MXPA00003255A

MXPA00003255A - Methods of modulating serine/threonine protein kinase function with 5-azaquinoxaline-based compounds

Info

Publication number: MXPA00003255A
Application number: MXPA/A/2000/003255A
Authority: MX
Inventors: Kutscher Bernhard; Gerald Mcmahon; Harald App; Eckhard Gunther
Original assignee: Zentaris Gmbh
Priority date: 1997-10-06
Filing date: 2000-04-03
Publication date: 2001-05-07

Abstract

The present invention is directed in part towards methods of modulating the function of serine/threonine protein kinases with 5-azaquinoxaline-based compounds. The methods incorporate cells that express a serine/threonine protein kinase, such as RAF. In addition, the invention describes methods of preventing and treating serine/threonine protein kinase-related abnormal conditions in organisms with a compound identified by the invention. Furthermore, the invention pertains to 5-azaquinoxaline compounds and pharmaceutical compositions comprising these compounds.

Description

DESCRIPTION METHODS TO MODULATE THE FUNCTION OF SERINE / TREONINE PROTEIN KINASE BY COMPOUNDS BASED ON 5-AZAQUINOXALINE BACKGROUND OF THE INVENTION The following description of the background of the invention is provided to help understand the invention, but is not admitted as prior art for the invention. The cellular signal transduction is a fundamental mechanism by which the external stimuli that regulate diverse cellular processes are transmitted to the interior of the cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which allows the regulation of the activity of mature proteins by altering their structure and function. The best characterized protein kinases in eukaryotes phosphorylate proteins in the alcohol moiety of the serine, threonine and tyrosine residues. These kinases are mainly found in two groups, those specific to phosphorylate serine and threonine and those specific to phosphorylate tyrosine. Some kinases, called "double specificity" kinases, are capable of phosphorylating tyrosine residues as well as serine / threonine.

Protein kinases can also be characterized by their position within the cell. Some kinases are transmembrane receptor proteins capable of binding ligands external to the cell membrane. The binding of the ligands alters the catalytic activity of the protein kinase receptor. Others are non-receptor proteins that lack a transmembrane domain. Non-receptor protein kinases can be found in various cell compartments from the inner surface of the cell membrane to the nucleus. Many kinases are involved in regulatory cascades where their substrates may include other kinases whose activities are regulated by their phosphorylation status. Finally, downstream effector activity is modulated by phosphorylation resulting from the activation of such a pathway. The serine / threonine kinase family includes members that regulate many stages of signaling cascades including cascades that control cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis and regulation of the cellular cycle. An example of a non-receptor protein kinase that phosphorylates target proteins on serine and threonine residues is RAF. RAF modulates the catalytic activity of other protein kinases, such as the protein kinase that phosphorylates and therefore activates the mitogen-activated protein kinase (MAPK).

The RAF itself is activated by the membrane-anchored RAS protein, which in turn is activated in response to a ligand that activates the receptor protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) receptor. of platelet-derived growth factor (PDGFR). The biological importance of RAF in the control of cellular events is underlined by the finding that the altered forms of RAF can cause cancer in organisms. Evidence of the importance of RAF in malignant cancers is provided by Monia et al., 1996, Nature Medicine 2: 668, incorporated herein by reference in its entirety and including all figures and tables. In an effort to discover novel treatments against cancer and other diseases, biomedical and chemical researchers have designed, synthesized and tested molecules that inhibit the function of protein kinases. Some small organic molecules of a class of compounds that modulate the function of protein kinases. Examples of molecules that have been reported to inhibit the function of protein kinases are bis-monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindol derivatives (PCT WO 94/14808), l-cyclopropyl-4- pyridylquinones (U.S. Patent No. 5,330,992), styryl compounds (by Levitzki et al., U.S. Patent No. 5,217,999 and entitled "Styril Compounds wich Inhibit EGF Receptor Protein Tyrosine Kinase, Lyon &; Lyon file No. 208/050), pyridyl compounds substituted with styryl (U.S. Patent No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 Al), seleoindoles and selenides (PCT WO 94/03427), polyhydroxy compounds tricyclics (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495). Compounds that can cross cell membranes and that are resistant to acid hydrolysis are potentially advantageous therapeutic substances because they can become highly bioavailable after being administered orally to patients. However, many of these protein kinase inhibitors only weakly inhibit the function of protein kinases. In addition, they can inhibit many protein kinases and therefore cause multiple side effects as therapeutic substances for diseases. Despite the significant progress that has been made in developing compounds for the treatment of cancer, there is a need in the art to identify particular structures and substitution patterns form the compounds capable of modulating the function of particular protein kinases.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed in part to methods for modulating the function of serine / threonine protein kinase with compounds based on 5-azaquinoxaline. The methods incorporate cells that express a serine / threonine protein kinase, such as RAF. In addition, the invention describes methods for preventing and treating abnormal conditions related to serine / threonine protein kinase in organisms with a compound identified by the methods described herein. In addition, the invention pertains to pharmaceutical compositions comprising compounds identified by methods of the invention.

I. Methods to analyze compounds that modulate the serine / threonine protein kinase function The methods of the present invention provide a means to modulate the function of the serine / threonine protein kinase both receptors and cytosol. These methods provide means to modulate the enzymes both in vitro and in vivo. For in vitro applications, the methods of the invention are related in part to the method for identifying compounds that modulate the function of the serine / threonine protein kinase. Therefore, in a first aspect, the invention describes a method for modulating the function of a serine / threonine protein kinase with a compound based on azabenzimidazole. The azabenzimidazole compound optionally is substituted with organic groups. The method comprises contacting cells that express the serine / threonine protein kinase with the compound.

The term "function" refers to the cellular role of a serine / threonine protein kinase. The serine / threonine protein kinase family includes members that regulate many stages in signaling cascades, including cascades that control cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis and regulation of the cell cycle. The term "catalytic activity" in the context of the invention defines the rate at which a protein kinase phosphorylates a substrate. The catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. Phosphorylation of a substrate occurs at the active site of a protein kinase. The active site is usually a cavity in which the substrate binds to the protein kinase and is phosphorylated. The term "substrate" as used herein refers to a molecule phosphorylated by a serine / threonine protein kinase. Preferably, the substrate is a peptide and more preferably a protein. In relation to the protein kinase RAF, the preferred substrates are MEK and the substrate of MEK, MAPK. The term "active" refers to increasing the cellular function of a protein kinase. The protein kinase function is preferably the interaction with a natural binding partner and more preferably with catalytic activity. The term "inhibits" refers to a decrease in the cellular function of a protein kinase. The function of protein kinara preferably is the interaction with a natural binding partner and more preferably with catalytic activity. The term "modulates" refers to altering the function of a protein kinase by increasing or decreasing the likelihood of a complex forming between a protein kinase and a natural binding partner. A modulator preferably increases the likelihood that such a complex forms between the protein kinase and the natural binding partner, and more preferably increases or decreases the likelihood that a complex forms between the protein kinase and the natural binding partner. , depending on the concentration of the compound to which the protein kinase is exposed, and more preferably decreases the likelihood of a complex being formed between the protein kinase and the natural binding partner. A modulator preferably activates the catalytic activity of a protein kinase, more preferably activates or inhibits the catalytic activity of a protein kinase depending on the concentration of the compound exposed to the protein kinase, or more preferably inhibits the catalytic activity of a protein kinase. The term "complex" refers to an assembly of at least two molecules bound together. Signal transduction complexes often contain at least two protein molecules linked together. For example, a protein tyrosine protein kinase receptor GRB2, SOS, RAF and RAS are assembled to form a signal transduction complex in response to a mitogenic ligand. The term "natural binding partner" refers to polypeptides that bind to a protein kinase in cells. Natural binding partners can play a role in the propagation of a signal in a transduction process of a protein kinase signal. A change in the interaction between a protein kinase and a natural binding partner can manifest itself as an increased or decreased likelihood of an interaction forming, or an increased or decreased concentration of the protein kinase / associated natural binding complex . A protein kinase and natural binding partner can bind to an intracellular region of a protein kinase with high affinity. A high affinity represents an equilibrium binding constant in the order of 10"6 M or less.In addition, a natural binding partner can also transiently interact with an intracellular region of protein kinase and chemically modify it. kinase are chosen from a group that includes, but is not limited to the SRC homology 2 (SH2) or 3 (SH3) domains, other phosphoryltyrosine binding domains (PTB), guanine nucleotide exchange factors, protein phosphatase and Other protein kinases Methods for determining changes in interactions between protein kinases and their natural binding partners are readily available in the art The term "serine / threonine protein kinase" refers to an enzyme with an amino acid sequence refers to a enzyme with an amino acid sequence with at least 10% amino acid identity with other enzymes that phosphorylate proteins on r wastes serine and threonine. A serine / threonine protein kinase catalyzes the addition of phosphate in proteins on serine and threonine residues. The serine / threonine protein kinase can exist as membrane bound proteins or as cytosolic proteins. The term "contacting", as used herein, refers to mixing a solution comprising a compound of the invention of 5-azaquinoxaline with a liquid medium bath with the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the 5-azaquinoxaline compound or compounds within the cells of the methods. The solution comprising the 5-azaquinoxaline compound can be added to the media bath of the cells using a delivery apparatus, such as a pipette-based device or a syringe-based device.

The term "5-azaquinoxaline-based compound" refers to an organic compound of 5-azaquinoxaline substituted with chemical substituents. The 5-azaquinoxaline compounds are of the general structure: The term "substituted", with reference to the invention, refers to a 5-azaquinoxaline compound that forms derivatives (is derivatized) with various chemical substituents. In a preferred embodiment, the invention relates to the method for modulating the function of a serine / threonine protein kinase, wherein the protein kinase is RAF. The RAF protein kinase phosphorylates target proteins in the serine or threonine residues. One such protein target is protein kinase (MEK) which phosphorylates and consequently activates the mitogen-activated protein kinase (MAPK). The RAF itself is activated by the membrane-bound guanine triphosphate hydrolyzing RAS enzyme in response to the mitogen-stimulated receptor protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and the growth factor receptor. platelet derivative (PDGFR). The methods of the present invention can detect compounds that modulate the function of the RAF protein kinase in cells. RAF phosphorylates a protein kinase (MEK) which in turn phosphorylates a mitogen-activated protein kinase (MAPK). The assays that only monitor the phosphorylation of MEK by RAF are not sensitive because the levels of MEK phosphorylation are not significant. To resolve this sensitivity dilemma, phosphorylation of both MEK and MAPK is followed in the assays of the present invention. The MAPK phosphorylation signal amplifies the MEK phosphorylation signal and allows RAF-dependent phosphorylation to be followed in assays, such as enzyme-linked immunosorbent assays. In addition, the assay of the invention is carried out in a high performance format so that many compounds can be monitored quickly in a short period of time. In another aspect, the invention describes a method for identifying compounds that modulate the serine / threonine protein kinase function, comprising the steps of contacting cells that express the serine / threonine protein kinase with the compound, and monitoring an effect on the cells The term "monitor" refers to observing the effect of adding the compound to the cells of the method. The effect can be manifested in a change in the cellular phenotype, cell proliferation, protein kinase catalytic activity or in the interaction between a protein kinase and a natural binding partner. The term "effect" describes a change or an absence of change in the cellular phenotype or cell proliferation. "Effect" can also describe a change or an absence of change in the catalytic activity of the protein kinase. "Effect" can also describe a change or absence of a change in an interaction between the protein kinase and a natural binding partner. A preferred embodiment of the invention relates to the method for identifying compounds that modulate the serine / threonine protein kinase function, wherein the effect is a change or an absence of a change in the cellular phenotype. The term "cell phenotype" refers to the outward appearance of a cell or tissue, or the function of the cell or tissue. Examples of cell phenotype are cell size (reduction or enlargement), cell proliferation (increased or decreased number of cells), cell differentiation (a change or absence of a change in cell shape), cell survival, apoptosis (cell death) ), or the use of a metabolic nutrient (for example, glucose uptake). The changes or the absence of changes in the cell phenotype is easily measured by techniques known in the art.

