from the group comprising compounds SABI (the band structures of compounds based on azobenzenes).

The term "connection SABI" means a group of compounds based on azobenzenes, having a structure represented by formula a or b, and numbered from A-1 to A-198 table 1.

II. Methods of prevention or treatment of pathological conditions

Another aspect of the invention is a method of prevention or treatment of pathological conditions of the body by ingestion of the compounds according to the invention, as defined in this text formulas I, II or III with any applicable restrictive conditions presented in this text.

The term "organism" refers to any living object, including at least one cell. The body can be a simple object, such as a eukaryotic cell or a complex organism such as a mammal. In a preferred variant embodiment of the invention the organism is a human Il is arnosti or eliminate the possibility of the occurrence or development of pathologic conditions.

The term "treatment "means a method according to the invention, allowing to obtain a therapeutic effect and at least partially remove or neutralize the pathological state of the organism.

The term "therapeutic effect" refers to the inhibition of cell growth that is caused by a pathological condition or contributes to pathological conditions. The term "therapeutic effect" refers to the inhibition of growth factors causing or contributing to the occurrence of pathological conditions (e.g., cancer). therapeutic effect is also to some extent alleviate one or more symptoms of pathological diseases. Relative to the treatment of cancer, the term "therapeutic effect" means one or more of the following effects: (a) reduction in tumor size; (b) inhibition (i.e., slowing or inhibition) of tumor metastasis; (C) suppression of tumor growth and (g) to some extent alleviate one or more symptoms of pathological conditions. Compounds showing efficacy against leukemia, can be defined, as specified in this text except the alifereti or growth of cells.

The term "pathological condition" refers to the function of cells or tissues that are different from the normal function of this body. The term "pathological condition" can refer to cell proliferation, cell differentiation or survival of cells.

Pathological conditions associated with impaired cell proliferation, include cancers such as fibrous and mesangial violations, violations of development and formation of blood vessels, wound healing, psoriasis, diabetes and inflammatory processes.

Pathological conditions associated with impaired differentiation include (but are not limited to neurodegenerative disorders) slow down the healing process and technique of implantation of tissues.

Pathological conditions associated with impaired survival of cells that belong to the conditions under which activated or broken paths programmed cell death (apoptosis). A number of protein kinases included in the process flow of apoptosis. Dysfunction of one of the protein kinases can lead to the immortalization of cells or to premature cell death.

In the art known simple ways DL is the number of cells or the appearance of cells under the microscope over time (for example, days).

The term "introduction" in the broad sense means providing body connection and more specifically a method of introducing a compound into cells or tissues of the body. Prevention or treatment of pathological conditions can be performed, if the cells or tissues of the body are inside the body or outside the body. Cells that exist outside the body, you can cultivate or grow in Petri dishes. In the art, the number of known methods of introducing the compounds into the cells existing in the body, including (but not limited to) oral, parenteral, dermal, injection and aerosol administration. In the art there is also known a number of ways of introducing the compounds into cells that exist outside of the body, including (but not limited to) methods of microinjection, transformation methods, and methods of using the media.

According to a preferred variant embodiment of the invention relates to a method for prevention or treatment of pathological conditions in which use connection-based azobenzenes, having a structure defined by formula I, II, and III, as specified in this text, or any appropriate subset of these modifications is relates to a method for prevention or treatment of pathological conditions of the body, in which connection on the basis of azobenzenes chosen from the group comprising compounds SABI.

According to another preferred embodiment of the invention relates to a method for prevention or treatment of pathological conditions in which the organism is a mammal.

The term "mammal" refers to such organisms as mice, rats, rabbits, Guinea pigs and goats, more preferably monkeys and primates, most preferably human.

According to another preferred embodiment of the invention relates to a method for prevention or treatment of pathological conditions of the body, in which the pathological condition is cancer or fibrotic disorders.

According to another preferred embodiment of the invention relates to a method for prevention or treatment of pathological conditions of the body where the cancer is chosen from the group comprising lung cancer, ovarian cancer, breast cancer, brain cancer, cancer vnutrivennogo brain cancer, colon cancer, prostate cancer, sarcoma, sarcoma of xeroderma, melanoma and glioma.

According to another preferred volumepoollist state are state, associated with the violation of the path of signal transduction, characterized by the interaction between the serine/threonine protein kinase and a natural binding partner.

The term "path of signal transduction" refers to the spread signal. Mainly external signal is passed through the cell membrane and is converted into an intracellular signal. Then this signal causes a cellular response. The term also includes signals that are distributed only inside cells. Typically, the polypeptides included in the processes of signal transduction, receptor are and preceptories protein kinase, receptor and preceptories proteinopathy, factors nucleotide metabolism and transcription factors.

The term "deviation" in combination with the process of signal transduction refers to a protein kinase, which is located in the body in overactivating or depressed. As a result of mutations of the enzyme has reduced or increased catalytic activity compared to proteinkinase activity of wild-type cells. As a result of mutations of the enzyme is not able to interact with the natural binding partner, and not modified by another protein kinase or proteinopathy.

According to another preferred embodiment of the invention relates to a method for prevention or treatment of pathological conditions of the body, in which the serine/threonine protein kinase is RAF.

III. Compounds and pharmaceutical compositions according to the invention

Another aspect of the invention are compounds based azobenzenes having the structure of formula I, II or III

where

(a) R1, R2, R3and R4independently selected from the group including

(i) hydrogen;

(ii) saturated or unsaturated alkyl;

(iii) N2X3where X2and X3independently selected from the group. including hydrogen, saturated or unsaturated alkyl and (v) a ketone of formula-CO-X4where X4choose from the group. comprising hydrogen, alkyl and remains homozygocity or heterocyclic rings;

(vi) a carboxylic acid of formula -(X5)n-COOH or ester of formula -(X6)n-COOH,-X7where X5X6and X7independently selected from the group including alkyl and remains homozygocity or heterocyclic ring, and n is 0 or 1;

(vii) an alcohol of formula (X8)n-OH, or alkoxylated formula(X8)4-O-X9where X8and X9independently selected from the group including hydrogen, saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings, where the ring for selection substituted by one or more substituents which are independently selected from the group including alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester residues, and n is 0 or 1;

(viii) an amide of formula NHCOX10where X10selected from the group including alkyl, hydroxyl and remains homozygocity or heterocyclic rings, where the ring for selection substituted by one or more substituents which are independently selected from the group including alkyl, alkoxy, halogen, trihalomethyl, carboxy which IMO is chosen from the group comprising hydrogen, alkyl and remains homozygocity or heterocyclic rings;

(x) the remainder homozygotesare or heterocyclic ring, optionally substituted one, two or three substituents, independently selected from the group including alkyl, alkoxy, halogen. trihalomethyl, carboxylate, nitro, and ester residues;

(xi) an aldehyde of the formula-CO-N

(xii) sulfon formula-SO2-X13where X13selected from the group comprising saturated or unsaturated alkyl and remains homozygotesare or heterocyclic ring;

(b) Z1and Z2independently selected from the group including nitrogen, sulfur, oxygen, NH and NR4provided that if one of Z1and Z2means nitrogen, NH or NR4then the other of Z1and Z2means nitrogen, sulfur, oxygen, NH or NR4and

(C) Z3and X1independently selected from the group including nitrogen, sulfur and oxygen.

The term "saturated alkyl" means an alkyl residue that does not contain any alkenovich or alkenovich residues. The alkyl residue may be branched or linear.

The term "unsaturated alkyl" means an alkyl residue, which contains p ptx2">

The term "amine" means a residue of the formula NR1R2where R1and R2independently selected from the group including hydrogen, saturated or unsaturated alkyl and remains homozygocity or heterocyclic ring, which ring can optionally be substituted by one or more substituents independently selected from the group including alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester residues.

The term "aryl" means an aromatic group containing at least one ring with conjugated system of n-electrons, and includes carbocyclic aryl (e.g. phenyl) and heterocyclic aryl group (e.g., pyridine). The term "carbocyclic" means a compound containing one or more covalently closed circular structures, consisting only of carbon atoms. Thus, the term "carbocyclic ring" differs from the term "heterocyclic ring", in which the ring structure contains at least one atom other than carbon. The term "heteroaryl" means an aryl group containing at least one heterocyclic ring.

The term "halogen" means an atom selected from the group vkluchaya the C group, including saturated and unsaturated alkyl and remains homozygocity or heterocyclic ring, and n is 0 or 1.

The term "carboxylic acid" means a residue of the formula -(R)n-COOH, where R is chosen from the group comprising saturated or unsaturated alkyl and remains homozygocity or heterocyclic ring, and n is 0 or 1.

The term "ester" means a residue of the formula -(R)n-COOR’, where R and R’ independently are selected from the group comprising saturated or unsaturated alkyl and remains homozygocity or heterocyclic ring, and n is 0 or 1.

The term "alcohol" means the Deputy formula ROH, where R is chosen from the group comprising hydrogen, saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings, and where the remainder of the ring for selection substituted by one or more substituents which are independently selected from the group including alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester residues.

The term "amide" means the Deputy formula-NHCOR, where R is chosen from the group comprising hydrogen, alkyl, hydroxyl and remains homozygocity or heterocyclic rings, where the remainder of the ring on the choice of substituted one or more Vice-ester residues.

The term "CNS balance" means the Deputy of the formula-OR, where R is hydrogen or a saturated or unsaturated alkyl residue.

The term "aldehyde" refers to the residue of the formula -(R)n-CHO, where R is chosen from the group comprising saturated or unsaturated alkyl and remains homozygocity or heterocyclic ring, and n is 0 or 1.

