Title of Invention	AZABENZIMIDAZOLE COMPOUNDS, METHODS OF MODULATING THE FUNCTION OF SERINE/THREONINE PROTEIN KINASES AND METHOD OF SYNTHESIS THEREOF
Abstract	The present invention provides an azabenzimidazole compound having the formula set forth in formula I: wherein, (a) R1, R2, and R3 are independently selected from the group consisting of: (i) unsaturated alkyl; (ii) —NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula -CO-X4, where X4, is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X6)n- COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and heter'ocyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O- X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or' more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii) an amide of formula —NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii) —SO2NX11X12, wherein Xn and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or' three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester' moieties; (x) an aldehyde of formula —CO-H; and (xi) a sulfone of formula --SO2-X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (b) Z1 and Z2 are NH; and (c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen.

Title of Invention

AZABENZIMIDAZOLE COMPOUNDS, METHODS OF MODULATING THE FUNCTION OF SERINE/THREONINE PROTEIN KINASES AND METHOD OF SYNTHESIS THEREOF

Abstract

The present invention provides an azabenzimidazole compound having the formula set forth in formula I: wherein, (a) R1, R2, and R3 are independently selected from the group consisting of: (i) unsaturated alkyl; (ii) —NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula -CO-X4, where X4, is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X6)n- COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and heter'ocyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O- X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or' more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii) an amide of formula —NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii) —SO2NX11X12, wherein Xn and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or' three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester' moieties; (x) an aldehyde of formula —CO-H; and (xi) a sulfone of formula --SO2-X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (b) Z1 and Z2 are NH; and (c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen.

Full Text	DESCRIPTION BACKGROUND OF THE INVENTION The following description of the background of the invention is provided to aid in understanding the invention but is not admitted to be prior art to the invention. Cellular signal transduction is a fundamental mechanism whereby external stimuli regulating diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosp.horylation of proteins, which enables regulation of the activity of mature proteins by altering their structure, and function. The best characterized protein kinases in eukaryotes phosphorylate proteins on the alcohol moiety of serine, threonine, and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylating serine and threonine, and those specific for phosphorylating tyrosine. Some kinases, referred to as "dual specificity" kinases, are able to phosphorylate on tyrosine as well as serine/threonine residues. Protein kinases can also be characterized by their location within the cell. Some kinases are transmembrane receptor proteins capable of binding ligands external to the cell membrane. Binding the ligands alters the receptor protein kinase's catalytic activity. Others are non- receptor proteins lacking a transmembrane domain. Non- receptor protein kinases can be found in a variety of cellular 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 state. Ultimately the activity of a downstream effector is modulated by phosphorylation resulting from activation of such a pathway. The serine/threonine kinase family includes members that regulate many steps of signaling cascades, including cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle. An example of a non-receptor protein kinase that phosphorylates protein targets on serine and threonine residues is RAF. RAF modulates the catalytic activity of other protein kinases, such as the protein kinase that phosphorylates and thereby activates mitogen activated protein kinase (MAPK). RAF itself is activated by the membrane anchored protein RAS, which in turn is activated in response to ligand activated tyrosine receptor protein kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR). The biological importance of RAF in controlling cellular events is underscored by the finding that altered forms of RAF can cause cancer in organisms. Evidence for importance of RAF in malignancies is provided in Monia et al., 1996, Nature Medicine 2: 668, incorporated herein by reference in its entirety including all figures and tables. In an effort to discover novel treatments for cancer and other diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein kinases. Some small organic molecules form 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-azaindole derivatives (PCT WO 94/14808), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Patent No. 5,330,992), styryl compounds (U.S. Patent No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Patent No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1) , seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660), and benzylphosphonic acid compounds (PCT WO 91/15495). The compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous therapeutics as 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, many inhibit a variety of protein kinases and will therefore cause multiple side-effects as therapeutics for diseases. SUMMARY OF THE INVENTION Accordingly, the present invention provides an azabenzimidazole compound having the formula set forth in formula I: wherein, (a) R1, R2, and R3 are independently selected from the group consisting of: (i) unsaturated alkyl; (ii) -NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (iii)halogen or trihalomethyl; (iv) a ketone of formula -CO-X4, where X4, is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (v) a carboxylic acid of formula -(X5)n-COOH or ester of formula - (X6) n-COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and heter'ocyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula -(X8)n-O-X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl,,. homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or' more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii)an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii) -SO2NX1 1X12, wherein Xn and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or' three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester' moieties; (x) an aldehyde of formula -CO-H; and (xi) a sulfone of formula -SO2-X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (b) Z1 and Z2 are NH; and (c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen. The present invention also provides an azabenzimida- zole compound having a structure set forth in formulas II or III: wherein (a) Rl, R2, R3, and R4 are independently selected from the group consisting of: (i) hydrogen; (ii) saturated or unsaturated alkyl; (ii) NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic or heterocyclic ring moieties; (iii)halogen or trihalomethyl; (iv) a ketone of formula -CO-X4, where X4 is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties and heterocyclic ring moieties; (v) a caxboxylic acid of formula -(X5)n-COOH or ester of formula - (X6) n-COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula -(X8)n-O-X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, txihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii)an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii)-SO2NX1 1X12, wherein X11 and X12 are selected from the group consisting of hydrogen, alkyl, and liomocyclic ring moieties, and beterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, car'boxylate, nitro, and ester moieties; (x) an aldehyde of formula -CO-H; and (xi) a sulfone of formula -SO2-X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (b) Z1 and Z2 are independently selected from the group . consisting of nitrogen,, NH, and NR4; and (c) Z3 and X1 are independently selected from the group consisting of NH, sulfur, and oxygen. The present invention further provides a method of modulating the function of a serine/threonine protein kina.se with an azabenzimidazole based compound in vitro , said compound having the formula set forth in formula I: wherein, (a) R1, R2, and R3 are independently selected from the group consisting of: (i) unsaturated alkyl; (ii)-NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula -CO-X4 where X4, is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (v) a carboxylic acid of formula (X5)n-COOH or ester of formula (X6) n-COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and hetero cyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula -(X8)n-O-X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii)an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii) -SO2NX1 1X12, wherein X11 and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties; (x) an aldehyde of formula -CO-H; and (xi) a sulfone of formula -SO2-X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, arid heterocyclic ring moieties; (b) Z1 and Z2 are NH; and (c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen, comprising the steps of contacting the cells expressing said serine/ threonine protein kinase with said azabenzimidazole based compound. The present invention also provides a method of modulating the function of a serine/threonine protein kinase with an azabenzimidazole-based compound in vitro, said compound having the formula as set forth in formula II or III: wherein (a) R1, R2, R3, and R4 are independently selected from the group consisting of: (i) hydrogen; (ii) saturated or unsaturated alkyl; (ii) NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic or heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula -CO-X4, where X4 is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties and heterocyclic ring moieties; (v) a carboxylic acid of formula -(X5)n-COOH or ester of formula - (X6) n-COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyciic ring moieties, and heterocyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula -(X8)n-O-X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii)an amide of formula -NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii)-SO2NX11X12, wherein X11 and X12 are selected from the group consisting of hydrogen, alkyl, and honiocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties; (x) an aldehyde of formula -CO-H; and (xi) a sulfone of formula -SO2 -X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (b) Z, and 4 are independently selected from the group consisting of nitrogen, NH, and NR,,; and (c) Z3 and X1 are independently selected from the group consisting of NH, sulfur, and oxygen, comprising the steps of contacting the cells expressing said serine/ threonine protein kinase with said azabenzimidazole based compound. The present invention further provides a method for synthesizing a compound having a structure set forth in formula I, II or III, as defined hereinbefore, said method comprising the steps of: (a) reacting 2-amino-6-chloro-3-nittOpyridine with a second reactant in a solvent, yielding a first intermediate, wherein said second reactant is selected from the group consisting of a substituted phenol, substituted thiophenol, and substituted aniline; (b) reducing the said first intermediate in the presence of a catalyst and a reducing agent, yielding a second intermediate; (c) reacting the second intermediate with a third reactant; and (d) purifying the compound, so obtained. I. Methods for Screening Compounds that Modulate Serine/Threonine Protein Kinase Function The methods of the present invention provide means for modulating the function of both receptor and cytosolic serine/threonine protein kinases. These methods provide umeans of modulating the enzymes both in vitro and in vivo. For in vitro applications, the methods of the invention relate in part to method of identifying compounds that modulate the function of serine/threonine protein kinases. Thus, in a first aspect, the invention features a method of modulating the function of a serine/threonine protein kinase with an azabenzimidazole-based compound. The azabenzimidazole compound is optionally substituted with organic groups. The method comprises comprises contacting cells expressing 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 steps in signaling cascades, including cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle. The term "modulates" refers to the ability of, a compound to alter the function of a protein kinase. 