In another embodiment, the invention relates to a method for identifying compounds that modulate the serine / threonine protein kinase function, wherein the effect is a change or an absence of change in cell proliferation. The term "cell proliferation" refers to the rate at which a group of cells is divided. The number of cells growing in a container can be quantified by a person skilled in the art when the person visually counts the number of cells in a defined volume using a common optical microscope. Alternatively, cell proliferation rates can be quantified by laboratory apparatus that optically or conductively measure cell density in an appropriate medium. In another preferred embodiment, the invention relates to a method for identifying compounds that modulate the serine / threonine protein kinase function, wherein the effect is a change or an absence of change in the interaction between serine / threonine protein kinase with an associated of natural union. The term "interaction", in the context of the invention, describes a complex formed between an intracellular region of a protein kinase and a natural binding partner or a compound. The term "interaction" can also be extended to a complex formed between a compound of the invention with intracellular regions and extracellular regions of the protein kinase under study. Although a cytosolic protein kinase will have no extracellular region, a protein kinase receptor will harbor an extracellular and an intracellular region. The term "intracellular region" as used herein, refers to the portion of a protein kinase which exists within a cell. The term "extracellular region", as used herein, refers to a portion of a protein kinase which exists outside the cell. In a preferred embodiment, the invention relates to a method for identifying compounds that modulate the serine / threonine protein kinase function and further comprising the following steps: (a) lysing the cells to obtain a lysate comprising serine / threonine protein kinase; (b) absorbing serine / threonine protein kinase to an antibody; (c) incubating the serine / threonine protein kinase adsorbed with a substrate or substrates; and (d) adsorbing the substrate or substrates to a solid support or antibody. The step of monitoring the effect on the cells comprises measuring the phosphate concentration of the substrate or substrates. The term "lysar", as used herein, refers to a method for breaking the integrity of a cell so that the contents of the interior are released. Cell lysis is carried out by many techniques known to those skilled in the art. The method is preferably carried out by sonication or cell disruption techniques and more preferably by detergent techniques. The term "antibody", as used herein, refers to a protein molecule having * specifically a protein kinase. An antibody preferably binds to a protein kinase class and more preferably binds specifically to the protein kinase RAF. The term "specifically binds", as used herein, refers to an antibody that binds to a protein kinase with greater affinity than to another protein kinase or cellular protein. An antibody that binds specifically to a protein kinase will bind to a higher concentration of the specific protein kinase than to another protein kinase or cellular protein. The term "adsorb" as used herein refers to the attachment of a molecule to the surface of an antibody or solid support. Examples of solid supports are chemically modified cellulose, such as phosphocellulose and nylon. The antibodies can be attached to solid supports using techniques well known to those of ordinary skill in the art. See for example Harlo & Lane, Antibodies, A Laboratory Manual, 1989, Cold Spring Harbor Laboratories. The term "measure phosphate concentration", as used herein, refers to techniques commonly known to those ordinarily skilled in the art.

These techniques may involve quantifying the concentration of phosphate concentrations within a substrate or determining the relative amounts of phosphate within a substrate. These techniques may include adsorbing the substrate to a membrane and detecting the amount of phosphate within the substrate by radioactive measurements. In another preferred embodiment, the invention relates to a method for identifying compounds that modulate the serine / threonine protein kinase function and that further comprise the following steps: (a) lysing the cells to obtain a lysate comprising RAF; (b) adsorbing the RAF to an antibody; (c) incubating the RAF adsorbed with MEK and MAPK; and (d) adsorbing MEK and MAPK to a solid support or an antibody or antibodies. The step of measuring the effect on the cells comprises monitoring the phosphate concentration of MEK and MAPK. In a preferred embodiment, the invention relates to the method for identifying compounds that modulate the function of serine / threonine protein kinase, wherein the compound based on 5-azaquinoxaline has the structure that is set forth in formula I as defined in present or any of the subgroups thereof established here. The term "compound" refers to the compound or a pharmaceutically acceptable salt, ester, amide, prodrug, isomer or metabolite thereof.

The term "pharmaceutically acceptable salt" refers to a formulation of a compound that does not abrogate the biological activity and properties of the compound. Pharmaceutical salts can be obtained by reacting a compound of the invention with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfadic acid, salicylic acid and Similar . The term "prodrug" refers to an agent that is converted to a parental drug in vivo. Medication may be easier to administer than parental medication than in some situations. For example, the prodrug may be available by oral administration, but the parental no, or the prodrug may involve solubility to allow its intravenous administration. In another preferred embodiment, the invention relates to a method for identifying compounds that modulate the function of serine / threonine protein kinase, wherein the compound based on 5-azaquinoxaline has the structure that is established in formula I, wherein a compound based on 5-azaquinoxaline from the group consisting of SAQAR compounds. The term "SAQAR compounds" refers to the group of compounds based on 5-azaquinoxaline having a structure which is set forth in formula I and which are numbered from A-1 to A-90 in the following table: (I) I_L_. Methods to avoid or treat abnormal conditions In another aspect, the invention describes a method for preventing or treating an abnormal condition in an organism. The method comprises the following steps: (a) administering a compound of the invention, as specified herein by formula I with any of the restrictions provided herein, to an organism; and (b) promote or interrupt the abnormal interaction. The term "organism" is related to any living entity that comprises at least one cell. An organism can be as simple as a eukaryotic cell or as complex as a mammal. In preferred embodiments, an organism refers to humans or mammals. The term "prevent" refers to the method of the invention that decreases the likelihood or eliminates the possibility of an organism contracting or developing an abnormal condition. The term "treat" refers to the method of the invention that has a therapeutic effect at least a partial relief or abrogation of the abnormal condition in the organism. The term "therapeutic effect" refers to the inhibition of cell growth caused or contributing to an abnormal condition (e.g. cancer). The term "therapeutic effect" also refers to the inhibition of the growth sectors that cause or contribute to the abnormal condition. A therapeutic effect releases to some extent one or more of the symptoms of the abnormal condition. With reference to the treatment of a cancer, a therapeutic effect refers to one or more of the following: (a) a reduction in the size of the tumor; (b) inhibition (i.e., braking or stopping) of the tumor metastasis; (c) inhibition of tumor growth, - and(d) release to some extent of one or more of the symptoms associated with the abnormal condition. Compounds demonstrating efficacy against leukemias can be identified as described herein, except that instead of inhibiting metastasis, the compounds can instead slow down or decrease cell proliferation or cell growth. The term "abnormal condition" refers to a function in the cells or tissues of an organism that deviates from its normal functions in an organism. An abnormal condition can be related to cell proliferation, cell differentiation or cell survival. Aberrant cell proliferative conditions include cancers such as fibrotic or mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus and inflammation. Aberrant differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing speeds and tissue grafting techniques. The survival conditions of aberrant cells are related to conditions in which programmed cell death pathways (apoptosis) are activated or abrogated. Many protein kinases are associated with the pathways of apoptosis. Aberrations in the function of any of the protein kinases can lead to cell immortality or premature cell death. Cell proliferation, differentiation and survival are phenomena measured simply by methods in the art. These methods may involve observing the number of cells or the appearance of the cells under the microscope with respect to time (for example days). The term "administer" is broadly related to the provision of an organism and more specifically to a method for incorporating a compound into the cells or tissues of an organism. An abnormal condition can be avoided or treated when the body's cells or tissues exist inside the body or outside the body. Cells that exist outside the body can be maintained or grown in a cell culture vessel. For cells harbored within the organism, there are many techniques in the art for administering compounds that include (but are not limited to) oral, parenteral, dermal, injection and aerosol application. For cells outside the organism, there are multiple techniques in the art to administer the compounds, which include (but are not limited to) cell microinjection techniques, transformation techniques and carrier techniques. In a preferred embodiment, the invention relates to a method for preventing or treating an abnormal condition in an organism, wherein the 5-azaquinoxaline-based compound has the structure that is set forth in formula I as defined herein or in any of the subgroups of the same that are established here. In other preferred embodiments, the invention relates to a method for preventing or treating an abnormal condition in an organism, wherein the 5-azaquinoxaline-based compound, having the structure set forth in formula I, is selected from the group consisting of consists of SAQAR compounds. In another preferred embodiment, the invention relates to a method for preventing or treating an abnormal condition in an organism wherein the organism is a mammal.

The term "mammal" preferably refers to organisms such as mice, rats, rabbits, guinea pigs and goats, more particularly to monkeys and apes, and more preferably to humans. In another additional preferred embodiment, the invention relates to a method for preventing or treating an abnormal condition in an organism, wherein the abnormal condition is cancer or a fibrotic disorder. In another preferred embodiment, the invention relates to a method for preventing or treating an abnormal condition in an organism, wherein the cancer is selected with a group consisting of lung cancer, ovarian cancer, breast cancer, brain cancer, intraaxial brain cancer, colon cancer, prostate cancer, sarcoma, Kaposi's sarcoma, melanoma and glioma. In another additional preferred embodiment, the invention relates to a method for preventing or treating an abnormal condition in an organism, wherein the method is applied to an abnormal condition and is associated with an aberration in a signal transduction pathway characterized by a interaction between a serine / threonine protein kinase and a natural binding partner. The term "signal transduction path" refers to the propagation of a signal. In general, an extracellular signal is transmitted through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response. The term also encompasses signals that are propagated completely within a cell. The polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein kinases, receptor and non-phosphatase phosphatase proteins, nucleotide exchange factors and transcription factors. The term "aberration", together with a signal transduction process, refers to a protein kinase that is overexpressed or underexpressed in an organism, mutated so that its catalytic activity is greater or less than the activity of a protein kinase of type wild, mutated so that it can no longer interact with a natural binding partner, nor can it be modified by another protein kinase or protein phosphatase, nor does it interact with a natural binding partner. The term "promote or interrupt the abnormal interaction" refers to a method that can be carried out by administering a compound of the invention to cells or tissues in an organism. A compound can promote an interaction between a protein kinase and the natural binding partners to form favorable interactions with multiple atoms at a complete interface. Alternatively, a compound can inhibit the interaction between a protein kinase and natural binding partners by compromising favorable interactions formed between atoms at the complete interface. In another preferred embodiment, the invention relates to a method for preventing or treating an abnormal condition in an organism when the serine / threonine protein kinase is RAF.