The term "sulfon" means the residue of the formula-SO3-R, where R is chosen from the group comprising saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where Z1and Z2independently selected from the group including nitrogen and NH.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where R1, R2, R3and R4independently selected from the group including hydrogen, saturated or unsaturated alkyl, substituted choice remains homozygocity or heterocyclic rings, where the remainder of the ring on the choice of substituted one, two or three Zam is XI, alkoxy, carboxylate, nitro, and ester residues; and the remains of homozygocity or heterocyclic rings, which for choosing substituted one, two or three substituents, independently selected from the group comprising alkyl, alkoxy, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, lonefire remains.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where R2and R3are hydrogen.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where R1is a phenyl group, optionally substituted by one. two or three substituents which are independently selected from the group including alkyl, alkoxy, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, or ester residues.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula or III, where R1selected from the group including Vice-SABI.

The term "Vice-SABI" means the group C is tylenil, 3-were, 4-were 2-forfinal, 3-forfinal, 4-forfinal, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 4-(trifluoromethyl)phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-carboxyphenyl, 3-carboxyphenyl and 4-carboxyphenyl.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where X1is grey.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where X1is oxygen.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where Z3is oxygen.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where R4selected from the group comprising methyl and ethyl.

In another preferred variant of embodiment of the invention described connection based azobenzenes having the structure of formula I, II or III, where the connection on the basis of azobenzenes the Kai composition, including a connection according to the invention, as defined in this text or its salt and a physiologically acceptable carrier or diluent.

Another aspect of the invention is a pharmaceutical composition comprising a compound having the structure of formula I, II or III as defined herein, or their subgroups described in this text.

In another preferred variant of embodiment of the invention describes a pharmaceutical composition in which the compound based on azobenzenes chosen from the group comprising compounds SABI.

The term "pharmaceutical composition" means a mixture of compounds azobenzenes according to the invention with other chemical components, such as diluents or carriers. The pharmaceutical composition helps to speed up the introduction of compounds into the body. In the art there are many ways of introducing compounds, including, but not limited to, oral, injection, aerosol, parenteral and local methods. Pharmaceutical compositions can also be produced by the interaction of the compounds with inorganic acids such as hydrochloric acid, Hydrobromic acid, seroshtanova acid, salicylic acid, etc.

The term "fiziologicheskii acceptable" refers to a carrier or diluent that does not cause reduction of biological activity and neutralization properties of the connection.

The term "carrier" refers to a chemical compound that facilitates the introduction of the compound into cells or tissues. For example, dimethylsulfoxide (DMSO) is a common carrier because it promotes the uptake of many organic compounds, cells or tissues of the body.

The term "diluent" refers to chemical compounds which in aqueous solution dissolving the active compound, as well as stabilize the biologically active form of connection. In the art as diluents used salt dissolved in the buffer solutions. One of the most widely used buffer solution is a phosphate buffer solution containing sodium chloride as specified buffer solution composition similar to the salt composition of human blood as salt buffer solution can maintain the pH of the solution at low concentrations, buffer diluent rarely modifies the biological activity of the compounds.

is inane based azobenzenes formula I, II and III, comprising the following stages:

(a) interaction of 2-amino-6-chloro-3-nitropyridine with the second reagent in a solvent to form a first intermediate compound, and the second reagent is a substituted aryl cycle; (b) restoration of the first intermediate compound in the presence of a catalyst and a reducing agent with the formation of the second intermediate compound; (C) the interaction of the second intermediate compound with a third reagent, and (d) purification of the compounds according to the invention.

According to a preferred variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which the substituted aryl cycle is substituted phenol, substituted thiophenol and substituted aniline.

According to a preferred variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which the substituted phenol, substituted thiophenol and substituted aniline is chosen from the group comprising reagents SABI.

The term "reagents SABI" refers to a group of chemicals that includes sodium salt of phenol, 2-NITROPHENOL, 3-NITROPHENOL, 4-NITROPHENOL, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2-cresol, 3-cresol, 4-cresol, 2-toxifera, 3-methoxyphenol, 4-methoxyphenol, 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, thiophenol, 2-nitrothiophene, 3-nitrothiophene, 4-nitrothiophene, 2-chlorothiophenol, 3-chlorothiophenol, 4-chlorothiophenol, 2-thiocresol, 3-thiocresol, 4-thiocresol, 2-portifino, 3-portifino, 4-portifino, 2-(trifluoromethyl)thiophenol, 3-(trifluoromethyl)thiophenol, 4-(trifluoromethyl)thiophenol, 2-methoxybenzoyl, 3-methoxybenzoyl, 4-methoxybenzoyl, 2-mercaptobenzoic acid, 3-mercaptobenzoic acid, 4-mercaptobenzoic acid, aniline, 2-nitroaniline, 3-nitroaniline, 4-nitroaniline, 2-Chloroaniline, 3-Chloroaniline, 4-Chloroaniline, 2-toluidine, 3-toluidine, 4-toluidine, 2-foranyone, 3-foranyone, 4-foranyone, 2-(trifluoromethyl)aniline, 3-(trifluoromethyl)aniline, 4-(trifluoromethyl)aniline, 2-anisidine, 3-anisidine, 4-anisidine, 2-aminobenzoic acid, 3-aminobenzoic acid and 4-aminobenzoic acid.

According to a preferred variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which the solvent is n-propanol.

According to another preferred variant embodiment of the invention using the method of synthesis swedenborgianism variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which the catalyst is Raney Nickel.

According to another preferred variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which the third reagent is methylisoleucine.

According to another preferred variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which the third reagent is the reaction product S-methylisothiourea sulfate and alkylphosphonate.

According to another preferred variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which alkylchlorosilanes is methylchloroform.

According to another preferred variant embodiment of the invention using the method of synthesis of the compounds according to the invention, in which alkylchlorosilanes is ethylchloride.

Description of the invention presented above, does not restrict the scope of the invention and other features and advantages of the invention will become apparent from the following description of the preferred embodiment variants of the invention and from the claims.

Information confirming the possibility of Eoin protein kinases using compounds based on azobenzenes. In addition, the invention in particular relates to methods of identifying compounds modulating the function of serine/threonine protein kinases. These methods include the use of cells expressing the serine/threonine a protein kinase, such as RAF kinase.

RAF is preceptory protein kinase, which is activated in the cell membrane in the process of binding to the activated enzyme RAS, gidrolizuut guaranteesthat. RAS is activated in the process of binding of the activated receptor tyrosine protein kinases, such as EGFR or DERIVED from a protein GRB2 adaptor and factor guaninnukleotidnyi exchange SOS. SOS it guaranteethat from RAS, replaces it on guaranteesthat, thereby activating RAS. Then RAS binds to RAF and thus activates RAF. Then RAF can fosforilirovanii other protein targets for residues of serine and threonine, such as kinase (MEK), which phosphorylates and thus activates the mitogen-activated protein kinase (MARK). Thus, the PAF is a mediator factor controlling mitogen-activated signal transduction.

Due to the important role of the RAF in cells life modifications in the amino acid sequence of RAF can lead to change is highlighted by the discovery of the relationship of mutations in the amino acid sequence of RAF with tumors and cancer. Because mutations in the molecule RAF. causing cancer in the cells that lead to the formation of molecules RAF with unregulated catalytic activity, the RAF inhibitors can reduce or even stop the proliferation of cells, causing cancer of these cells.

The methods according to the present invention can be used to identify compounds that modulates the function of the protein kinase RAF in cells. RAF phosphorylates MEK, which in turn phosphorylates mitogen-activated protein kinase (MARK). Methods of determining only the phosphorylation of MEK using RAF are not sensitive enough, as the levels of phosphorylation of MEK low. To overcome this problem, in the detection method according to the invention includes a definition of phosphorylation of both MEK and MARK. The phosphorylation signal MARK amplifies the signal of the phosphorylation of MEK and allows to determine the level of RAF-dependent phosphorylation method immuno-enzymatic assay (ELISA). Furthermore, the method according to the invention is characterized by high productivity, and thus you can quickly analyze many compounds in a short period of time.

With the help of the F. Such compounds are derived on the basis of azobenzenes.

Although derived on the basis of azobenzenes well studied with respect to their ability to inhibit the enzymes included in the synthesis of nucleotides in bacteria, many of these compounds are not yet sufficiently investigated in relation to inhibition of protein kinases.

Since RAF is characterized by high amino acid homology with other serine/threonine-protein kinases, one can assume that the connection on the basis of azobenzenes will inhibit other serine/threonine protein kinase that is different from the RAF, including receptor and preceptories serine/threonine protein kinase.

The methods according to the invention also apply to other compounds that modulate the function of RAF in cells, as the high performance of these methods allows to analyze a large number of molecules in a short period of time. Therefore, by using the methods according to the invention it is possible to identify a number of existing molecules not included in the scope of the present invention and modulating the function of STK.

I. Biological activity of compounds based on azobenzenes

Connection-based incense RAF. Biological methods of analysis and results of evaluation of the inhibitory capacity presented in this text descriptions. For measurement of modulating the function of protein kinases by the compounds based on azobenzenes use methods with high performance, similar to published by Tang et al. in the application for the grant of a U.S. patent, cep. No. 08/702232 "Indolinone Combinatorial Libraries and Related Products and Methods for Treatment of Disease" (a Combinatorial library indoline-containing and similar products, methods of treatment of diseases), Lyon & Lyon Docket No. 221/187, filed August 23, 1996. Application No. 08/702232 fully included in this description of the invention, including any figures.