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 most preferably inhibits the catalytic activity of a protein kinase. The term "catalytic activity", in the context of the invention, defines the rate at which a protein kinase phosphorylates a substrate. 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 normally 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. The substrate is preferably a peptide and more preferably a protein. In relation to the protein kinase RAF, preferred substrates are MEK and the MEK substrate MAPK. The term "activates" refers to increasing the cellular function of a protein kinase. The protein kinase function is preferably the interaction with a natural binding partner and most preferably catalytic activity. The term "inhibit" refers to decreasing the cellular function of a protein kinase. The protein kinase function is preferably the interaction with a natural binding partner and most preferably catalytic activity. The term "modulates" also refers to altering the function of a protein kinase by increasing or decreasing the probability that a complex forms between a protein kinase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the protein kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the protein kinase and the natural binding partner depending on the concentration of the compound exposed to the protein kinase, and most preferably decreases the probability that a complex forms between the protein kinase and the natural binding partner. The term "complex" refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another. For instance, a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble 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 propagating a signal in a protein kinase signal transduction process. A change in the interaction between a protein kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of the protein kinase/natural binding partner complex. A protein kinase natural binding partner can bind to a protein kinase's intracellular region with high affinity. High affinity represents an equilibrium binding constant on the order of 10"6 M or less. In addition, a natural binding partner can also transiently interact with a protein kinase intracellular region and chemically modify it. Protein kinase natural binding partners are chosen from a group that includes, but is not limited to, SRC homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding (PTB) domains, guanine nucleotide exchange factors, protein phosphatases, and other protein kinases. Methods of 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 with at least 10% amino acid identity to other enzymes that phosphorylate proteins on serine and threonine residues. A serine/threonine protein kinase catalyzes the addition of phosphate onto proteins on serine and threonine residues. Serine/threonine protein kinases can exist as membrane bound proteins or cytosolic protins. The term "contacting" as used herein refers to mixing a solution comprising an azabenzimidazole compound of the invention with a liquid medium bathing the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethylsulfoxide (DMSO), which facilitates the uptake of the azabenzimidazole compound or compounds into the cells of the methods. The solution comprising the azabenzimidazole compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device. The term "azabenzimidazole-based compound" refers to an azabenzimidazole organic compound substituted with chemical substituents. Azabenzimidazole compounds are of the general structure: The term "substituted", in reference to the invention, refers to an azabenzimidazole compound that is derivatized with any number of chemical substituents. In a preferred embodiment, the invention relates to the method of modulating the function of a serine/threonine protein kinase, where the protein kinase is RAF. The RAF protein kinase phosphorylates protein targets on serine or threonine residues. One such protein target is the protein kinase (MEK) that phosphorylates and consequently activates mitogen activated protein kinase (MAPK). RAF itself is activated by the membrane-bound guanine triphosphate hydrolyzing enzyme RAS in response to mitogen-stimulated receptor protein tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet- derived growth factor receptor (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 mitogen-activated protein kinase (MAPK). Assays that monitor only the phosphorylation of MEK by RAF are not sensitive because the phosphorylation levels of MEK are not significant. To overcome this sensitivity dilemma, the phosphorylation of both MEK and MAPK are 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 immunosorbant assays. In addition, the assay of the invention is performed in a high throughput format such that many compounds can be rapidly monitored in a short period of time. In another aspect, the invention describes a method of identifying compounds that modulate the function of serine/threonine protein kinase, comprising the steps of contacting cells expressing the serine/threonine protein kinase with the compound, and monitoring an effect upon the cells. The term "monitoring" refers to observing the effect of adding the compound to the cells of the method. The effect can be manifested in a change in cell 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 a change in cell phenotype or cell proliferation. "Effect" can also describe a change or an absence of a change in the catalytic activity of the protein kinase. "Effect" can also describe a change or an 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 of identifying compounds that modulate the function of serine/threonine protein kinase, where the effect is a change or an absence of a change in cell 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 numbers of cells), cell differentiation (a change or absence of a change in cell shape), cell survival, apoptosis (cell death), or the utilization of a metabolic nutrient (e.g., glucose uptake). Changes or the absence of changes in cell phenotype are readily measured by techniques known in the art. In another preferred embodiment, the invention relates to the method of identifying compounds that modulate the function of serine/threonine