III, Compounds and pharmaceutical compositions of the invention In another aspect, the characteristics of the invention of the 5-azaquinoxaline compounds having the structures established in formula I: (I) wherein (a) Rx, R2 and R6 are independently selected from the group consisting of (i) hydrogen; (ii) saturated or unsaturated alkyl; (iii) an amine of formula NX2X3, wherein X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five or six membered aryl or heteroaryl ring or heteroaryl ring portions; (iv) halogen or trialomethyl; (v) a ketone of formula -CO-X4, wherein X4 is selected from the group consisting of hydrogen, alkyl and five or six membered aryl or heteroaryl portions; (vi) a carboxylic acid of formula - (X5) n -COOH or ester of formula - (X6) n -COO-X7, wherein Xs, X6 and X7 are independently selected from the group consisting of alkyl and aryl or heteroaryl portions of five members or six members and where n is 0 or 1; (vii) an alcohol of formula (X8) n-OH or a portion of formula - (Xt) n-0-X9, wherein X "and X9 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl and portions thereof of a five-membered or six-membered aryl or heteroaryl ring, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trialomethyl, carboxylate, nitro and ester, and wherein n is 0 or 1; (viii) an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, and a five-membered or six-membered aryl or heteroaryl ring portion, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro or ester; (ix), wherein XX1 and X? a are selected from the group consisting of hydrogen, alkyl, and five or six membered aryl or heteroaryl ring portions, - (x) a five membered aryl or heteroaryl ring portion or six members optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester portions; (xi) an aldehyde of formula -CO-H; and (xii) a sulfone of the formula -S02-X13, wherein X13 is selected from the group consisting of saturated or unsaturated alkyl and five or six membered aryl or heteroaryl portions; and (b) X-. it is selected from the group consisting of nitrogen, sulfur and oxygen. The term "saturated alkyl" refers to an alkyl portion that does not contain any alkene or alkyne portion. The alkyne portion may be branched or unbranched. The term "unsaturated alkyl" refers to an alkyl portion that contains at least one alkene or alkyl portion. The alkyl portion may be branched or unbranched. The term "amine" refers to a chemical moiety of formula NRjR2 wherein Rx and R2 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five or six membered aryl or heteroaryl ring portions, wherein the ring is optionally substituted with one or more substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester portions. The term "aryl" refers to an aromatic group which has at least one ring having a system of conjugated pi electrons and which includes both carbocyclic aryl groups (for example phenyl) and heterocyclic aryl groups (for example pyridine). The term "carbocyclic" refers to a compound which contains one or more covalently saturated ring structures and wherein the atoms that form the main structure of the ring are all carbon atoms. Therefore, the term differentiates carbocyclic rings from heterocyclics in which the main ring structure contains at least one atom which is different from carbon. The term "heteroaryl" refers to an aryl group which contains at least one heterocyclic ring. The term "halogen" refers to an atom that is selected from the group consisting of fluorine, chlorine, bromine and iodine.

The term "ketone" refers to a chemical moiety with the formula - (R) n-CO-R ', wherein R and R' are selected from the group consisting of saturated or unsaturated alkyl, and aryl or heteroaryl portions of five members or six members, and wherein n is 0 or 1. The term "carboxylic acid" refers to a chemical moiety with the formula - (R) n -COOH, wherein R is selected from the group consisting of saturated alkyl or unsaturated, and the aryl or heteroaryl portions of five members or six members, and wherein n is zero or one. The term "ester" refers to a chemical moiety with the formula - (R) n-COOR ', wherein R and R' are independently selected from the group consisting of saturated or unsaturated alkyl and of five-membered aryl or heteroaryl portions or six members and wherein n is 0 or 1. The term "alcohol" refers to a chemical substituent of the formula -ROH, wherein R is selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and aryl ring portions or five-membered or six-membered heteroaryl wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester moieties. The term "amide" refers to a chemical substituent of the formula -NHCOR, wherein R is selected from the group consisting of hydrogen, alkyl, hydroxyl, and five or six membered aryl or heteroaryl ring portions, wherein the ring is optionally substituted by one or more substituents, which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro or ester. The term "alkoxy moiety" refers to a chemical substituent of the formula -OR, wherein R is hydrogen or a saturated or unsaturated alkyl moiety. The term "aldehyde" refers to a chemical moiety with formula - (R) n-CHO wherein R is selected from the group consisting of saturated or unsaturated alkyl and aryl or heteroaryl portions of five members or six members and wherein n is 0 or 1. The term "sulfone" refers to a chemical moiety with the formula -S02-R, wherein R is selected from the group consisting of saturated or unsaturated alkyl and five or six membered aryl or heteroaryl portions. In another preferred embodiment, the invention relates to a 5-azaquinoxaline-based compound having a structure as set forth in formula I wherein R 3 and R 4 are independently selected from the group consisting of hydrogen and saturated or unsaturated alkyl. In another further preferred embodiment, the invention relates to a compound based on 5-azaquinoxaline having a structure that is set forth in formula I wherein R 3 and R 4 are hydrogen. In other preferred embodiments, the invention relates to a compound based on 5-azaquinoxaline having a structure that is set forth in formula I, wherein R- and R 2 are selected from the group consisting of hydrogen, saturated or unsaturated alkyl, a A five or six member aryl or heteroaryl ring portion which is optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro and ester portions. In still further preferred embodiments, the invention relates to a 5-azaquinoxaline-based compound having a structure which is set forth in formula I wherein R x is phenyl optionally substituted with one, two or three substituents which are independently selected from the group It consists of alkyl, halogen, hydroxy and alkoxy portions. In another preferred embodiment, the invention relates to a compound based on 5-azaquinoxaline having a structure that is set forth in formula I wherein R 1 is phenyl. In another preferred embodiment, the invention relates to a 5-azaquinoxaline-based compound having a structure that is set forth in formula I, where Rx is -hydroxyphenyl. In other preferred embodiments, the invention relates to a 5-azaquinoxaline-based taxcompound having the structure set forth in formula I, wherein R 2 is selected from the group consisting of hydrogen, saturated or unsaturated alkyl and optionally substituted phenyl with one, two or three substituents that are independently selected from the group consisting of alkyl, halogen, hydroxy and alkoxy portions. In another preferred embodiment, the invention relates to a compound based on 5-azaquinoxaline having the structure set forth in formula I, wherein R 2 is hydrogen. In another preferred embodiment, the invention relates to a compound based on 5-azaquinoxaline having the structure set forth in formula I, wherein R 2 is methyl. In another preferred embodiment, the invention relates to a compound based on 5-azaquinoxaline having the structure set forth in formula I, wherein R 2 is phenyl. In another preferred embodiment, the invention relates to a 5-azaquinoxaline-based compound having the structure set forth in formula I, wherein R2 is E In another preferred embodiment, the invention relates to a compound based on azaquinoxalina that has the structure that is established in the formula I, where Xj. It is nitrogen or oxygen. In another preferred embodiment, the invention relates to a compound based on 5-azaquinoxaline having the structure set forth in formula I, wherein Xi is oxygen. In another preferred embodiment, the invention relates to a compound based on 5-azaquinoxaline having the structure set forth in formula I, wherein X- is nitrogen. In other preferred embodiments, the invention relates to a compound based on 5-azaquinoxaline having the structure set forth in formula I, wherein Rs is selected from the group consisting of hydrogen; optional saturated or unsaturated alkyl substituted with a five-membered or six-membered aryl or heteroaryl ring portion optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro and ester; and a five or six member aryl or heteroaryl ring portion optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro and ester portions. In further preferred embodiments, the invention relates to a 5-azaquinoxaline-based compound having the structure set forth in formula I, wherein the R6 and X1 portions (taken together, form a compound which is selected from the group which consists of the SAQAR substituents The term "SAQAR substituents" refers to the group of substituents consisting of methoxy, benzylamino, 4-fluoro-benzylamino, 2-carboxybenzylamino, 3-carboxybenzylamino, 4-carboxybenzylamino, 2-nitrobenzylamino, 3- Nitrobenzylamino, 4-nitrobenzylamino, 2-methylbenzylamino, 3-methylbenzylamino, 4-methylbenzylamino, 2-chlorobenzylamino, 3-chlorobenzylamino, 4-chlorobenzylamine, 2-fluorobenzylamino, 3-fluorobenzylamino, 4-fluorobenzylamino, 2 - (trifluoromethyl) benzylamine , 3- (trifluoromethyl) benzylamino, 4- (trifluoromethyl) benzylamino, phenethyl-1-amino, phenylamino, 2-carboxy-phenylamino, 3-carboxyphenylamino, 4-carboxyphenylamino, 2-nitrophenylamino, 3-nitrophenylamino, 4-nitrophen ilamino, 2-methylphenylamino, 3-methylphenylamino, 4-methylphenylamino, 2-chlorophenylamino, 3-chlorophenylamino, 4-chlorophenylamino, 2-fluorophenylamino, 3-fluorophenylamino, 4-fluorophenylamino, 2- (trifluoromethyl) phenylamino, 3 - (trifluoromethyl) phenylamino, 4- (trifluoromethyl) phenylamino, pyrid-2-amino-pyrid-3-amino, pyrid-4-yne and pyrid-2-ylamino. The term "benzylamino" refers to a group that has the structure that is established in the following formula: wherein the aryl ring may be optionally substituted in the 2, 3 or 4 position. The term "phenylamino" refers to a group having the structure that is set forth in the following formula: wherein the aryl ring may be optionally substituted at the 2, 3 or 4 position.

The term "phenethyl-1-amino" refers to a group having a structure as set forth in the following formula: The term "pyrid-2-amino" refers to a pyridine ring which is substituted with an NH group at position 2. Similarly, the terms "pyrid-3-amino" and "pyrid-4-amino" refer to a pyridine ring which is substituted with an NH group at positions 3 and 4, respectively. In another preferred embodiment, the invention relates to a 5-azaquinoxaline-based compound having a structure as set forth in formula I, wherein the 5-azaquinoxaline-based compound is selected from the group consisting of SAQAR compounds.

IV. Methods of synthesis of the invention In another aspect, the invention describes a pharmaceutical composition comprising a compound having a structure of formula I as defined herein, any of the subgroups thereof set forth herein, or its salt, and a carrier or physiologically acceptable diluent. In a preferred embodiment, the invention relates to a pharmaceutical composition wherein the 5-azaquinoxaline-based compound is selected from the group consisting of SAQAR compounds. The term "pharmaceutical composition" refers to a mixture of a 5-azaquinoxaline compound of the invention with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. There are multiple techniques in the art for administering a compound that include, but are not limited to, oral, injection, aerosol, parenteral and topical administration. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. The term "physiologically acceptable" defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.

The term "carrier" defines a chemical compound that facilitates incorporation into compound in cells or tissues. For example, dimethyl sulfoxide (DMSO) is commonly used as a carrier that facilitates the uptake of many organic compounds into the cells or tissues of an organism. The term "diluent" defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffer solutions are used as diluents in the art. A buffer solution commonly used is phosphate buffered saline because it mimics the saline conditions of human blood. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound. In still another aspect, the invention describes a method for synthesizing a compound of the invention, comprising the steps of: (a) reacting two amino-6-chloro-3-nitropyridine with a second reagent in a solvent and in the presence of a base, wherein the second reagent is selected from the group consisting of an alcohol and an amine to provide the first intermediate; (b) reacting the first intermediate with a 1,2-dione in the presence of a catalyst and a reducing agent and (d) purifying the final product.

The term "1,2-dione" refers to a chemical moiety of the formula Rx-C (0) C (0) -R2, independently selected from the group consisting of hydrogen, - saturated or unsaturated alkyl optionally substituted with a five-membered or six-membered aryl or heteroaryl ring portion optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy moieties , carboxylate, nitro and ester, - and a five or six membered aryl or heteroaryl ring portion optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy moieties , carboxylate, nitro and ester. In a preferred embodiment, the invention relates to a method for synthesizing a compound of the invention wherein the solvent is n-butanol. In another preferred embodiment, the invention relates to a method for synthesizing a compound of the invention wherein the base is pulverized potassium carbonate. In another preferred embodiment, the invention relates to the method for synthesizing a compound of the invention wherein the second reagent is selected from the group consisting of SAQAR reagents.