II. Disease target for treating the compounds based on azobenzenes

Developed in this invention, the methods, compounds and pharmaceutical compositions intended for the inhibition of disorders of cell proliferation by modulating the function of protein kinase RAF. Disorders of cell proliferation leading to unwanted cell proliferation of one or more subsets of cells in a multicellular organism, which in turn causes harm to the body. The methods, compounds and pharmaceutical compositions described herein, can is installed with premature cell death (i.e. neurological diseases) or inflammatory process. Such violations can result from inappropriate function of the molecules RAF or inappropriate function of molecules such as protein kinase RAF.

Changing functions of the molecules of the RAF protein kinase or kinases that are similar to RAF may result in an increase or decrease in cell proliferation observed in certain diseases. Abnormal condition of cell proliferation include neoplastic diseases, fibrotic disorders, mesangial disorders, abnormal formation and development of blood vessels, wound healing, psoriasis, restenosis, and inflammatory processes.

Fibrotic disorders related to abnormal formation of the extracellular matrix of cells. An example of fibrotic disorders is cirrhosis of the liver. This disease is characterized by an increase in the concentration of components of the extracellular matrix that leads to scarring in the liver, and then to the liver disease.

Proliferative disorders mesangial cells occur due to abnormal differentiation mesangial cells and includes a variety of renal diseases in humans, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic syndromes of microen the problems which can be subjected to treatment using the methods and compounds of the invention are lung cancer, ovarian cancer, breast cancer, brain cancer, cancer vnutrivennogo brain cancer, colon cancer, prostate cancer, sarcoma xeroderma, melanoma and glioma. Data confirming the effectiveness of the use of methods and compounds according to the invention for retaining or changing the direction of the proliferation of cancer cells, presented in this text descriptions in the form of links.

Violations of the formation and development of blood vessels are the result of excessive proliferation of blood vessels. Proliferation of blood vessels necessary for a number of normal processes, such as embryonic development, the formation of the yellow body, the healing of wounds and regeneration of organs. However, the proliferation of blood vessels also plays a significant role in the development of cancerous tumors. Other examples of violations of proliferation of blood vessels include arthritis, where new capillary blood vessels invade the joint and destroy cartilage. In addition, diseases associated with proliferation of blood vessels, include eye diseases such as diabetic retinopathy, when kutasanee with narrowing, spasm or occlusion of blood vessels, such as restenosis, are also the result of a negative impact on the regulation of protein kinases.

Moreover, the processes of formation and development of blood vessels associated with the growth of malignant tumors and metastasis. To continue the growth of emerging cancer requires a supply of oxygen-rich blood with nutrients. As a result, in most cases, simultaneously with the growth of the tumor is formed abnormally large number of capillary blood vessels supplying the tumor. In addition to providing the tumor with nutrients new blood vessels embedded in the tumor, serve as the gateway for cancer cells, which enables the tumor cells in the bloodstream and their metastasis in distant parts of the body (Article Folkman, J. Natl. Cancer Inst., vol.82, page 4-6, 1990).

Inappropriate activity of RAF can stimulate the violation of cell proliferation. It is shown that molecules that specifically modulates the function of the protein kinase RAF, inhibit cell proliferation. More specifically, molecules, antisense nucleic acids, which are intended for binding brushout cells transformation A in vitro (Article Monia et al., Nature Medicine, vol.2, page 688, 1996). This article is fully included in the text of the present invention as a reference, including all figures and tables. Cells A mean malignant human cells.

These studies aimed at RAF antisense suggests that compounds based azobenzenes according to the invention, which modulate the function of protein kinases RAF, can reduce and, apparently, to pay the proliferation of malignant cells in the body. The connection data on the basis of azobenzenes can be analyzed in vitro using the methods described in the example. Moreover, these compounds on the basis of azobenzenes can be analyzed in vivo no effect on tumor cells using xenotransplantation methods, also described in the example.

There are at least two ways in which inappropriate activity of RAF may cause unwanted cell proliferation of a particular cell type: (1) direct stimulation of the growth of individual cells or (2) an increase in tumor blood vessels in specific areas, such as tumor tissue, thus promoting tissue growth.

The use of the present invention will be more effectice violations identified. it is possible to identify patients suffering from such disorders, by analyzing the symptoms using procedures well-known to physicians or veterinarians who are specialists in this field of technology. Such patients can be treated, as explained in the description text.

Determining the dependence of the impairment of cell proliferation from the RAF kinase can be done by determining the first level of RAF activity in a cell or in a particular part of the body of the patient. For example, in the case of cancer cells, the level of activity of one or more RAF can be compared with the level of activity in the cell independent of the RAF and caused RAF cancer. If the tumor cells have higher levels of activity of RAF than in cells with dependent RAF cancer, preferably equal to or more compared to dependent on RAF cancer, these cells are a target for treatment with the use of modulating RAF methods and compounds according to the invention.

In the case of disorders of cell proliferation resulting from unwanted proliferation of non-cancer cells, the level of RAF activity compared with the level of assets is different with the exception of people or animals, suffering from disorders of cell proliferation). If the violation is associated with unwanted cell proliferation, characterized by a higher level of RAF activity compared with the observed level in the General (main) population, this violation is subject to treatment using the described modulation of RAF methods and compounds according to the invention.

III. Pharmaceutical compositions and the introduction of the compound based on azobenzenes

Methods of obtaining pharmaceutical compositions containing the compounds, methods for determining quantities of compounds for introduction patients and methods introduction compounds in the organism described by Tang et al. in the application for the grant of a U.S. patent cep. No. 08/702232 "Indolinone Combinatorial Libraries and Related Products and Methods for Treatment of Disease" (a Combinatorial library indoline-containing and similar products, methods of treatment of diseases), Lyon & Lyon Docket No. 221/187, filed August 23, 1996, and Buzzetti et al. in the published international patent application WO 96/22976 "Hydrosoluble 3-Arylidone-2-Oxindol Derivatives as Tyrosine Kinase Inhibitors" (water-Soluble derivatives of 3-arylidene-2-oxindole as tyrosine kinase inhibitors), published August 1, 1996, Both publications are fully incorporated in this description izobreteniya can be used in the invention and can be easily adapted for use in the invention.

Examples

The following examples do not limit the scope of the invention and represent different aspects and features of the present invention. In the examples described methods of synthesis of compounds of the present invention and methods of measuring the effect of the compound on the function of the protein kinase RAF.

Used in methods cells are commercially available. Vectors of nucleic acids contained in the cells, are also available, and the sequences of genes of different protein kinases can easily be identified using database sequences. Thus, the specialist in the art can easily obtain a cell line using a combination of industrial products of cells, vectors, nucleic acids and genes protein kinases, using methods that are easily accessible to a person skilled in this technical field.

Example 1: Methods of synthesis isopentenyltransferase compounds of the present invention

The present invention will be illustrated in the following examples, without limitation, which, if not specifically covered:

(i) carry out evaporation on a rotary evaporator in a vacuum;

(ii) processes are given p is ografia (HPLC) performed on brasindiana silica gel Merck LiChrosorb RP-18 (Merck, Darmstadt, Germany);

(iv) the outputs of the compounds are given for illustration only and are not necessarily the maximum possible;

(v) the melting temperature is given without amendment and were determined using a digital device for determining the melting temperature HWS Mainz SG 2000;

(vi) the structures of all the compounds of formulas I, II and III of the present invention, podtverjdeny spectroscopy proton nuclear magnetic resonance spectrophotometer Bruker AMX500-NMR, elemental analysis and, in certain cases, mass spectroscopy;

(vii) the purity of the compounds was determined by thin-layer chromatography (TLC) on silica gel (Merck Silica Gel 60 F254) or by HPLC;

(vii) intermediate compounds usually do not fully characterize and assess purity TLC or HPLC.

Methods of synthesis

The compound A-90:

2 methoxycarbonylamino-6-(phenylmercaptan)-3H-imidazo[4,5-b]pyridine

2-Amino-3-nitro-6-(phenylmercaptan)pyridine obtained by boiling under reflux with 2-amino-6-chloro-3-nitropyridine (84,0 g, 0,484 mol) and thiophenolate sodium (Fluka) (72,0 g, 0,545 mol) in 2-propanol (1500 ml) within 2 hours After cooling to room temperature the resulting suspense dried in vacuum at 50C, you get 109,1 g (yield 95%) of 2-amino-3-nitro-6-(phenylmercaptan)pyridine, T. pl. 148-S.

2,3-Diamino-6-(phenylmercaptan)pyridine obtained by hydrogenation of 2-amino-3-nitro-6-(phenylmercaptan)pyridine (107,1 g, 0,433 mol) under a pressure of 5 MPa in an atmosphere of H2in the presence of 30 g of Ni-Raney in 1200 ml of 2-propanol at 70 ° C. After 4 h (29,1 l of hydrogen), the reaction mixture was cooled to 4C with constant stirring. The residue is collected by filtration in vacuo, washed with 2-propanol and dried at 50C in a vacuum. The combined filtrates are concentrated under reduced pressure and recrystallized from 2-propanol. After double washing device for hydrogenation of 1000 ml of THF, evaporation under reduced pressure and recrystallization from 2-propanol precipitate is collected and dried at 50C in a vacuum, you get 80.4 g (yield 87.1 percent) of 2,3-diamino-6-(phenylmercaptan)pyridine, T. pl. 119-S.

2 Methoxycarbonylamino-6-(phenylmercaptan)-3H-imidazo[4,5-b]pyridine obtained by adding dropwise methylcarbamate (34 ml, 0.44 mol) in chilled (5-15C) sulfate solution S-methylisothiourea (53 g, to 0.19 mol) (Aldrich) in 68 ml of water at a temperature below 20C. Then gently add the aqueous solution of sodium hydroxide (116 g, 25% NaOH), while a white precipitate is formed. After 20 NML add a solution of 2,3-diamino-6-(phenylmercaptan) pyridine (37,8 g, 0,174 mol) in 210 ml of ethanol and heated at 85-90 ° C within 2 hours After cooling the reaction mixture during the night the precipitate are filtered, washed with warm water (1000 ml), dried and recrystallized from acetic acid and ethanol at 4C. Precipitates are filtered, washed with methanol and dried at 50C in a vacuum, you get a 30 g (yield 57,4%) 2 methoxycarbonylamino-6-(phenylmercaptan)-3H-imidazo[4,5-b]pyridine, T. pl. 269-C (decomp.).

Compound a-3: 2-Methoxycarbonylamino-6-phenoxy-3H-imidazo[4,5-b]pyridine

2 Methoxycarbonylamino-6-phenoxy-3H-imidazo[4,5-b]pyridine receive a similar procedure as in example A-90, replacing thiophenolate of sodium phenolate sodium, so pl.>280S (decomp.).

Compound a-4: 2 ethoxycarbonyl-6-phenoxy-3H-imidazo[4,5-b]pyridine

2 Ethoxycarbonyl-6-phenoxy-3H-imidazo[4,5-b]pyridine receive is similar to the method shown in example a-3, substituting methyl chloroformiate on ethylchloride, so pl.>280S (decomp.).

The compound a-1: 2-oxo-6-phenoxy-3H-imidazo[4,5-b]pyridine

2-Oxo-6-phenoxy-3H-imidazo[4,5-b]pyridine receive is similar to the method shown in example a-3 using direct interaction O-methylisoleucine with 2,3-diamino-6-(finalmark the P>Compound a-2: 2-Oxo-6-phenylmercaptan-3H-imidazo[4,5-b]pyridine

2-Oxo-6-phenylmercaptan-3H-imidazo[4,5-b]pyridine receive is similar to the method shown in example a-1, replacing the phenolate and sodium thiophenolate sodium, so pl. 253-S.

Connection A-5-a-25

The following connections get the same methodology specified in example a-1, replacing the sodium phenolate to the appropriate thiophenol sodium. In examples a-23, A-24 and a-25 carboxyl group protects methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-5 2-oxo-6-(2-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-6 2-oxo-6-(3-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-7 2-oxo-6-(4-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-8 2-oxo-6-(2-chlorophenoxy)-3H-imidazo[4,5-b]pyridine

A-9 2-oxo-6-(3-chlorophenoxy)-3H-imidazo [4,5-b]pyridine

A-10 2-oxo-6-(4-chlorphenoxy)-3H-imidazo[4,5-b]pyridine

A-11 2-oxo-6-(2-methylphenoxy)-3H-imidazo[4,5-b]pyridine

A-12 2-oxo-6-(3-methylphenoxy)-3H-imidazo[4,5-b]pyridine

A-13 2-oxo-6-(4-methylphenoxy)-3H-imidazo[4,5-b]pyridine

A-14 2-oxo-6-(2-pertenece)-3 is noxy)-3H-imidazo[4,5-b]pyridine

A-17 2-oxo-6-[2-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine

A-18 2-oxo-6-[3-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine

A-19 2-oxo-6-[4-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine

A-20 2-oxo-6-(2-methoxyphenoxy)-3H-imidazo[4,5-b]pyridine

A-21 2-oxo-6-(3-methoxyphenoxy)-3H-imidazo[4,5-b]pyridine

A-22 2-oxo-6-(4-methoxyphenoxy)-3H-imidazo[4,5-b]pyridine

A-23 2-oxo-6-(2-carboxyphenoxy)-3H-imidazo[4,5-b]pyridine

A-24 2-oxo-6-(3-carboxyphenoxy)-3H-imidazo[4,5-b]pyridine

A-25 2-oxo-6-(4-carboxyphenoxy)-3H-imidazo[4,5-b]pyridine

Compound A-26-A-46

The following connections get the same methodology specified in example a-2, replacing thiophenolate sodium on the appropriate thiophenolate. In examples A-44, A-45 and a-46 carboxyl group protects methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-26 2-oxo-6-(2-nitrophenylamino)-3H-imidazo[4,5-b]pyridine

A-27 2-oxo-6-(3-nitrophenylamino)-3H-imidazo[4,5-b]pyridine

A-28 2-oxo-6-(4-nitrophenylamino)-3H-imidazo[4,5-b]pyridine

A-29 2-oxo-6-(2-chlorp the

A-31 2-oxo-6-(4-chlorophenylurea)-3H-imidazo[4,5-b]pyridine

A-32 2-oxo-6-(2-methylphenylimino)-3H-imidazo[4,5-b]pyridine

A-33 2-oxo-6-(3-methylphenylimino)-3H-imidazo[4,5-b]pyridine

A-34 2-oxo-6-(4-methylphenylimino)-3H-imidazo[4,5-b]pyridine

A-35 2-oxo-6-(2-performancepro)-3H-imidazo[4,5-b]pyridine

A-36 2-oxo-6-(3-performancepro)-3H-imidazo[4,5-b]pyridine

A-37 2-oxo-6-(4-performancepro)-3H-imidazo[4,5-b]pyridine

A-38 2-oxo-6-[2-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]pyridine

A-39 2-oxo-6-[3-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]pyridine

A-40 2-oxo-6-[4-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]pyridine

A-41 2-oxo-6-(2-methoxyphenylacetyl)-3H-imidazo[4,5-b]pyridine

A-42 2-oxo-6-(3-methoxyphenylacetyl)-3H-imidazo[4,5-b]pyridine

A-43 2-oxo-6-(4-methoxyphenylacetyl)-3H-imidazo[4,5-b]pyridine

A-44 2-oxo-6-(2-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-45 2-oxo-6-(3-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-46 2-oxo-6-(4-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

Compound A-47-A-68

The following connections get the same methodology specified in example assaut methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-47 2-oxo-6-phenylamino-3H-imidazo[4,5-b]pyridine

A-48 2-oxo-6-(2-nitrophenylamino)-3H-imidazo[4,5-b]pyridine

A-49 2-oxo-6-(3-nitrophenylamino)-3H-imidazo[4,5-b]pyridine

A-50 2-oxo-6-(4-nitrophenylamino)-3H-imidazo[4,5-b]pyridine

A-51 2-oxo-6-(2-chlorpheniramine)-3H-imidazo[4,5-b]pyridine

A-52 2-oxo-6-(3-chlorpheniramine)-3H-imidazo[4,5-b]pyridine

A-53 2-oxo-6-(4-chlorpheniramine)-3H-imidazo[4,5-b]pyridine

A-54 2-oxo-6-(2-methylphenylimino)-3H-imides [4,5-b]pyridine

A-55 2-oxo-6-(3-methylphenylimino)-3H-imidazo[4,5-b]pyridine

A-56 2-oxo-6-(4-methylphenylimino)-3H-imidazo[4,5-b]pyridine

A-57 2-oxo-6-(2-forgenerating)-3H-imidazo[4,5-b]pyridine

A-58 2-oxo-6-(3-forgenerating)-3H-imidazo[4,5-b]pyridine

A-59 2-oxo-6-(4-forgenerating)-3H-imidazo[4,5-b]pyridine

A-60 2-oxo-6-[(2-trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-61 2-oxo-6-[(3-trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-62 2-oxo-6-[(4-trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-63 2-oxo-6-(2-m the ptx2">

A-65 2-oxo-6-(4-methoxybenzylamine)-3H-imidazo[4,5-b]pyridine

A-66 2-oxo-6-(2-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-67 2-oxo-6-(3-carboxymethylamino)-3H-imidazo[4,5-b]pyridine

A-68 2-oxo-6-(4-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

Compounds A-69-a-89

The following connections get the same methodology specified in example a-3, replacing the sodium phenolate to the corresponding phenolate. In examples A-87, A-88 A-89 a carboxyl group protects methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-69 2-methoxycarbonylamino-6-(2-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-70 2-methoxycarbonylamino-6-(3-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-71 2-methoxycarbonylamino-6-(4-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-72 2-methoxycarbonylamino-6-(2-chlorophenoxy)-3H-imidazo[4,5-b]pyridine

A-73 2-methoxycarbonylamino-6-(3-chlorophenoxy)-3H-imidazo[4,5-b]pyridine

A-74 2-methoxycarbonylamino-6-(4-chlorphenoxy)-3H-imidazo[4,5-b]pyridine

A-75 2-methoxycarbonylamino-6-(2-methylphenoxy)-3H-imidazo[4,5-b]-pyridine

A-is phenoxy)-3H-imidazo[4,5-b]-pyridine

A-78 2-methoxycarbonylamino-6-(2-pertenece)-3H-imidazo[4,5-b]-pyridine

A-79 2-methoxycarbonylamino-6-(3-pertenece)-3H-imidazo[4,5-b]-pyridine

A-80 2-methoxycarbonylamino-6-(4-pertenece)-3H-imidazo[4,5-b]-pyridine

A-81 2-methoxycarbonylamino-6-[2-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]-pyridine

A-82 2-methoxycarbonylamino-6-[3-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]-pyridine

A-83 2-methoxycarbonylamino-6-[4-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine

A-84 2-methoxycarbonylamino-6-(2-methoxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-85 2-methoxycarbonylamino-6-(3-methoxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-86 2-methoxycarbonylamino-6-(4-methoxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-87 2-methoxycarbonylamino-6-(2-carboxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-88 2-methoxycarbonylamino-6-(3-carboxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-89 2-methoxycarbonylamino-6-(4-carboxyphenoxy)-3H-imidazo[4,5-b]-pyridine

Compounds a-91-A-111

The following connections will receive is similar to the method shown in example a-90, replacing thiophenolate sodium on the appropriate thiophenolate. In examples A-109, A-110 and a-111 carboxyl group protects mate the Hai remove the protection with the formation of the desired compounds.