The term "SAQAR reagents" refers to the group of reagents consisting of methanol, benzylamine, 4-fluorobenzylamine, 2-carboxybenzylamine, 3-carboxybenzylamine, 4-carboxybenzylamine, 2-nitrobenzylamine, 3-nitrobenzylamine, 4-nitrobenzylamine, 2-methylbenzylamine , 3-methylbenzylamine, 4-methylbenzylamine, 2-chlorobenzylamine, 3-chlorobenzylamine, 4-chlorobenzylamine, 2-fluorobenzylamine, 3-fluorobenzylamine, 4-fluorobenzylamine, 2- (trifluoromethyl) benzylamine, 3- (trifluoromethyl) benzylamine, 4- ( trifluoromethyl) benzylamine, phenethyl-1-amine, aniline, 2-carboxyaniline, 3-carboxyaniline, 4-carboxyaniline, 2-nitroaniline, 3-nitroaniline, 4-nitroaniline, 2-toluidine, 3-toluidine, 4-toluidine, 2- chloroaniline, 3-chloroaniline, 4-chloroaniline, 2-fluoroaniline, 3-fluoroaniline, 4-f luoroani 1 ina, 2 - (trif 1 or orne ti 1) ani 1 i na, 3-8trifluoromethyl) aniline, 4 - (trifluoromethyl) ) aniline, 2-aminopyridine, 3-aminopyridine, 4-aminopyridine and 2-methylaminopyridine. In another further preferred embodiment, the invention relates to a method for synthesizing a compound of the invention wherein the third reagent is selected from the group consisting of 4-hydroxyphenylglyoxal, 1-phenyl-1,2-propanedione and benzyl. The term "catalyst" as used herein, refers to a chemical molecule, which when added to a group of reagents, can increase the rate at which the reactants react to form products. Many types of catalysts are well known to those of ordinary skill in the art. In a preferred embodiment, the invention relates to methods for synthesizing compounds of the invention, wherein the reducing agent is hydrogen. In another preferred embodiment, the invention relates to methods for synthesizing compounds of the invention, wherein the catalyst is Raney nickel. The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments and the claims.

DESCRIPTION OF THE PREFERRED MODALITIES The present invention is directed in part to a method for modulating the serine / threonine protein kinase function with 5-azaquinoxaline-based compounds. In addition, the invention is related in part to methods for identifying compounds that modulate the serine / threonine protein kinase function. The methods incorporate cells that express a serine / threonine protein kinase, such as RAF.

RAF is a protein kinase without receptor that is recruited to the cell membrane when bound to activated RAS, a hydrolyzing enzyme of guanine triphosphate. RAS is activated when the activated receptor tyrosine kinase protein such as EGFR or PDGFR is activated, binding to an adapter protein, GRB2 and a guanine nucleotide exchange factor, SOS. SOS removes guanine diphosphate from RAS, replaces it with guanine triphosphate and thus activates RAS. RAS then joins RAF and consequently activates RAF. RAF can then phosphorylate other target proteins in the serine and threonine residues, such as the kinase (MEK) that phosphorylates and consequently activates the mitogen-activated protein kinase (MAPK). Therefore, RAF serves as an intermediate controlling factor in signal transduction activated by mitogen. Due to the important regulatory role of RAF in cells, modifications to the amino acid sequence of RAF can alter its function and consequently modify cellular behavior. The role of RAF in cell proliferation is understood by the observation that mutations to the amino acid sequence of RAF have been associated with tumors and cancers. Because mutations to RAF result in cancer in cells that lead to RAF molecules that exhibit unregulated catalytic activity, RAF inhibitors can alleviate or even abrogate the cell proliferation that leads to cancer in these cells.

The methods of the present invention can detect compounds that modulate the function of the RAF protein kinase in cells. RAF phosphorylates a protein kinase (MEK) which in turn phosphorylates a mitogen-activated protein kinase (MAPK). The assays that only monitor the phosphorylation of MEK by RAF are not sensitive because the levels of MEK phosphorylation are not significant. To resolve this sensitivity dilemma, phosphorylation of both MEK and MAPK is followed in the assays of the present invention. The MAPK phosphorylation signal amplifies the MEK phosphorylation signal and allows RAF-dependent phosphorylation to be followed in enzyme-linked immunosorbent assays. In addition, the assay of the invention is preferably carried out in a high throughput format so that many compounds can be monitored quickly in a short period of time. The methods of the present invention have identified compounds that inhibit the function of RAF protein kinase. These compounds are derivatives based on 5-azaquinoxaline. Although derivatives based on 5-azaquinoxaline have been tested for their ability to inhibit the enzymes involved in the synthesis of nucleotides in bacteria, many of these compounds have not yet been explored significantly with respect to inhibition of protein kinase.

Because RAF shows significant amino acid homology to other serine / threonine protein kinase, the 5-azaquinoxaline-based compounds of the invention can similarly inhibit serine / threonine protein kinase other than RAF. Therefore, the methods of the invention are also related to serine / threonine protein kinase different from RAF, which include serine / threonine protein kinase with receptor and without receptor. The methods of the invention also belong to other compounds that modulate the function of RAF in cells as the high throughput aspect of the methods which allows a broad array of molecules that can be tested in a short period of time. Therefore, the methods of the invention can identify existing molecules not described in the present invention that modulate the RAF function.

I. Biological activity of the compounds based on 5- azaauinoxaline The 5-azaquinoxaline-based compounds of the present invention are tested for their ability to inhibit the function of RAF protein kinase. The biological assays and the results of these inhibition studies are reported here. The methods used to inhibit the modulation of 5-azaquinoxaline-based compounds of protein kinase function are similar to those described in the U.S. application. Serial No. 08 / 702,232 by Tang et al., And entitled "Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease," (Lyon &Lyon File No. 221/187), filed on August 23, 1996, regarding the high performance aspect of the method. The application 08 / 702,232 is hereby incorporated by reference in its entirety, including any drawings.

II. Target diseases to be treated by compounds based on 1., 5-triazanaphthalene The methods, compounds and pharmaceutical compositions described herein are designed to inhibit cell proliferative disorders by modulating the function of the RAF protein kinase. Proliferative disorders result in unwanted cell proliferation of one or more subsets of cells in a multicellular organism resulting in damage to the organism. The methods, compounds and pharmaceutical compositions described herein may also be useful for treating and preventing other disorders in organisms, such as disorders related to premature cell death (i.e., neurological diseases) or inflammation. These disorders can be a result of RAF molecules that function inappropriately or as a result of protein kinase molecules related to RAF that function inappropriately. Alterations in the function of RAF protein kinase or protein kinases related to RAF can lead to cell proliferative conditions increased or decreased eluants in certain diseases. Aberrant cell proliferative conditions include cancers, fibrotic disorders, mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, restenosis and inflammation. Fibrotic disorders are related to an abnormal formation of cellular extracellular matrix. An example of a fibrotic disorder is liver cirrhosis. Liver cirrhosis is characterized by an increased concentration of the extracellular matrix constituent resulting in the formation of a hepatic scar. Liver cirrhosis can cause diseases such as cirrhosis of the liver. Mesangial cell proliferative disorders occur due to the abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human kidney diseases such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndrome, transplant rejection and glomerulopathies.

Preferred types of cancers that can be treated by the methods and compounds of the invention are lung cancer, ovarian cancer, breast cancer, brain cancer, intraaxial brain cancer, colon cancer, prostate cancer, Kaposi's sarcoma, melanoma and glioma. Evidence that the compounds and methods of the invention can be used effectively to support and reverse the proliferation of cancer cells is provided herein as a reference. Angiogenic and vasculogenic disorders result from excessive proliferation of blood vessels. The proliferation of blood vessels is necessary in various normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. However, the proliferation of blood vessels is also essential in the development of cancerous tumors. Other examples of blood vessel proliferative disorders include arthritis, where new capillary blood vessels invade the joints and describe the cartilage. In addition, proliferative diseases of blood vessels include other diseases such as diabetic retinopathy, where new capillaries in the retina invade the vitreous humor, cause effusions and cause blindness. Conversely, disorders related to shrinkage, contraction or closure of blood vessels, such as restenosis, are also implicated in the adverse regulation of protein kinases. In addition, vasculogenesis and angiogenesis are associated with the growth of malignant solid tumors and metastases. A cancer tumor that grows vigorously requires a supply of nutrients and blood rich in oxygen to continue growing. As a result, an abnormally large number of capillary blood vessels often grow in concert with the tumor and act as delivery pipes for the tumor. In addition to supplying nutrients to the tumor, the same blood vessels embedded in the tumor provide a way for tumor cells to enter circulation and metastasize to distant sites in the body. Folkman, 1990, J. Nati. Cancer Inst. 82: 4-6. Inappropriate activity of RAF can stimulate cell proliferative disorders. Molecules designed specifically to modulate the function of the RAF protein kinase have been shown to inhibit cell proliferation. Specifically, antisense nucleic acid molecules, which are designed both to bind messenger RNA encoding the RAF protein kinase and block the translation of that message, effectively reverse the transformation of A549 cells in vi tro. Monia et al., 1996, Nature Medicine 2: 688, incorporated herein by reference in its entirety including all figures and tables. A549 cells are human malignant cells. These antisense studies directed to RAF provide evidence that the 5-azaquinoxaline molecules of the invention, which modulate the function of the RAF protein kinase, can sustain and likewise reverse the proliferation of malignant cells in an organism. These 5-azaquinoxaline compounds can be tested in the in vitro methods that are provided herein as an example. In addition, the 5-azaquinoxaline compounds can be tested for their effect on tumor cells in vivo by xenograft methods which are also provided herein as an example. There are at least two pathways in which an inappropriate activity of RAF can stimulate the proliferation of unwanted cells of a particular cell type: (1) directly stimulate the growth of the particular cell, or (2) increase the vascularity of a cell. particular area, such as a tumor tissue, whereby tissue growth is facilitated. The use of the present invention is facilitated by first identifying whether a cell proliferation disorder is being carried out by RAF. Once such disorders are identified, patients suffering from such disorder can be identified by analysis of their symptoms using procedures well known to physicians or veterinarians usually skilled in the art. Such patients can be treated later as described in, the present. Determining whether the cell proliferation disorder is driven by RAF can be carried out by first determining the level of RAF activity that occurs in the cell or at a particular position in the patient's body. For example, in the case of cancer cells, the level of one or more RAF activities can be compared to cancers activated without RAF and cancers activated with RAF. If the cancer cells have a higher level of RAF activity compared to the RAF-activated cancers, preferably equal to or greater than the RAF-activated cancers, then they are candidates for treatment using the described RAF modulator methods, and the compounds of the invention. In the case of cellular proliferative disorders that arise due to unwanted proliferation of non-cancerous cells, the level of RAF activity is compared to the level that occurs in the general population (for example, the average level that occurs in the general population of people or animals excluding persons or animals suffering from a cell proliferative disorder). If the unwanted cell proliferative disorder is characterized by a higher level of RAF than that which occurs in the general population, then the disorder is a candidate for treatment using the described RAF modulation methods and the compounds of the invention.