A-91 2-methoxycarbonylamino-6-(2-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-92 2-methoxycarbonylamino-6-(3-nitrophenylamino)-3H-imidazo [4,5-b]-pyridine

A-93 2-methoxycarbonylamino-6-(4-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-94 2-methoxycarbonylamino-6-(2-chlorophenylurea)-3H-imidazo[4,5-b]-pyridine

A-95 2-methoxycarbonylamino-6-(3-chlorophenylurea)-3H-imidazo[4,5-b]-pyridine

A-96 2-methoxycarbonylamino-6-(4-chlorophenylurea)-3H-imidazo[4,5-b]-pyridine

A-97 2-methoxycarbonylamino-6-(2-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-98 2-methoxycarbonylamino-6-(3-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-99 2-methoxycarbonylamino-6-(4-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-100 2-methoxycarbonylamino-6-(2-performancepro)-3H-imidazo[4,5-b]-pyridine

A-101 2-methoxycarbonylamino-6-(3-performancepro)-3H-imidazo[4,5-b]-pyridine

A-102 2-methoxycarbonylamino-6-(4-performancepro)-3H-imidazo[4,5-b]-pyridine

A-103 2-methoxycarbonylamino-6-[2-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]pyridine

A-104 2-methoxycarbonylamino-6-[3-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]pyridine

A-105 2-local-6-(2-methoxyphenylacetyl)-3H-imidazo[4,5-b]-pyridine

A-107 2-methoxycarbonylamino-6-(3-methoxyphenylacetyl)-3H-imidazo[4,5-b] pyridine

A-108 2-methoxycarbonylamino-6-(4-methoxyphenylacetyl)-3H-imidazo[4,5-b]pyridine

A-109 2-methoxycarbonylamino-6-(2-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-110 2-methoxycarbonylamino-6-(3-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-111 2-methoxycarbonylamino-6-(4-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

Compounds A-112 A-133

The following connections get the same methodology specified in example a-3, replacing the sodium phenolate to the corresponding aniline salt. In examples A-131, A-132 a-133 a carboxyl group protects methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-112 2-methoxycarbonylamino-6-phenylamino-3H-imidazo[4,5-b]pyridine

A-113 2-methoxycarbonylamino-6-(2-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-114 2-methoxycarbonylamino-6-(3-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-115 2-methoxycarbonylamino-6-(4-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-116 2-methoxycarbonylamino-6-(2-chlorphen the n

A-118 2-methoxycarbonylamino-6-(4-chlorpheniramine)-3H-imidazo[4,5-b]-pyridine

A-119 2-methoxycarbonylamino-6-(2-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-120 2-methoxycarbonylamino-6-(3-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-121 2-methoxycarbonylamino-6-(4-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-122 2-methoxycarbonylamino-6-(2-forgenerating)-3H-imidazo[4,5-b]-pyridine

A-123 2-methoxycarbonylamino-6-(3-forgenerating)-3H-imidazo[4,5-b]-pyridine

A-124 2-methoxycarbonylamino-6-(4-forgenerating)-3H-imidazo[4,5-b]-pyridine

A-125 2-methoxycarbonylamino-6-[2-(trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-126 2-methoxycarbonylamino-6-[3-(trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-127 2-methoxycarbonylamino-6-[4-(trifluoromethyl)phenylamino]-3H-imidazo [4,5-b]pyridine

A-128 2-methoxycarbonylamino-6-(2-methoxyphenylazo)-3H-imidazo[4,5-b]-pyridine

A-129 2-methoxycarbonylamino-6-(3-methoxybenzylamine)-3H-imidazo[4,5-b]-pyridine

A-130 2-methoxycarbonylamino-6-(4-methoxybenzylamine)-3H-imidazo[4,5-b]-pyridine

A-131 2-methoxycarbonylamino-6-(2-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-132 2-methoxycarbonylamino-6-(3 is idazo[4,5-b]pyridine

Compounds A-134-A-154

The following connections get the same methodology specified in example a-4, replacing the sodium phenolate to the corresponding phenolate. In examples A-152, A-153 a-154 a carboxyl group protects methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-134 2-ethoxycarbonyl-6-(2-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-135 2-ethoxycarbonyl-6-(3-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-136 2-ethoxycarbonyl-6-(4-nitrophenoxy)-3H-imidazo[4,5-b]pyridine

A-137 2-ethoxycarbonyl-6-(2-chlorophenoxy)-3H-imidazo[4,5-b]pyridine

A-138 2-ethoxycarbonyl-6-(3-chlorophenoxy)-3H-imidazo[4,5-b]pyridine

A-139 2-ethoxycarbonyl-6-(4-chlorphenoxy)-3H-imidazo[4,5-b]pyridine

A-140 2-ethoxycarbonyl-6-(2-methylphenoxy)-3H-imidazo[4,5-b]pyridine

A-141 2-ethoxycarbonyl-6-(3-methylphenoxy)-3H-imidazo[4,5-b]pyridine

A-142 2-ethoxycarbonyl-6-(4-methylphenoxy)-3H-imidazo[4,5-b]pyridine

A-143 2-ethoxycarbonyl-6-(2-pertenece)-3H-imidazo[4,5-b]pyridine

A-144 2-ethoxycarbonyl-6-(3-perfino the

A-146 2-ethoxycarbonyl-6-[2-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine

A-147 2-ethoxycarbonyl-6-[3-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine

A-148 2-ethoxycarbonyl-6-[4-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]-pyridine

A-149 2-ethoxycarbonyl-6-(2-methoxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-150 2-ethoxycarbonyl-6-(3-methoxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-151 2-ethoxycarbonyl-6-(4-methoxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-152 2-ethoxycarbonyl-6-(2-carboxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-153 2-ethoxycarbonyl-6-(3-carboxyphenoxy)-3H-imidazo[4,5-b]-pyridine

A-154 2-ethoxycarbonyl-6-(4-carboxyphenoxy)-3H-imidazo[4,5-b]-pyridine

Compounds A-155-A-176

The following examples get the same methodology specified in example a-4, replacing the sodium phenolate to the appropriate thiophenolate. In examples A-174, A-175 a-176 a carboxyl group protects methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-155 2-ethoxycarbonyl-6-phenylmercaptan-3H-imidazo[4,5-b]pyridine

A-158 2-ethoxycarbonyl-6-(4-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-159 2-ethoxycarbonyl-6-(2-chlorophenylurea)-3H-imidazo[4,5-b]-pyridine

A-160 2-ethoxycarbonyl-6-(3-chlorophenylurea)-3H-imidazo[4,5-b]-pyridine

A-161 2-ethoxycarbonyl-6-(4-chlorophenylurea)-3H-imidazo[4,5-b]-pyridine

A-162 2-ethoxycarbonyl-6-(2-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-163 2-ethoxycarbonyl-6-(3-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-164 2-ethoxycarbonyl-6-(4-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-165 2-ethoxycarbonyl-6-(2-performancepro)-3H-imidazo[4,5-b]-pyridine

A-166 2-ethoxycarbonyl-6-(3-performancepro)-3H-imidazo[4,5-b]-pyridine

A-167 2 ethoxycarbonyl-6-(4-performancepro)-3H-imidazo[4,5-b]-pyridine

A-168 2-ethoxycarbonyl-6-[2-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]-pyridine

A-169 2-ethoxycarbonyl-6-[3-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]pyridine

A-170 2-ethoxycarbonyl-6-[4-(trifluoromethyl)phenylmercaptan]-3H-imidazo[4,5-b]pyridine

A-171 2-ethoxycarbonyl-6-(2-methoxyphenylacetyl)-3H-imidazo[4,5-b]pyridine

A-172 2-Atomik)-3H-imidazo[4,5-b]pyridine

A-174 2-ethoxycarbonyl-6-(2-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-175 2-ethoxycarbonyl-6-(3-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-176 2-ethoxycarbonyl-6-(4-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

Compounds A-177-198

The following examples get the same methodology specified in example a-4, replacing the sodium phenolate to the corresponding aniline salt. In examples A-196, A-197 a-198 a carboxyl group protects methyl, ethyl, benzyl, tert-butyl, or other suitable esters and then removed at the last stage protection with the formation of the desired compounds.

A-177 2-ethoxycarbonyl-6-phenylamino-3H-imidazo[4,5-b]pyridine

A-178 2-ethoxycarbonyl-6-(2-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-179 2-ethoxycarbonyl-6-(3-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-180 2-ethoxycarbonyl-6-(4-nitrophenylamino)-3H-imidazo[4,5-b]-pyridine

A-181 2-ethoxycarbonyl-6-(2-chlorpheniramine)-3H-imidazo[4,5-b]-pyridine

A-182 2-ethoxycarbonyl-6-(3-chlorpheniramine)-3H-imidazo[4,5-b]-pyridine

A-183 2-ethoxycarbonyl-6-(4-chlorpheniramine)-3H-imidazo[4,5-b]-pirronello-6-(3-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-186 2-ethoxycarbonyl-6-(4-methylphenylimino)-3H-imidazo[4,5-b]-pyridine

A-187 2-ethoxycarbonyl-6-(2-forgenerating)-3H-imidazo[4,5-b]-pyridine

A-188 2-ethoxycarbonyl-6-(3-forgenerating)-3H-imidazo[4,5-b]-pyridine

A-189 2-ethoxycarbonyl-6-(4-forgenerating)-3H-imidazo[4,5-b]-pyridine

A-190 2-ethoxycarbonyl-6-[2-(trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-191 2-ethoxycarbonyl-6-[3-(trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-192 2-ethoxycarbonyl-6-[4-(trifluoromethyl)phenylamino]-3H-imidazo[4,5-b]pyridine

A-193 2-ethoxycarbonyl-6-(2-methoxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-194 2-ethoxycarbonyl-6-(3-methoxybenzylamine)-3H-imidazo[4,5-b]pyridine

A-195 2-ethoxycarbonyl-6-(4-methoxybenzylamine)-3H-imidazo[4,5-b]pyridine

A-196 2-ethoxycarbonyl-6-(2-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

A-197 2-ethoxycarbonyl-6-(3-carboxymethylamino)-3H-imidazo[4,5-b]pyridine

A-198 2-ethoxycarbonyl-6-(4-carboxyphenylazo)-3H-imidazo[4,5-b]pyridine

Example 2: Method for the determination of fosforiliruyusciye functions RAF

This method of analysis allows to determine the degree katal the ka-target protein kinase MEK). The sequence of the gene RAF described in article c Bonner et al., 1985, Molec. Cell. Biol., 5:1400-1407, and can be obtained in different databases on gene sequences. The design of the vector nucleic acid and cell lines used for this part of the invention, is fully described in the article by Morrison et al., 1988, Proc. Natl. Acad. Sci. USA, 85:8855-8859.

Materials and reagents

1. Cells Sf9 (Spodoptera frugiperda); GIBCO-BRL, Gaithersburg, MD.

2. The RIPA buffer: 20 mm Tris/HCl pH 7.4, 137 mm NaCl, 10% solution of glycerol, 1 mm PMSF, 5 mg/l Aprotinin, 0,5% Triton X-100.

3. Hybrid protein thioredoxin-MEK (T-MEK): expression and purification of T-MEK affinity chromatography is carried out according to the manufacturer's recommendations (the directory To 350-01 and R 350-40, Invitrogen Corp., San Diego. CA).

4. His-MAPK (ERK 2); the expression of His-containing MARK is carried out in cells XL1 Blue, transformed by the vector pUC18, encoding His-MAPK. His-MAPK purified by Ni-affinity chromatography by well-known methods (catalogue 27-4949-01, Pharmacia, Alameda, CA).

5. The sheep antibodies against mouse lgG: Jackson laboratories, West Grove, PA (catalogue 515-006-008, Lot# 28563).

6. Antibodies specific to the protein kinase protein RAF-1: URP2653 company UBI.

7. The buffer for application: PBS, a solution of sodium chloride in phosphate buffer firm GIBCO-BRL, Gaithersburg, MD.

10. DMSO, the company Sigma, St. Louis, MO.

11. Buffer for kinase reaction (KB): 20 mm Hepes/HCl pH to 7.2, 150 mm NaCl, 0.1% Triton X-100, 1 mm PMSF, 5 mg/l Aprotinin, 75 µm orthovanadate sodium, 0.5 mm DTT, and 10 mm MgCl2.

12. A mixture of ATP: 100 mm MgCl2, 300 μm ATP, 10 µci -33P ATP (Dupont-NEN)/ml.

13. The solution to stop reaction: 1% phosphoric acid firm Fisher, Pittsburgh, PA.

14. Strips of filter paper-based phosphate cellulose Walla firm Wallac, Turku, Finland.

15. The rinse solution of filters: 1% phosphoric acid; firm, Fisher, Pittsburgh, PA.

16. The device Plate Tomtec Harvester, the company Wallac, Turku, Finland.

17. Reader Wallac beta plate, Turku, Finland.

18. Polypropylene tablets for ELISA with a V-shaped bottom 96-wells company NUNC (Applied Scientific Catalog, AS-72092).

Methodology

All the subsequent stage is carried out at room temperature unless otherwise stated.

1. The application of antibodies: wells balance 100 µl purified by affinity chromatography specific anticorodal sheep against mouse antibodies (1 μg/100 μl of buffer for drawing) overnight at 4C. Tablets can be used for 2 weeks storage at 4C.

2. Turning the tablet and remove the liquid. Di washed 4 times with buffer for washing. Place the Cup on a paper towel and remove the excess liquid.

4. To each well add 1 µg of Titel specific to RAF-1, and incubated for 1 h then Washed as described in stage 3.

5. Thawed lysates of Sf9 cells infected with RAS/RAF, and dilute them with TBST buffer to a concentration of 10 μg/100 μl. In wells add 10 µg of diluted lysate and incubated for 1 h during incubation tablets shaken. In negative control lysate is added. Lysates from insect cells SF9 infected with RAS/RAF will receive after infection of cells with recombinant baculoviruses (5 Mol and each virus) and collection of cells after 48 hours the Cells are washed 1 time in PBS and are lysed in RIPA buffer. Insoluble components are removed by centrifugation (5 min at 10000g). Aliquots of lysates frozen in a mixture of dry ice/ethanol and stored at-80C until use.

6. Unbound components are removed and washed as described above (stage 3).

7. To each well add 2 μg of T-MEK and 2 μg His-MAPK and the volume was adjusted to 40 μl of buffer for kinase reactions. Methods of purification of T-MEK and MARK from cell extracts as described below in this example.

8. Connection (standard concentrated dissolve the pre-diluted compounds/extracts described in stage 6. Incubated for 20 min In the control of medicinal substances are missing.

9. Kinase reaction activated by adding 5 μl of a mixture of ATP during incubation tablet shaken on a shaker for tablets ELISA.

10. Kinase reaction is stopped after 60 min by the addition to each well 30 μl of a buffer to stop the reaction.

11. Phosphocellulose filter paper strips and the plate is placed in the device Tomtec. Carry out the process of collecting and filter wash solution for washing the filters in accordance with the recommendations of the suppliers. Strips of filter paper is dried. Filter paper strips sealed, and place them in the holder. The holder is placed in the device for determining the radioactivity in the filters determine the amount of radioactive phosphorus.

Another method: aliquots of 40 ál of the individual wells can be transferred to the appropriate position phosphocellulose strips of filter paper. After drying, the filters in the air they are placed in the pallet. The pan gently shake and change the wash solution every 15 min for 1 h Strips of filter paper and air-dried. Strips of filter paper is scah. The holder is placed in the meter and strips determine the amount of radioactive phosphorus.

Value lC50determined in accordance with the methodology for the corresponding compounds based on azobenzenes ELISA-RAF-1:

The value of lC50the concentration of the inhibitor on the basis of azobenzenes, which leads to a decrease in the amount of phosphorylated protein target or cell growth by 50%. Value lC50defined by the degree of phosphorylation of RAF-1 by ELISA are presented in table 2.

Example 3: Purification of MARK and MEK

Proteins MARK and MEK easily expressed in cells by sublimirovanny gene encoding the sequence data of proteins in industrial output vector, which expressive proteins with polyhistidine free end. The genes encoding the sequence of these proteins, can be obtained in the respective laboratories, where usually work with the data of proteins, or by using the cloning of these genes in cells containing cDNA library.

Libraries are released by firms and professionals in the art can easily create probes of nucleic acids, A nucleic acid MEK and MARK, available in various databases of genes, such as Genbank. Cloning of the gene can be carried out very quickly with the use of techniques that are easily accessible for professionals in this field.

Purification of proteins MEK and MARK from cell extracts can be performed using the following modified method described in the article by Robbins et al., 1993, J. Biol. Chem., 268: 5097-5106:

1. Cells are lysed by the methods of scoring, osmotic shock or homogenizer type French Press available to professionals in this field. Suitable buffer for reading aloud described below.

2. Solid media, previously associated with Nickel ions or cobalt, the balance in the equilibrium buffer, described below. Polyhistidine free end specifically binds with atoms of cobalt or Nickel on solid media. Balancing is achieved 3x washing the resin with 10 volumes of the equilibrium buffer in relation to the amount of solid carrier. Solid media are easily accessible for professionals in this field.

3. Cell lysates are added to solid media, and balance in the vessel for a certain period of time. In another way, the solid carrier can podyjí carrier is washed with a buffer for washing, described below.

5 Proteins MEK and MARK elute the required amount of buffer for washing (described below) for the elution of the principal amount of protein from solid media.

Buffer for reading aloud

50 mm sodium phosphate pH 8.0

0.3 M sodium chloride

10 mm-mercaptoethanol

1% NP40

10 mm NaF

0.5 mm Pefablock

Equilibrating buffer

50 mm sodium phosphate pH 8.0

0.3 M sodium chloride

10 mm-mercaptoethanol

1% NP40

10 mm NaF

1 mm imidazole

Buffer for washing

50 mm sodium phosphate pH 8.0

0.3 M sodium chloride

10 mm-mercaptoethanol

1% NP40

10 mm NaF

10 mm imidazole

Buffer for elution

50 mm sodium phosphate pH 8.0

0.3 M sodium chloride

10 mm-mercaptoethanol

1% NP40

10 mm NaF

10-500 mm imidazole

Example 4. The method of determining fosforiliruyusciye functions of the EGF receptor

Kinase activity of the EGF receptor (method of analysis of the EGFR-NIH3T3) in whole cells determined as described in detail in PCT Publication WO 9640116 filed June 5, 1996, Tang et al. "Indolinone Compounds for the Treatment of Disease" góra.