III, Pharmaceutical compositions and administration of 5-azaquinoxaline-based compounds Methods for preparing pharmaceutical formulations of the compounds, methods for determining the amounts of compounds to be administered to a patient and ways of administering the compounds to an organism are described in the U.S. application. Serial No. 08 / 702,232 by Tang et al., And entitled "Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease", (Lyon &Lyon File No. 221/187), filed on August 23, 1996, and International Patent Publication No. WO 96/22976, by Buzzetti et al., And entitled "Hydrosoluble 3-Arylidene-2-Oxindole Derivatives as Tyrosine Kinase Inhibitors", published August 1, 1996, both of which they are incorporated herein by reference in their entirety, including any drawings. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.

Examples The examples below are non-limiting and are only representative of the various aspects and features of the present invention. The examples describe methods for synthesizing compounds of the invention and methods for measuring an effect of a compound on the function of the RAF protein kinase. The cells used in the methods are commercially available. The nucleic acid vectors harbored by the cells are also initially available and the gene sequences for the various protein kinases are easily accessible in the sequence data banks. Therefore, a person ordinarily skilled in the art can easily re-create cell lines in a time-appropriate manner by combining commercially available cells, commercially available nucleic acid vectors and genes for protein kinase using readily available techniques. for people usually skilled in the art.

Example 1; Procedure to synthesize compounds based on 5-asaguj-noyali-na qe la. igvengióii The invention is now illustrated in the following non-limiting examples in which, unless stated otherwise: (i) the evaporations are carried out by rotary evaporation in vacuo; (ii) the operations are carried out under an atmosphere of inert gas such as nitrogen; (iii) high-performance liquid chromatography (CLAP) is performed in Merck LiChrosorb RP-18 reverse phase silica obtained from E. Merck, Darmstadt, Germany. (iv) The yields are provided for illustration only and are not necessarily the maximum obtainable, - (v) The melting points are uncorrected and are determined using a HWS Mainz SG 2000 digital melting point apparatus; (vi) The structures of all the compounds of the formula (I) of this invention are confirmed by magnetic resonance spectroscopy of the proton in a Brucker spectrophotometer.

AMX500-NMR by elemental microanalysis and, in certain cases, by mass spectroscopy; (vii) the purity of the structures is determined by thin layer chromatography (CCD) using silica gel (Merck Silica Gel 60 F254) or by CLAP; and (viii) intermediates in general are not fully characterized and purity is determined by thin layer chromatography (CCD) or by CLAP.

Synthesis procedures Compound A-2: 6-benzylamino-3, 4-hydroxyphenyl-5-azaquinoxaline 4-Hydroxyphenylglyoxal is prepared from 4-hydroxyacetophenone (Lancaster, Acros) according to the published method (J. Amer. Chem. Soc., 71, 1045 (1949)). 2-Amino-6-benzylamino-3-nitropyridine is prepared from 2-amino-3-nitropyridine as follows: 2-amino-6-chloro-3-nitropyridine (17.35 g, 0.10 mol), benzylamine (Fluka) (10.72 g, 0.10 moles) and powdered potassium carbonate (10.4 g, 0. 035 moles) in 100 ml of n-butanol are heated under reflux for 2 hours. The suspension is filtered and after cooling to room temperature, the solid is collected by filtration, washed with butanol and dried at 50 ° C in vacuo to give 6-amino-6-benzylamino-3-nitropyridine (22.2 g, 91%). %, mp 145-146 ° C). 6-Benzylamino-3, 4-hydroxyphenyl-5-azaquinoxaline is prepared from 2-amino-6-benzylamino-3-nitropyridine as follows: 2-amino-6-benzylamino-3-nitropyridine is hydrogenated (25 g, 0.10 moles) under 5.5 bar of H2 in the presence of 10 g of Raney nickel in 400 ml of dioxane at 60 ° C. After 2 hours, the reaction mixture is cooled to room temperature, filtered and 4-hydroxyphenylglyoxal is added and stirred for 2 hours under an argon atmosphere. The suspension is then diluted with water, the solid is collected by filtration, washed with water, recrystallized from 2-propanol and dried in vacuo at 50 ° C to provide 6-benzylamino-3- (4-hydroxyphenyl) -5 -azaquinoxaline (8 g, 24.4%, mp 271-273 ° C).

Compound A-1: 6-phenylamino-3- (4-hydroxyphenyl) -5-azaquinoxaline When replacing phenylamine in place of benzylamine in the process for compound A-2, the identical process provides 6-phenylamino-3- (4-hydroxyphenyl) -5-azaquinoxaline.

Compound A-3: 6-methoxy-2-methyl-3-phenyl-5-azaquinoxaline When replacing 1-phenyl-1, 2-propanedione instead of 4-hydroxyphenylglyoxal and methanol in place of benzylamine in the process for compound A-2, the identical process provides 6-methoxy-2-methyl-3-phenyl-5 -azaquinoxaline.

Compound A-4: 6-methoxy-2,3-diphenyl-5-azaquinoxaline When replacing benzyl in place of 4-hydroxyphenylglyoxal and methanol in place of benzylamine in the process for compound A-2, the identical process provides 6-methoxy-2,3-diphenyl-5-azaquinoxaline.

Compound A-5: 6- (4-fluorobenzylamino) -2-methyl-3-phenyl-5-azquingx lin When replacing 1-phenyl-1, 2-propanedione in place of 4-hydroxyphenylglyoxal and 4-fluorobenzylamine in the process for compound A-2, the identical process provides 6- (4-fluorobenzylamino) -2-methyl-3-phenyl -5-azaquinoxaline.

Compound A-6: 2, 3-diphenyl-6- (4-fluorobenzylamino) -5-azaquinoxaline When replacing benzyl instead of 4-hydroxyphenylglyoxal and 4-fluorobenzylamine instead of benzylamine in the process for compound A-2, the identical process provides 2,3-diphenyl-6- (4-fluorobenzylamino) -5-azaquinoxaline.

Compound A-7: 3-phenyl-6-phenylamino-5-azaquinoxaline When replacing phenylglyoxal instead of 4-hydroxyphenylglyoxal and aniline in place of benzylamine the procedure for compound A-2, the identical process provides 3-phenyl-6-phenylamino-5-azaquinoxaline.

Compounds A-8 - A-26 When replacing the appropriate substituted benzylamine in place of benzylamine in the process of compound A-2, identical processes provide the following compounds: Compound A-8: 6- (2-carboxybenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-9: 6- (3-carboxybenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A -10: 6- (4-carboxybenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-ll: 3- (4-hydroxyphenyl) -6- (2-nitrobenzylamino) -5-azaquinoxaline, Compound A- 12: 3 - (4-hydroxyphenyl) -6- (3-n-robenzylamino) -5-azquinoxaline Compound A-13: 3- (4-hydroxyphenyl) -6- (4-nitrobenzylamino) -5-azaquinoxaline Compound A- 14: 3- (4-hydroxyphenyl) -6- (2-methylbenzylamino) -5-azaquinoxaline Compound A-15: 3- (4-hydroxyphenyl) -6- (3-methylbenzylamino) -5-azaquinoxaline * Compound A-16 : 3- (4-hydroxyphenyl) -6- (4-methylbenzylamino) -5-azaquinoxaline Compound A-17: 6- (2-chlorobenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-18: 6 - (3-chlorobenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-19: 6- (4-chlorobenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-20: 6- ( 2-fluorobenz lamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-21: 6- (3-fluorobenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-22-. 6- (4-Fluorobenzylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-23: 3 - (4 -hi roxyphenyl) -6- [2 - (trifluoromethyl) benzylamino] -5 -azaquinoxaline Compound A-24: 3 - (4-Hydroxy-enyl) -6- [3 - (trifluoromethyl) -benzylamino] -5-azaquinoxaline Compound A-25: 3 - (4-hydroxy-enyl) -6- [4 - (trifluoromethyl) ) benzylamino] -5-azaquinoxaline Compound A-26: 3- (4-hydroxyphenyl) -6-phenethyl-l-amino) -5-azaquinoxaline Compounds A-27 - A-48 When replacing the appropriate substituted aniline in place of benzylamine in the process of compound A-2, the identical process provides the following compounds: Compound A-27: 6- (2-carboxyphenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-28: 6- (3-carboxyphenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A -29: 6- (4-carboxyphenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-30: 3- (4-hydroxyphenyl) -6- (2-nitrophenylamino) -5-azaquinoxaline Compound A-31 : 3- (4-hydroxyphenyl) -6- (3-nitrophenylamino) -5-azaquinoxaline Compound A-32: 3- (4-hydroxyphenyl) -6- (4-nitrophenylamino) -5-azaquinoxaline Compound A-33: 3 - (4-hydroxyphenyl) -6- (2-methylphenylamino) -5-azaquinoxaline Compound A-34: 3- (4-hydroxifeinl) -6- (3-methylphenylamino) -5-azaquinoxaline Compound A-35: 3- ( 4-hydroxyphenyl) -6- (4-methylphenylamino) -5-azaquinoxaline Compound A-36: 6- (2-chlorophenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-37: 6- (3- chlorophenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-38: 6- (4-chlorophenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-39: 6- (2-fluorophenylamino) -3- (4-hydroxy phenyl) -5-azaquinoxaline Compound A-40: 6- (3-fluorophenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-41: 6- (4-fluorophenylamino) -3- (4-hydroxyphenyl) -5-azaquinoxaline Compound A-42: 3- (4-hydroxyphenyl) -6- [(2-trifluoromethyl) -phenylamino] -5-azaquinoxaline Compound A-43: 3- (4-hydroxyphenyl) -6- [(3 -trifluoromethyl) -phenylamino] -5-azaquinoxaline Compound A-44: 3- (4-hydroxyphenyl) -6- [(4-trifluoromethyl) -phenylamino] -5-azaquinoxaline Compound A-45: 3- (4-hydroxyphenyl) -6- (pyrid-2-amino) -5-azaquinoxaline Compound A-46: 3- (4-hydroxyphenyl) -6- (pyrid-3-amino] -5-azaquinoxaline Compound A-47: 3- (4- hydroxyphenyl) -6- (pyrid-4-amino] -5-azaquinoxaline Compound A-48: 3- (4-hydroxyphenyl) -6- (pyrid-2-methylamino] -5-azaquinoxaline Compounds A ~ 49 - A-67 When replacing the appropriate substituted benzylamine in place of benzylamine and phenylglyoxal instead of 4-hydroxyphenylglyoxal in the process of compound A-2, the identical process provides the following compounds: Compound A-49: 6- (2-carboxybenzylamino) -3-phenyl-5-azaquinoxaline Compound A-50: 6- (3-carboxybenzylamino) -3-phenyl-5-azaquinoxaline Compound A-51: 6- (4-carboxybenzylamino) - 3-phenyl-5-azaquinoxaline Compound A-52 6- (2-nitrobenzylamino-3-phenyl) -5-azaquinoxaline Compound A-53 6- (3-nitrobenzylamino) -3-phenyl-5-azaquinoxaline Compound A- 54 6- (4-Nitrobenzylamino) -3-phenyl-5-azaquinoxaline Compound A-55 6- (2-methylbenzylamino) -3-phenyl-5-azaquinoxaline Compound A-56 6- (3-methylbenzylamino) -3 phenyl-5-azaquinoxaline Compound A-57 6- (4-methylbenzylamino) -3-phenyl-5-azaquinoxaline Compound A-58 6- (2-chlorobenzylamino) -3-phenyl-5-azaquinoxaline Compound A- 59 6- (3-chlorobenzylamino) -3-phenyl- • 5-azaquinoxaline Compound A-60 6- (4-chlorobenzylamino) -3-phenyl-5-azaquinoxaline Compound A-61: 6- (2-fluorobenzylamino) -3 -phenyl-5-azaquinoxaline Compound A-62: 6- (3-fluorobenzylamino) -3-phenyl-5-azaquinoxaline Compound A-63: 6- (4-fluorobenzylamino) -3-phenyl-5-azaqu Inoxaline Compound A-64: 3-phenyl-6- [2- (trifluoromethyl) benzylamino] -5-azaquinoxaline Compound A-65: 3-phenyl-6- [3- (trifluoromethyl) enylamino] -5-azaquinoxaline Compound A- 66: 3-phenyl-6- [4- (trifluoromethyl) enylamino] -5-azaquinoxaline Compound A-67: 3-phenyl-6- (phenethyl-1-amino) -5-azaquinoxaline Compounds A-68 - A-89 By replacing the appropriate substituted aniline in place of benzylamine and phenylglyoxal instead of 4-hydroxyphenylglyoxal in the process of compound A-2, the identical process provides the following compounds: Compound A-68 6- (2-carboxyphenylamino) -3-phenyl-5-azaquinoxaline Compound A-69 6- (3-carboxyphenylamino) -3-phenyl-5-azaquinoxaline Compound A-70 6- (4-carboxyphenylamino) - 3-phenyl-5-azaquinoxaline Compound A-71 6- (2-nitrophenylamino) -3-phenyl-5-azaquinoxaline Compound A-72 6- (3-nitrophenylamino) -3-phenyl-5-azaquinoxaline Compound A-73 6- (4-nitrophenylamino) -3-phenyl-5-azaquinoxaline Compound A- 74 6- (2-methylphenylamino) -3-phenyl-5-azaquinoxaline Compound A-75 6- (3-Methylphenylamino) -3-phenyl-5-azaquinoxaline Compound A-76: 6- (-methylphenylamino) -3-phenyl -5-azaquinoxaline Compound A-77: 6- (2-chlorophenylamino) -3-phenyl-5-azaquinoxaline Compound A-78: 6- (3-chlorophenylamino) -3-phenyl-5-azaquinoxaline Compound A-79: 6 - (4-chlorophenylamino) -3-phenyl-5-azaquinoxaline Compound A-80: 6- (2-fluorophenylamino) -3-phenyl-5-azaquinoxaline Compound A-81: 6- (3-fluorophenylamino) -3-phenyl -5-azaquinoxaline Compound A-82: 6- (4-fluorophenylamino) -3-phenyl-5-azaquinoxaline Compound A-83: 3-phenyl-6- [(2-trifluoromethyl) phenylamino] -5-azaquinoxaline Compound A- 84: 3-phenyl-6- [(3-trifluoromethyl) phenylamino] -5-azaquinoxaline Compound A-85: 3-phenyl-6- [(4-trifluoromethyl) phenylamino] -5-azaquinoxaline Compound A- 86 3-phenyl-6- (pyrid-2-amino) -5-azaquinoxaline Compound A-87 3-phenyl-6- (pyrid-3-amino) -5-azaquinoxaline Compound A-88 3-phenyl-6- (pyrid-4-amino] -5-azaquinoxaline Compound A-89 3-phenyl-6- (pyrid-2-methylamino] -5-azaquinoxaline Compound A-90: 6-phenylamino-3- (4-methoxyphenyl) -5 -azaquinoxaline When replacing 4-methoxyphenyl in place of 4-hydroxyphenyl in the process for compound A-1, the identical process provides 6-phenylamino-3- (4-methoxyphenyl) -5-azaquinoxaline.

Example 2: Test that measures the phosphorylation function of RAF By following the assay reports, the amount of phosphorylation catalyzed by RAF of its MEK target protein as well as the target of MEK, MAPK. The gene sequence for RAF is described in Bonner et al., 1985, Molec. Cell. Biol. 5: 1400-1407, and is easily accessible in multiple gene sequence data banks. The construction of the nucleic acid vector and the cell lines used for this portion of the invention are fully described in Morrison et al., 1988, Proc. Nati Acad. Sci. USA 85: 8855-8859.

Materials and reagents 1. Sf9 cells (Spodoptera frugiperda); GIBCO-BRL, Gaithersburg, MD. 2. RIPA buffer: 20 mM Tris / HCl pH 7.4, NaCl 137 mM, 10% glycerol, 1 mM PMSF, 5 mg / l aprotenin, Triton X-100 0.5%; 3. Thioredoxin-MEK fusion protein (T-MEK): expression and purification of T-MEK by affinity chromatography which is carried out according to the manufacturer's procedures. Catalog # K 350-01 and R 350-40, Invitrogen Corp., San Diego, CA 4. His-MAPK (ERK 2); His-tagged MAPK which is expressed in XLl Blue cells transformed with the pUCld vector encoding His-MAPK. His-MAPK is purified by Ni affinity chromatography. Cat # 27-4949-01, Pharmacia, Alameda, CA, as described herein. 5. Goat IgG against mouse, - (West Grove, PA. Catalog, # 515-006-008, Lot # 28563. 6. Specific antibody to RAF-1 protein kinase: URB2653 from UBI 7. Coating buffer: PBS phosphate buffered saline, GIBCO-BRL, Gaithersburg, MD 8. Washing buffer: TBST - 50 mM Tris / HCl, pH 7.2, 150 mM NaCl, 0.1% Triton X-100 9. Blocking damper: TBST , ethanolamine 0.1%, pH 7.4 10. DMSO, Sigma, St. Louis, MO 11. Kinease buffer (KB): 20 mM Hepes / HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100, PMSF 1 mM, 5 mg / l of aprotenin, sodium orthovanadate 75 μM, DTT 0.5 mM and MgCl210 mM 12. ATP mixture: MgCl2100 mM, ATP 300 μM, 10 μCi of β "P ATP (Dupont-NEN) / ml. 13. Detention solution: phosphoric acid 1%; Fisher, Pittsburgh, PA. 14. Cellulose phosphate filter pads Wallac, Turku, Finland.

. Filter wash solution: 1% phosphoric acid, Fisher, Pittsburgh, PA. 16. Tomtec plate harvester, Wallac, Turku, Finland. 17. Wallac beta plate reader # 1205, Wallac, Turku, Finland. 18. NUNC 96-well V-bottom polypropylene plates for composites and Applied Scientific Catalog # AS-72092.

Process All of the following steps were carried out at room temperature unless otherwise indicated. 1. ELISA plate coating: ELISA wells were coated with 100 μl of goat anti-affinity purified mouse antiserum (1 μg / 100 μl coating buffer) overnight at 4 ° C. ELISA plates were used for two weeks and then stored at 4 ° C. 2. The plate is inverted and the liquid is eliminated. 100 μl of blocking solution is added and incubated for min. 3. The blocking solution is removed and washed four times with washing buffer. Dry the plate on a paper towel to remove excess liquid. 4. 1 μg of RAF-1 specific antibody is added to each well and incubated for 1 hour. Washing is performed as described in step 3. 5. The lysates of Sf9 cells infected with RAS / RAF are reheated and diluted with TBST at 10 μg / 100 μl. 10 μg of diluted lysate is added to the wells and incubated for 1 hour. The plate is shaken during incubation. Negative controls do not receive a lysate. Lysates of Sf9 insect cells infected with RAS / RAF are prepared after the cells are infected with recombinant baculovirus at an MOI of 5 for each virus, and are harvested 48 hours later. The cells are washed once with PBS and lysed in RIPA buffer. The insoluble material is removed by centrifugation (5 min at 10,000 x g). The aliquots of the lysates are frozen in dry ice / ethanol and stored at -80 ° C until use. 6. Removal of unbound and washed material as indicated above (step 3). 7. Add 2 μg of T-MEK and 2 μg of His-MAEPK per well and adjust the volume to 40 μl with buffer kinase. Methods for purifying T-MEK and MAPK from cell extracts are provided herein as an example. 8. Prediluted compounds (10 mg / ml concentrated solution of DMSO) or extracts 20 times in TBST plus DMSO 1%. 5 μl of the prediluted compounds / extracts are added to the wells described in step 6. Incubate for 20 min. The controls do not receive medication. 9. The kinase reaction is started by adding 5 μl of the ATP mixture; The plates are shaken on an ELISA plate shaker during incubation. 10. The kinase reaction is stopped after 60 min by the addition of 30 μl of stop solution to each well. 11. Place the phosphocellulose pad and the ELISA plate on a Tomtec plate harvester. Harvesting and washing is filtered with the filter wash solution according to the manufacturer's recommendation. The filter pads are dried. The filter pads are sealed and placed on a support. The support is inserted into a radioactive detection apparatus and the radioactive phosphorus is quantified in the filter pads. Alternatively, aliquots of 40 μl of individual wells can be transferred from the assay plate to the corresponding positions on the phosphocellulose filter pad. After air drying the filters, the filters are placed in a tray. The tray is shaken gently, changing the washing solution at 15-minute intervals for 1 hour. The filter pads are air dried. The filter pads are sealed and placed in a suitable holder for reading the radioactive phosphorus in the samples. The support is inserted into a detection device and the radioactive phosphorus is quantified on the filter pads. The CIS0 values are measured according to the protocol for the following compounds based on 5-azaquinoxaline in the RAF-1 ELISA assay: (To the) (A-2) (A-7) (A-36) (A-77) (A-90) A CIS0 value is the concentration of the 5-azaquinoxaline-based inhibitor necessary to decrease the maximum amount of phosphorylated target protein or cell growth by 50%.

The IC50 values are measured in the phosphorylation assay of RAF-1 and are shown in Table 1: TABLE 1 Example 3: Purification of Mapk and Mek The MAPK and MEK proteins are readily expressed in cells by subcloning a gene encoding these proteins into the commercially available vector that expresses proteins with a poly-histidine tag. Genes coding for these proteins are readily available from laboratories that normally work with these proteins or by cloning these genes from cells containing cDNA libraries. Libraries are commercially available with ease and a person skilled in the art can easily design nucleic acid probes homologous to the cDNA molecules that code for MEK or MAPK from the MEK and MAPK nucleic acid sequences, available in bases from Multiple gene data such as Genbank. The cloning of a gene can be carried out in a short period of time using techniques currently available to persons skilled in the art. The purification of MEK and MAPK from cell extracts can be carried out using the following protocol, which is adapted from Robbins et al., 1993, J. Biol. Chem. 268: 5097-5106: 1. Cells are lysed by sonication, osmotic tension or readily available French pressure techniques for those skilled in the art. A suitable sonication damper is provided below. 2. A solid support is equilibrated which is conjugated with nickel or cobalt with the equilibrium buffer described below. The poly-histidine tag binds specifically to the nickel and cobalt atoms in the solid support. The equilibrium can be obtained by washing the resin three times with a volume of the equilibrium buffer equal to ten times the volume of the solid support. The solid support is readily available to persons usually skilled in the art. 3. Add the cell lysate to the solid support and equilibrate in a container for a certain period of time. Alternatively, the solid support can be packed into a protein chromatography column and the lysate can be flowed through the solid support. 4. Wash the solid support with the wash buffer described above. 5. Elute the MEK or MAPK protein from the solid support with an amount of elution buffer (given below) that removes a significant portion of the protein from the solid support.