Value lC50measured by the method of determining fosforiliruyusciye functions of the EGF receptor are presented in table 3.

Example 5: the Method of determining the effects of compounds on the basis of azobenzenes on the growth of cells expressing RAS

The following method is designed to measure the growth rates of cells NIH-3T3, ekspressiruyushchikh RAS. The purpose of the method is to determine the effect of compounds on the growth of cells NIH-3T3 expressing H-Ras.

Materials

Sterile tablets with 96 wells flat-bottomed

Sterile tablets with 96 wells with round bottoms

A sterile container with a volume of 25 ml or 100 ml

Pipettes, multi-channel pipetman

Sterile pipette tips

Sterile tubes with a volume of 15 ml and 50 ml

Reagents

of 0.4% SRB in 1% acetic acid

10 mm Tris-base

10% THU (trichloroacetic acid)

1% acetic acid

Sterile DMSO (Sigma)

The compound in DMSO (100 mm or less standard solution)

Trypsin-EDTA (GIBCO BRL)

Cell line

T/H-Ras (clone 7 cells NIH ZTZ expressing genomic fragment of the gene it is deruosi sequence of Ras, subcloning in industrial output vector, which will be consistently transfectional cells NIH-3T3. This fragment comes from genetically transformed allele cHa-ras.

2. Cells NIH-3T3 transfection subcloned vector hospatility method. Cells expressing Ras are selected in 2% serum in DMEM. Visible colonies observed after 2 weeks. Transformed cells are collected to create a stable transformed cell line.

The growth media

2% calf serum/DMEM + 2 mm glutamine, Pen/Strep

Method:

Day 0: the Deposition of cells on the tablets:

This phase of the method is carried out in the kelp.

1. Cells treated with trypsin. 200 μl of the cell suspension is transferred into a 10 ml isotonic buffer. The number of cells counted using an instrument Coulter Counter.

2. Cells were diluted to a concentration of 60,000 cells/ml of medium for growth. To each well of the plate with 96 wells flat bottom transfer 100 ál of cells, i.e., at 6000 cells per well.

3. For each connection, use half a tablet (four rows) and four wells for each concentration of the compound. Four holes left to control the environment.

< is in the incubator, containing 10% CO2.

Day 1: adding a connection:

This stage is carried out under the kelp.

1. In the tablet with 96 wells round bottom in each well of vertical rows (columns) 1 to 11 add 120 ál environment for growth, containing DMSO, with a concentration in 2 times (2x) compared to the final concentration of DMSO defined for the highest concentration of the tested compounds. For example, if the highest concentration is 100 μm, which was obtained from the concentrated standard solution (100 mm), the concentration of 1x DMSO is 0.1%, and then 2 DMSO will be 0.2%. This tablet is used for titration of compound, four rows on 1 connection.

2. In the sterile tube with a volume of 15 ml of the solution prepared with the highest concentration of compounds in the growth medium + 2 DMSO. Requires 1 ml 1 cell line. The initial concentration is usually 100 μm, but may vary depending on the solubility of the compounds.

3. Four wells of column 12 of the plate with 96 holes with a round bottom is placed in 240 ál of solution 2 with the initial concentration. Conduct a series of dilutions of 1:2 in the direction to the right patient the NCI column 2. Then transfer 100 μl of the dilutions of the compounds and 100 ál of medium from the column 1 into the appropriate wells of the plate with 96 wells flat bottom, which contained 100 μl of cell suspension. The total volume in the well is 200 μl.

4. Again put the tablet in the incubator and incubated for 3 days.

Day 4: cell Staining

This stage is performed on a lab table.

1. The environment is removed. To each well was added 200 μl of a cold solution of 10% THU to fix the cells. Tablet incubated at least for 60 min at 4C.

2. THU remove and wash the wells with tap water 5 times. The tablet is dried by placing it side on paper towels.

3. Cells stained by adding to each well 100 μl of 0.4% SRB solution and inquira tablet within 10 minutes

4. The SRB solution is removed and the wells washed with 1% acetic acid 5 times. The tablet is dried by placing it side on paper towels.

5. Dye solubilizers, adding to each well 100 ál of 10 mm solution of Tris-base and inquira for 5-10 min while mixing on a rocking chair.

6. The absorption was measured at 570 nm compared with 630 nm, placing the regulating Ras, as shown in table 4.

Example 6: the Method of determining the effects of compounds on the basis of azobenzenes on cell growth A.

The following method is designed to measure the speed of cell growth A. The purpose of the method is to determine the effect of compounds on cell growth A lung carcinoma person. These cells can be obtained in commercial and industrial enterprises, for example in the collection of ATS (CCL185).

Materials

Sterile tablets with 96 wells flat-bottomed

Sterile tablets with 96 wells with round bottoms

A sterile container with a volume of 25 ml or 100 ml

Pipettes, multi-channel pipetman

Sterile pipette tips

Sterile tubes with a volume of 15 ml and 50 ml

Reagents

of 0.4% SRB in 1% acetic acid

10 mm Tris-base

10% THU

1% acetic acid

Sterile DMSO (Sigma)

The compound in DMSO (original standard solution with a concentration of 100 mm or less)

Trypsin-EDTA (GIBCO BRL)

Cell line and growth medium

Cells A lung carcinoma person (ATSS CCL185)

10% serum embryo is th stage is carried out in the kelp.

1. Cells treated with trypsin. 200 μl of the cell suspension is transferred into a 10 ml istoricheskogo buffer. The number of cells counted using an instrument Coulter Counter.

2. Cells were diluted to a concentration of 20,000 cells/ml of medium for growth. To each well of the plate with 96 wells flat bottom transfer 100 ál of cells, i.e., at 2000 cells per well.

3. For each connection, use half a tablet (four rows) and four wells for each concentration of the compound. Four holes left to control the environment.

4. Gently utrachivayut tablets for uniform binding to cells.

5. Tablets incubated at C in the incubator containing 10% CO2.

Day 1: adding a connection:

This stage is carried out under the kelp.

1. In the tablet with 96 wells round bottom in each well of vertical rows (columns) 1 to 1 1 add 120 ál environment for growth, containing DMSO, with a concentration in 2 times (2x) compared to the final concentration of DMSO defined for the highest concentration of the tested compounds. For example, if the highest concentration is 100 μm, which was obtained from a concentrated solution (100 mm), concentrationtime series on 1 connection.

2. In the sterile tube with a volume of 15 ml of the solution prepared with the highest concentration of compounds in the growth medium + 2 DMSO. Requires 1 ml 1 cell line. The initial concentration is usually 100 μm, but may vary depending on the solubility of the compounds.

3. Four wells of column 12 of the plate with 96 holes with a round bottom is placed in 240 ál of solution 2 with the initial concentration. Conduct a series of dilutions of 1:2 in the direction from right to left, carrying 120 μl from the wells of column 12 in the hole of the column 11 from column 11 to column 10 and so on up to column 2. Then transfer 100 μl of the dilutions of the compounds and 100 ál of medium from the column 1 into the appropriate wells of the plate with 96 wells flat bottom, which contained 100 μl of medium of cell suspension. The total volume in the well is 200 μl.

4. Again put the tablet in the incubator and incubated for 3 days.

Day 5: cell Staining

This stage is performed on a lab table.

1. The environment is removed. To each well was added 200 μl of a cold solution of 10% THU to fix the cells. Tablet incubated at least for 60 min at 4C.

2. THU removed.

3. Cells stained by adding to each well 100 μl of 0.4% SRB solution and inquira tablet within 10 minutes

4. The SRB solution is removed and the wells washed with 1% acetic acid 5 times. The tablet is dried by placing it side on paper towels.

5. Dye solubilizers, adding to each well 100 ál of 10 mm solution of Tris-base and inquira for 5-10 min while mixing on a rocking chair.

6. The absorption was measured at 570 nm compared with 630 nm, placing the tablet in the reader Dynatech ELISA.

Selected compounds, inhibiting the growth rate of cells A, as presented in table 5.

Example 7: the Method of determining the biological activity of modulators RAF in vivo

Xenotransplantation analysis is used to measure the inhibitory ability of compounds according to the invention in relation to the cell tumors of the ovary, melanoma, prostate, lung and breast cancer. The method of analysis described by Tang et al. in PCT publication WO 9640116 filed June 5, 1996, "Indolinone Compounds for the Treatment of Disease" (Connection indolinone for the treatment of diseases) and fully included in the text of the present description by reference, including any figures.

Illust any element or elements, limitations or restrictions that are not described in detail in this text. Used in this text, the terms and expressions should be considered as descriptive and not limiting terms, and should not assume that the use of such terms and expressions excluded any equivalents considered or described or portions thereof. However, it should be understood that within the scope of the claimed invention various modifications and variations. Thus, it should be understood that although the present invention is described in detail in the form preferred options embodiments of the invention and optional features, specialists in the art may use modifications and variations are described in this text, concepts, and that such modifications and variations will be within the scope of the invention claimed in the claims.

Specialist in the art it is evident that the objects of the present invention is easy to reproduce, and to achieve the mentioned objectives, advantages and characteristics of the subject invention. Described in the present invention the molecular complexes and the methods, techniques, methods Le is positive variants of embodiment of the invention and do not limit the scope of the invention. Changes in these characteristics and other use cases that will be obvious to a person skilled in the art and defined in the claims, are within the invention.

Specialist in the art it is obvious that the change of deputies and modifications described in this text, is only possible within the scope and essence of the invention.

Everything mentioned in this text patents and publications define the state of the art to which the invention relates. All patents and publications incorporated in the text of this specification as references to the same extent as if each particular publication was specifically mentioned as included in this description by reference.