Shock absorber Sodium phosphate 50 mM, pH 8.0 Sodium chloride 0.3 M 10 mM ß-mercaptoethanol NP40 1% 10 mM NaF Pefablock 0.5 mM Balance cushion 50 mM Sodium Phosphate, pH 8.0 Sodium Chloride »0.3 M 10 mM β-mercaptoethanol NP40 1% 10 mM NaF 1 mM Imidazole Shock absorber 50 mM Sodium Phosphate, pH 8.0 Sodium Chloride 0.3 M 10 mM ß-mercaptoethanol NP40 1% 10 mM NaF 10 mM Imidazole Elution buffer 50 mM sodium phosphate, pH 8.0 Sodium chloride 0.3. 10 mM M β-mercaptoethanol NP40 1% 10 mM NaF Imidazole 10 -500 mM Example 4: Test that measures the phosphorylation function of the EGF receptor The kinase activity of the EGF receptor (EGFR-NIH3T3 assay) is measured in whole cells, as described in detail in PCT publication WO 9640116, filed on June 5, 1996, by Tang et al., And entitled "Indolinone". Compounds for the Treatment of Disease ", incorporated herein by reference in its entirety, including any drawings. Table 2 shows the CIS0 values measured in the EGF receptor phosphorylation assay: TABLE 2 Example 5: Test that measures the effect of 5-aza-based compounds uinoxaline on the growth of cells expressing RAS The following assay measures the growth rates of NIH-3T3 cells expressing RAS. The purpose of the assay is to determine the effects of the compounds on the growth of NIH 3T3 cells overexpressing H-Ras. materials Sterile 96-well flat bottom plates Sterile 96-well round bottom plates Sterile 25 ml or 100 ml containers Pipettes, multi-channel pipetman Sterile pipette tips Sterile 15 ml and 50 ml tubes Reagents SRB 0.4% in acetic acid 1% Base Tris 10 mM TCA 10% Acetic acid 1% Sterile DMSO (Sigma) Compound in DMSO (100 mM or less of the concentrated solution) Tri sina-EDTA (GIBCO BRL) Cellphone line 3T3 / H-Ras (NIH 3T3 cells, clone 7, expressing the genomic fragment of oncogenic H-Ras). The cells can be constructed using the following protocol: 1. A fragment of a gene encoding Ras is subcloned into a commercially available vector that will stably transfect NIH-3T3 cells. The fragment is from the genomic transforming allele of cHa-ras. 2. NIH-3T3 cells are transfected with the subcloned vector by a calcium phosphate method. The cells expressing the Ras construct in serum in 2% in DMEM are selected. Visible foci are observed after 2 weeks. Transformed cells accumulate to generate a stably transformed cell line.

Growth medium: 2% bovine serum / DMEM + 2 mM glutamine, Pen / Strep Protocol: Day 0: Planting in cell plate: This part of the test is carried out in a laminar flow hood. 1. Tripsinization of cells. 200 μl of the cell suspension is transferred to 10 ml of isotone. Cellular accounts are performed with a Coulter Counter. 2. Cells are diluted in growth medium at 60,000 cells / ml. 100 μl of the cells are transferred to each well in a 96 well flat bottom plate to provide 6000 cells / well. 3. Half of the plate (4 rows) is used for each compound and wells in quadruplicate for each concentration of compound, and a set of 4 wells for the control medium. 4. The plates are gently shaken to allow uniform union of the cells. 5. The plates are incubated at 37 ° C in a 10% C02 incubator. pía i; Addition < a $? com This part of the test is carried out in a laminar flow hood. 1. In a 96-well round bottom plate, 120 μl of growth medium containing 2X final DMSO% found with the highest analysis concentration of the compound for columns 1 to 11 is added. For example, if the highest concentration is 100 μl, and this is made from a 100 mM concentrate, IX DMSO is 0.1%, so 2X DMSO is 0.2%. This plate is used to title the compound, 4 rows per compound. 2. In a sterile 15 ml tube, a 2X solution of the highest analysis concentration of the compound and growth medium plus 2X DMSO is made. 1 ml per cell line is needed. The initial concentration of the compound is usually 100 μm, but this concentration can vary based on the solubility of the compound. 3. Transfer 240 μl of the 2X starting compound solution to quadruple the wells in column 12 of the 96 well round bottom plate Serial 1: 2 dilutions are made through the plate from right to left by transferring 12 μl from column 12 to column 11, from column to 10 and so on up to column 2. Transfer 100 μl of the dilutions of the compound, and 100 μl of the medium in column 1, onto 100 μl of medium in cells in the corresponding wells of the 96-well flat bottom plate The total volume per well should be 200 μl. 4. The plate is returned to the incubator and incubated for 3 days.

Day 4: Trial development This part of the test is carried out in a cabinet. 1. The medium is aspirated or eliminated. 200 μl of cold 10% TCA is added to each well to fix the cells. The plate is incubated for at least 60 min at 4 ° C. 2. TCA is discarded and the wells are rinsed 5 times with running water. The plates are dried upside down on paper towels. 3. Cells are stained with 100 μl / well of SRB 0.4% for 10 ml. 4. SRB is removed and the wells are rinsed 5 times with 1% acetic acid. The dry plates are completely flipped over onto paper towels. 5. The dye is solubilized with 100 μl / well of base Tris 10 mM for 5-10 min on a shaker. 6. The plates are read on a Dynatech plate reader ELISA at 570 nm with reference to 630 nm. The selected compounds inhibit the growth rate of the cells overexpressing RAS, as illustrated in Table 3.

TABLE 3 Example 6: Test that measures the effect of 5-azaquinoxaline-based compounds on the growth of A549 cells The following assay measures the growth rates for A549 cells. The purpose of the assay is to determine the effects of the compounds on the growth of human lung carcinoma cells A549. A549 cells are readily available from commercial sources, such as ATCC (CCL185).

Materials: Sterile 96-well flat bottom plates Sterile 96-well round bottom plates Sterile 25 ml or 100 ml containers Pipettes, multi-channel pipetman Sterile pipette tips Sterile 15 ml and 50 ml tubes Reagents SRB 0.4% acetic acid 1% Base Tris 10 mM TCA 10% Acetic acid 1% Sterile DMSO (Sigma) Compound in DMSO (100 mM or less of the concentrated solution) Trypsin-EDTA (GIBCO BRL) Cell line and growth medium: Human lung carcinoma cells A549 (ATCC CCL185) 10% Fetal bovine serum in Ham F12-K Protocol: Day 0: plaque placement of the cells: This part of the test is carried out in a laminar flow hood. 1. The cells are trypsinized. 200 μl of the cell suspension is transferred to 10 ml of isotonic solution. Cell counts are performed with a Coulter Counter 2. The cells are diluted in growth medium to 20,000 cells / ml. 100 μl of cells are transferred to each well in a 96 well flat bottom plate to provide 2000 cells / well. 3. The use of half of the plate (4 rows) of each compound and the wells in quadruplicate for each concentration of compound, and a set of 4 wells for the control medium. 4. The plates are gently shaken to allow uniform union of the cells. 5. Plates are incubated at 37 ° C in a C02 incubator % Day 1: Addition of the compound: This part of the test is carried out in a laminar flow hood. 1. In a 96-well round bottom plate, add 120 μL of growth medium containing final 2X DMSO% which is in the highest analysis concentration of the compound for columns 1 to 11. For example, if the highest analysis concentration is 100 μM, and this is made from a 100 mM concentration, IX DMSO is 0.1%, so 2X DMSO is 0.2%. This plate is used to title the compound, 4 rows per compound. 2. In a sterile 15 ml tube, a 2X solution of the highest analysis concentration of the compound is made in growth medium plus 2X DMSO. 1 ml per cell line is needed. The initial concentration of the compound is usually 100 μM, but this concentration can vary depending on the solubility of the compound. 3. Transfer 240 μl of the 2X starting compound solution to quadrupled wells in column 12 of the 96-well round bottom plate. Serial 1: 2 dilutions are made through the plate from right to left by transferring 120 μl from column 12 to column 11, column 11 to column 10, and so on up to column 2. Transfer of 100 μl of the dilutions of compound and 100 μl of the medium in column 1, in 100 μl of medium in cells in the corresponding wells of the 96-well flat bottom plate. The total volume per well should be 200 μl. 4. Return the plate to the incubator and incubate for 3 days.

Day 5: Development of the test This part of the test is carried out in the cabinet. 1. The medium is aspirated or eliminated by pouring. 200 μl of cold 10% TCA is added to each well to fix the cells. The plate is incubated for at least 60 min at 4 ° C. 2. TCA is discarded and the wells are rinsed 5 times with running water. The plates are dried by turning them over on paper towels. 3. Cells are stained with 100 μl / well of SRB 0.4% for 10 min. 4. It is removed by pouring SRB and the wells are rinsed 5 times with 1% acetic acid. Dry the plates completely by turning them upside down on paper towels. 5. Dye is solubilized with 100 μl / well base Tris 10 mM for 5-10 min, in a shaker. 6. The plates are read on a Dynatech ELISA plate reader at 570 nm with reference to 630 nm. Compounds that inhibit the growth rates of A549 cells are selected as illustrated in Table 4.

TABLE 4 Example 7: Method for determining the biological activity of RAF modulators in vivo Xenograft studies can be used to monitor the effect of the compounds of the invention on the inhibition of ovarian, melanoma, prostate, lung and breast tumor tumor cells. The protocol for the assay is described in detail in PCT publication WO9640116, filed on June 5, 1996 by Tang et al., And entitled "Indolinone".

Compounds for the Treatment of Disease ", incorporated herein by reference in its entirety, including the drawings The invention is described illustratively herein may be carried out in the absence of any element or elements, limitation or limitations the which are not specifically described in this document The terms and expressions which have been used are those used as terms of description and not as limitation, and there is no intention that in the use of such terms and expressions any equivalent to the features that are shown and described or portions thereof, but it must be recognized that various modifications are possible within the scope of the invention as claimed.Therefore, it should be understood that although the present invention has been specifically described by preferred modalities and optional features, modifications and variation can be made the concepts described herein by those skilled in the art and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims. All references not previously incorporated herein by reference, including both patent and non-patent references, are expressly incorporated by reference herein to all purposes. The other embodiments are within the following claims.

Claims

1. A method for modulating the function of a serine / threonine protein kinase with a compound based on 5-azaquinoxaline, comprising the step of contacting cells expressing serine / threonine protein kinase with the compound.

2. The method as described in claim 1, wherein serine / threonine protein kinase is RAF.

3. A method for identifying compounds that modulate the serine / threonine protein kinase function, comprising the following steps: (a) contacting cells that express serine / threonine protein kinase with the compound; and (b) monitor the "effect on cells.

4. The method as described in claim 3, wherein the effect is a change or an absence of a change in the cellular phenotype.

5. The method as described in claim 3, wherein the effect is a change or an absence of a change in cell proliferation.

6. The method as described in claim 3, wherein the effect is a change or absence of a change in the catalytic activity of the serine / threonine protein kinase.

7. The method as described in claim 3, wherein the effect is a change or absence of a change in the interaction between serine / threonine protein kinase with a natural binding partner, as described herein.

8. The method as described in claim 3, comprising the following steps: (a) lysing the cells to obtain a lysate comprising serine / threonine protein kinase; (b) adsorbing the serine / threonine protein kinase to an antibody; (c) incubating the serine / threonine protein kinase adsorbed with a substrate or substrates; and (d) adsorbing the substrate or substrates to a solid support or antibody; wherein the step of monitoring the effect on the cells comprises measuring the phosphate concentration of the substrate or substrates.