The invention illustratively described herein, can be used appropriately in the absence of any element or elements, limitation or limitations which is not described in detail in this text. Thus, for example, any of the used in this text the terms "including", "containing essentially of" and "consisting of" may be replaced by two others. Used in this text terminal, when the use of such terms and expressions excluded any equivalents considered or described or portions thereof. It should be understood that various modifications are possible within the inventive scope of the invention. Thus, it should be understood that although the present invention is described in detail in the form preferred options embodiments of the invention and optional features, specialists in the art may use modifications and variations are described in this text, concepts, and that such modifications and variations will fall within the scope of the invention claimed in the claims.

In addition, if the characteristics or aspects of the invention are described in terms of Markush groups, for specialists in the art it is obvious that in the invention described thus any individual representative or subgroup representatives of the Markush formula. For example, if X is described as being selected from the group including bromine, chlorine and iodine, the points in which X is bromine, and the points in which X is bromine or chlorine, is fully described.

Those links that were not previously included in the description text as links, VK is in many ways the embodiment of the invention claimed in the following claims.

1. Connection-based azobenzenes, having a structure represented by formula I, II or III

where (a) R1, R2, R3and R4independently selected from the group comprising (i) hydrogen; (ii) saturated or unsaturated alkyl; (iii) NX2X3where X2and X3independently selected from the group comprising hydrogen, saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings; (iv) halogen or trihalomethyl; (v) a ketone of formula-CO-X4where X4selected from the group comprising hydrogen, alkyl and remains homozygocity or heterocyclic rings; (vi) carboxylic acid of formula -(X5)n-COOH or ester of formula -(X6)n-COOH-X7where X5, X6and X7independently selected from the group comprising alkyl and remains homozygocity or heterocyclic ring, and n is 0 or 1; (vii) an alcohol of formula (X8)n-OH, or an alkoxy residue of formula(X8)n-O-X9where X8and X9independently selected from the group comprising hydrogen, saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings, where the ring is substituted by choice one is methyl, carboxylate, nitro, and ester residues, and n is 0 or 1; (viii) an amide of formula NHCOX10where X10selected from the group comprising alkyl, hydroxyl and remains homozygocity or heterocyclic rings, where the ring for selection substituted by one or more substituents which are independently selected from the group comprising alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester residues; (ix) -SO2NX11X12where X11and X12selected from the group comprising hydrogen, alkyl and remains homozygocity or heterocyclic rings; (x) the remainder homozygotesare or heterocyclic ring, optionally substituted one, two or three substituents, independently selected from the group comprising alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester residues; (xi) an aldehyde of the formula-CO-N and (xii) sulfon formula-SO2-X13where X13selected from the group comprising saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings;

(b) Z1and Z2independently selected from the group comprising nitrogen, sulfur, oxygen, NH and NR4provided that if one of Z1and Z2means nitrogen, NH or NR4then the other is

(C) Z3and X1independently selected from the group comprising nitrogen, sulfur and oxygen.

2. Connection on p. 1, characterized in that the said Z1and Z2independently selected from the group comprising nitrogen and NH.

3. Connection on p. 2, wherein R1, R2, R3and R4independently selected from the group comprising (i) hydrogen; (ii) saturated or unsaturated alkyl, optionally substituted residues homozygocity or heterocyclic ring, or polycyclic residue rings, in which the mentioned cyclic residue on the choice of substituted one, two or three substituents which are independently selected from the group comprising alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, and ester residue, and (iii) the balance homozygotesare or heterocyclic ring, optionally substituted one, two or three substituents which are independently selected from the group comprising alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, and ester residue.

4. Connection on p. 3, characterized in that the said R2and R3are hydrogen.

5. Connection on p. 4, characterized in that the said R1aaany from the group comprising alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, or ester residue.

6. Connection on p. 5, characterized in that the said R1selected from the group consisting of substituents SABI.

7. Connection on p. 6, characterized in that the mentioned X1selected from the group comprising sulfur, oxygen and NH.

8. Connection on p. 7, characterized in that the said Z3is oxygen.

9. Connection on p. 8, characterized in that the said R4selected from the group comprising methyl and ethyl.

10. Connection on p. 9, characterized in that the said asianshemale compound selected from the group consisting of compounds SABI.

11. The method of modulating the function of serine/threonine protein kinase with the compound based on azobenzenes on PP.1-10, characterized in that the cells expressing mentioned serine/threonine a protein kinase, is brought into contact with the said connection.

12. The method according to p. 11, characterized in that the said serine/threonine protein kinase is RAF.

13. The method according to p. 11, characterized in that the said connection on the basis of azobenzenes chosen from the group comprising compounds SA is different, however, what does the contacting of the cells expressing mentioned serine/threonine the protein kinase with a compound according to PP.1-10 and monitor the effect on these cells.

15. The method according to p. 14, characterized in that the said effect is a change or absence of change in cell phenotype.

16. The method according to p. 14, characterized in that the said effect is a change or absence of change of cell proliferation.

17. The method according to p. 14, characterized in that the said effect is a change or absence of change of catalytic activity referred to serine/threonine protein kinase.

18. The method according to p. 14, characterized in that the said effect is a change or absence of change of interaction between these serine/threonine protein kinase and a natural binding partner.

19. The method according to p. 14, characterized in that the mentioned are lysed cells with obtaining a lysate containing serine/threonine a protein kinase, adsorb mentioned serine/threonine the protein kinase on the antibody, incubated adsorbed serine/threonine the protein kinase with a substrate or substrates and adsorb the above-mentioned substrate or substrates on solid the ation of phosphate in the above-mentioned substrate or substrates.

20. The method according to p. 14, characterized in that, as mentioned serine/threonine protein kinase is chosen RAF, these cells are lysed to obtain a lysate containing RAF, adsorb mentioned RAF on the antibody, incubated adsorbed RAF to MEK and MARK and adsorb mentioned MEK and MARK on a solid medium or antibodies, and the effect on these cells was measured by monitoring the concentration of phosphate in the above-mentioned MEK and MARK.

21. A method of preventing or treating a pathological condition associated with abnormal ways of signal transduction, characterized by the interaction between the serine/threonine protein kinase and a natural binding partner in the body, cancer or fibrotic disorders, including the introduction of the mentioned body connection-based azobenzenes formulas I, II and III

where (a) R1, R2, R3and R4independently selected from the group comprising (i) hydrogen; (ii) saturated or unsaturated alkyl; (iii) NX2X3where X2and X3independently selected from the group including hydrogen, saturated or unsaturated alkyl and remains homozygocity or heterocy, comprising hydrogen, alkyl and remains homozygocity or heterocyclic rings; (vi) carboxylic acid of formula -(X5)n-COOH or ester of formula -(X6)n-COOH-X7where X5, X6and X7independently selected from the group including alkyl and remains homozygocity or heterocyclic ring, and n is 0 or 1; (vii) an alcohol of formula (X8)n-OH, or an alkoxy residue of formula(X8)n-O-X9where X8and X9independently selected from the group including hydrogen, saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings, where the ring for selection substituted by one or more substituents which are independently selected from the group including alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester residues, and n is 0 or 1; (viii) an amide of formula NHCOX10where X10selected from the group including alkyl, hydroxyl and remains homozygocity or heterocyclic rings, where the ring for selection substituted by one or more substituents which are independently selected from the group including alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester residues; (ix) -SO2NX11X12where ical rings; (x) the remainder homozygotesare or heterocyclic ring, optionally substituted one, two or three substituents, independently selected from the group comprising alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester residues; (xi) an aldehyde of the formula-CO-N and (xii) sulfon formula-SO2-X13where X13selected from the group comprising saturated or unsaturated alkyl and remains homozygocity or heterocyclic rings;

(b) Z1and Z2independently selected from the group including nitrogen, sulfur, oxygen, NH and NR4provided that if one of Z1and Z2means nitrogen, NH or NR4then the other of Z1and Z2means nitrogen, sulfur, oxygen, NH or NR4where R4defined above, and

(C) Z3and X1independently selected from the group including nitrogen, sulfur and oxygen.

22. The method according to p. 21, characterized in that the said organism is a mammal.

23. The method according to p. 21, characterized in that said cancer is chosen from the group comprising lung cancer, ovarian cancer, breast cancer, brain cancer, cancer vnutrivennogo brain cancer, colon cancer, prostate cancer, sarcomas is Oh serine/threonine protein kinase chosen by RAF.

25. A pharmaceutical composition comprising a compound based on azobenzenes according to any one of paragraphs.1-10 and a physiologically acceptable carrier or diluent.

26. The method of synthesis of compounds of formula (I) under item 1, characterized in that 2-amino-6-chloro-3-nitropyridine enter into reaction with substituted aryl cycle in a solvent to form a first intermediate compound, restores the received first intermediate compound in the presence of a catalyst and a reducing agent with the formation of a second intermediate compound, which is then injected into the reaction with O-methylisoleucine or the reaction product of sulfate S-methylisothiazoline and alkylphosphonate to obtain the corresponding target product, which is then cleaned.

27. The method according to p. 26, characterized in that as the solvent using n-propanol.

28. The method according to p. 27, characterized in that the said substituted aryl cycle is chosen from the group consisting of substituted phenol, substituted thiophenol and substituted aniline.

29. The method according to p. 28, characterized in that the said substituted phenol, substituted thiophenol and substituted aniline is selected from the group of compounds SABI.

30LASS="ptx2">

31. The method according to p. 26, wherein as the catalyst use Raney Nickel.

32. The method according to p. 26, characterized in that the above-mentioned alkyl use of methyl or ethyl group.

 

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