9. The method as described in claim 3, wherein serine / threonine protein kinase is RAF and comprises the following steps: (a) lysing the cells to obtain a lysate comprising RAF; (b) adsorbing RAF to an antibody; (c) incubate RAF adsorbed with MEK and MAPK; and (d) adsorbing MEK or MAPK to a solid support or antibody or antibodies; wherein the step of measuring the effect of the cells comprises monitoring the phosphate concentration of MEK and MAPK.

10. The method as described in claim 1, wherein the compound based on 5-azaquinoxaline has the formula set forth in structure I: (I) wherein (a) Rlf R2, R3, R4 and R6, are independently selected from the group consisting of (i) hydrogen, - (ii) saturated or unsaturated alkyl optionally substituted with a five-membered aryl or heteroaryl ring portion or six members, wherein the ring portion is optionally substituted with one, two, or three substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester portions; (iii) an amine of formula NX2X3, wherein X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five or six membered aryl portions or heteroaryl ring; (iv) halogen or trialomethyl; (v) a ketone of formula -CO-X4, wherein X4 is selected from the group consisting of hydrogen, alkyl and five or six membered aryl or heteroaryl portions; (vi) a carboxylic acid of formula - (Xs) n-C00H or ester of formula - (X6) n-COO-X7, wherein X5, X6 and X7 are independently selected from the group consisting of alkyl and aryl or heteroaryl moieties of five members or six members and where n is 0 or 1; (vii) an alcohol of formula (Xß) n-0H or a portion of formula - (Xß) n-0-X9, wherein Xß and X9 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl and portions of a five-membered or six-membered aryl or heteroaryl ring, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester, and wherein n is 0 or 1; (viii) an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, and a five-membered or six-membered aryl or heteroaryl ring portion, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro or ester; (ix) -S03NX.11X.12, wherein XX1 and X12 are selected from the group consisting of hydrogen, alkyl, and five or six membered aryl or heteroaryl ring portions, - (x) an aryl or heteroaryl ring moiety of five members or six members optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester portions; (xi) an aldehyde of formula -CO-H; and (xii) a sulfone of the formula -S02-X13, wherein X13 is selected from the group consisting of saturated or unsaturated alkyl and five or six membered aryl or heteroaryl portions; and (b) Xx is selected from the group consisting of nitrogen, sulfur and oxygen.

11. The method as described in claim 10, wherein Rlf R2, R3, R4 and R6 are independently selected from the group consisting of: (i) hydrogen; (ii) saturated or unsaturated alkyl optionally substituted with a five or six member aryl or heteroaryl ring portion, wherein the ring portion is optionally substituted with one, two, or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro and ester portions; (iii) a five or six member aryl or heteroaryl ring portion, optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro and ester.

12. The method as described in claim 11, wherein Rx and R2 are independently selected from the group consisting of: (i) hydrogen; and (ii) phenyl optionally substituted with the substituent selected from the group consisting of alkyl, halogen, trihalomethyl, nitro, carboxylate, hydroxy and alkoxy portions.

13. The method as described in claim 12, wherein Xx is nitrogen or oxygen.

14. The method as described in claim 13, wherein the substituents X6 and Xlr taken together, form a portion that are selected from the group consisting of SAQAR substituents, as defined herein.

15. The method as described in claim 14, wherein the compound based on 5-Azaquinoxaline is selected from the group consisting of SAQAR compounds, as defined herein.

16. A method for preventing or treating an abnormal condition in an organism, comprising the step of administering a 5-azaquinoxaline-based compound of formula I to the organism: (I) wherein (a) Rt, R2, R3, R4 and Rs are independently selected from the group consisting of (i) hydrogen; (ii) saturated or unsaturated alkyl optionally substituted with a five or six member aryl or heteroaryl ring portion, wherein the ring portion is optionally substituted with one, two, or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester portions; (iii) an amine of formula NX2X3, wherein X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five or six membered aryl portions or heteroaryl ring; (iv) halogen or trialomethyl; (v) a ketone of formula -C0-X4, wherein X4 is selected from the group consisting of hydrogen, alkyl and five or six membered aryl or heteroaryl portions, - (vi) a carboxylic acid of the formula - (Xs) n-C00H or ester of formula - (X6) n-C00-X7, wherein Xs, Xs and X7 are independently selected from the group consisting of alkyl and aryl or heteroaryl portions of five members or six members and wherein n is 0 or 1; (vii) an alcohol of formula (X ") n-0H or a portion of formula - (Xß) n-0-X9, wherein Xβ and X, are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl and five-membered or six-membered aryl or heteroaryl ring portions, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester, and wherein n is 0 or 1; (viii) an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, and a five-membered or six-membered aryl or heteroaryl ring portion, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro or ester; (ix) -S02NX1: LX12, wherein XX1 and X12 are selected from the group consisting of hydrogen, alkyl and five or six membered aryl or heteroaryl ring portions; (x) a five or six member aryl or heteroaryl ring portion optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester, - (xi) ) an aldehyde of formula -CO-H; and (xii) a sulfone of the formula -S02-X13, wherein X13 is selected from the group consisting of saturated or unsaturated alkyl and five or six membered aryl or heteroaryl portions; and (b) Xx is selected from the group consisting of nitrogen, sulfur and oxygen.

17. The method as described in claim 16, wherein R 1 t R 2, R 3, R 4 and R 6 are independently selected from the group consisting of: (i) hydrogen; (ii) saturated or unsaturated alkyl optionally substituted with a five or six member aryl or heteroaryl ring portion, wherein the ring portion is optionally substituted with one, two, or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro and ester portions; and (iii) a five-membered or six-membered aryl or heteroaryl ring portion, optionally substituted with one, two or three substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro portions and ester.

18. The method as described in claim 17, wherein Rx and R2 are independently selected from the group consisting of: (i) methyl optionally substituted with phenyl optionally substituted with a substituent selected from the group consisting of alkyl, halogen portions , hydroxy and alkoxy; and (ii) phenyl optionally substituted with substituents which are selected from the group consisting of alkyl, halogen, hydroxy and alkoxy portions.

19. The method as described in claim 18, wherein Xx is nitrogen or oxygen.

20. The method as described in claim 19, wherein the Rβ and Xlf substituents taken together, form a portion that is selected from the group consisting of SAQAR substituents, as defined herein.

21. The method as described in claim 20, wherein the compound based on 5-Azaquinoxaline is selected from the group consisting of SAQAR compounds, as defined herein.

22. The method as described in claim 16, wherein the organism is a mammal.

23. The method as described in claim 16, wherein the abnormal condition is cancer or a fibrotic disorder.

24. The method as described in claim 23, wherein the abnormal condition is a cancer that is selected from the group consisting of lung cancer, ovarian cancer, breast cancer, brain cancer, intraaxial brain cancer, colon cancer, cancer of prostate, sarcoma, Kaposi's sarcoma, melanoma and glioma.

25. The method as described in claim 16, wherein the abnormal condition is associated with an aberration in a signal transduction pathway characterized by an interaction between a serine / threonine protein kinase and a natural binding partner.

26. The method as described in claim 25, wherein the serine / threonine protein kinase is RAF.

27. A compound based on 5-azaquinoxaline, which has a structure as stated in formula I: (I) wherein (a) R i t R a, R 3, R 4 and R b are independently selected from the group consisting of (i) hydrogen; (ii) saturated or unsaturated alkyl optionally substituted with a five or six member aryl or heteroaryl ring portion, wherein the ring portion is optionally substituted with one, two, or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester portions; (iii) an amine of formula NX2X3, wherein X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and five or six membered aryl or heteroaryl ring portions. (iv) halogen or trialomethyl; (v) a ketone of formula -C0-X4, wherein X4 is selected from the group consisting of hydrogen, alkyl and five or six membered aryl or heteroaryl portions, - (vi) a carboxylic acid of formula - (X n -COOH or ester of formula - (X6) n -COO-X7, wherein Xs, Xβ and X7 are independently selected from the group consisting of alkyl and aryl or heteroaryl portions of five members or six members and wherein n is 0 or 1; (vii) an alcohol of formula (Xß) n-OH or a portion of formula - (X8) n-0-X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl and portions of a five-membered or six-membered aryl or heteroaryl ring, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester, and wherein n is 0 or 1; (viii) an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, and a five-membered or six-membered aryl or heteroaryl ring portion, wherein the ring is optionally substituted with one or more substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro or ester; (ix) -S02NX ?: X12, wherein XX1 and X12 are selected from the group consisting of hydrogen, alkyl and five or six membered aryl or heteroaryl ring portions; (x) a five or six member aryl or heteroaryl ring portion optionally substituted with one, two or three substituents which are independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro and ester moieties; (xi) an aldehyde of formula -CO-H; and (xii) a sulfone of the formula -S02-X13, wherein X13 is selected from the group consisting of saturated or unsaturated alkyl and five or six membered aryl or heteroaryl portions; and (b) Xx is selected from the group consisting of nitrogen, sulfur and oxygen.

28. The method as described in claim 27, wherein Rlf R2, R3, R4 and R6 are independently selected from the group consisting of: (i) hydrogen, - (ii) saturated or unsaturated alkyl optionally substituted with a ring portion five or six membered aryl or heteroaryl, wherein the ring portion is optionally substituted with one, two, or three substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro portions and ester; and (iii) a five-membered or six-membered aryl or heteroaryl ring portion, optionally substituted with one, two or three substituents that are independently selected from the group consisting of alkyl, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro portions and ester.

29. The compound as described in claim 28, wherein R3 and R4 are hydrogen.

30. The compound as described in claim 29, wherein Rx and R2 are independently selected from the group consisting of: (i) methyl optionally substituted with phenyl optionally substituted with a substituent selected from the group consisting of alkyl, halogen portions , hydroxy and alkoxy; and (ii) phenyl optionally substituted with substituents which are selected from the group consisting of alkyl, halogen, hydroxy and alkoxy portions.

31. The compound as described in claim 29, wherein R x and R 2 are independently selected from the group consisting of hydrogen, methyl, phenyl and 4-hydroxyphenyl.

32. The compound as described in claim 30, wherein Xx is nitrogen or oxygen.

33. The compound as described in claim 32, wherein the Rβ and Xx substituents, taken together, form a portion that is selected from the group consisting of SAQAR substituents, as defined herein.

34. The method as described in claim 33, wherein the compound based on 5-Azaquinoxaline is selected from the group consisting of SAQAR compounds, as defined herein.

35. A pharmaceutical composition comprising a 5-azaquinoxaline compound of any of claims 26-33 or a salt thereof, and a physiologically acceptable carrier or diluent.

36. A method for synthesizing a compound as described in claim 27, comprising the steps of: (a) reacting a first reagent with a second reagent in a solvent and in the presence of a base, wherein the first reagent is 2- amino-6-chloro-3-nitropyridine and wherein the second reactant is an alcohol or an amine, which provides a first intermediate; (b) reacting the first intermediate with a third reagent, in the presence of a catalyst, and a reducing agent, wherein the third reagent is 1,2-dione; and (c) purifying the compound of claim 27.

37. The method as described in claim 36, wherein the second reagent is selected from groups consisting of SAQAR reagents, as defined herein.

38. The method as described in claim 36, wherein the third reagent is selected from the group consisting of 4-hydroxyphenylglyoxal, l-phenyl-1,2-propanedione and benzyl.

39. The method as described in claim 36, wherein the reducing agent is hydrogen.

40. The method as described in claim 36, wherein the catalyst is Raney nickel.