protein kinase, where the effect is a change or an absence of a change in cell proliferation. The term "cell proliferation" refers to the rate at which a group of cells divides. The number of cells growing in a vessel can be quantified by a person skilled in the art when that person visually counts the number of cells in a defined volume using a common light microscope. Alternatively, cell proliferation rates can be quantified by laboratory apparatae that optically or conductively measure the density of cells in an appropriate medium. In another preferred embodiment, the invention relates to the method of identifying compounds that modulate the function of serine/threonine protein kinase, where the effect is a change or an absence of a change in the interaction between the serine/threonine protein kinase with a natural binding partner. The term "interaction", in the context of the invention, describes a complex formed between a protein kinase's intracellular region and a natural binding partner or compound. The term "interaction" can also extend 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 receptor protein kinase will harbor both an extracellular and an intracellular region. The term "intracellular region" as used herein refers to the portion of a protein kinase which exists inside a cell. The term "extracellular region" as used herein refers to a portion of a protein kinase which exists outside of the cell. In a preferred embodiment, the invention relates to the method of identifying compounds that modulate the function of serine/threonine protein kinase that further comprises the following steps:(a) lysing the cells to render a lysate comprising serine/threonine protein kinase; (b) adsorbing the serine/threonine protein kinase to an antibody; (c) incubating the adsorbed serine/threonine protein kinase 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 "lysing" as used herein refers to a method of disrupting the integrity of a cell such that its interior contents are liberated. Cell lysis is accomplished by many techniques known to persons skilled in the art. The method is accomplished preferably by sonication or cell sheering techniques and more preferably by detergent techniques. The term "antibody" as used herein refers to a protein molecule that specifically binds a protein kinase. An antibody preferably binds to one class of protein kinase and more preferably specifically binds to the RAF protein kinase. The term "specifically binds" as used herein refers to an antibody that binds a protein kinase with higher affinity than another protein kinase or cellular protein. An antibody that specifically binds to a protein kinase will bind a higher concentration of the specific protein kinase than any other protein kinase or cellular protein. The term "adsorbing" as used herein refers to the binding 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. Antibodies can be linked to solid supports using techniques well known to individuals of ordinary skill in the art. See, e.g., Harlo & Lane, Antibodies, A Laboratory Manual, 1989, Cold Spring Harbor Laboratories. The term "measuring the phosphate concentration" as used herein refers to techniques commonly known to persons of ordinary skill in the art. These techniques can involve quantifying the concentration of phosphate concentrations within a substrate or determining relative amounts of phosphate within a substrate. These techniques can 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 the method of identifying compounds that modulate the function of serine/threonine protein kinase that further comprises the following steps: (a) lysing the cells to render a lysate comprising RAF; (b) adsorbing the RAF to an antibody; (c) incubating the adsorbed RAF with MEK and MAPK; and (d) adsorbing the MEK and MAPK to a solid support or antibody or antibodies. The step of measuring the effect on the cells comprises monitoring the phosphate concentration of said MEK and MAPK. In a preferred embodiment, the invention relates to the method of identifying compounds that modulate the function of serine/threonine protein kinase, where the azabenzimidazole-based compound has a structure set forth in formula I, II, or III as defined herein or any of the subgroups thereof set forth herein. 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-toluenesulfonic acid, salicylic acid and the like. The term "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs may be easier to administer than the parent drug in some situations. For example, the prodrug may be bioavailable by oral administration but the parent is not, or the prodrug may improve solubility to allow for intravenous administration. In another preferred embodiment, the invention relates to the method of identifying compounds that modulate the function of serine/threonine protein kinase, where the azabenzimidazole-based compound has a structure set forth in formula I, II, or III, where the azabenzimidazole compound is selected from the group consisting of SABI compounds. The term "SABI compounds" refers to the group of azabenzimidazole-based compounds having a structure set forth in formula A or B, and numbered A-l through A-198 in the following table: In another aspect, the invention features a method of preventing or treating an abnormal condition in an organism by administering a compound of the invention, as specified herein by formula I, II, or III, with any of the constraints provided herein, to an organism. The term "organism" relates to any living entity comprising at least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. In preferred embodiments, an organism refers to humans or mamals. The term "preventing" refers to the method of the invention decreasing the probability, or eliminating the possibility, that an organism contracts or develops the abnormal condition. The term "treating" refers to the method of the invention having a therapeutic effect and at least partially alleviating or abrogating the abnormal condition in the organism. The term "therapeutic effect" refers to the inhibition of cell growth causing or contributing to an abnormal condition. The term "therapeutic effect" also refers to the inhibition of growth factors causing or contributing to the abnormal condition (e.g. cancer). A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of a cancer, a therapeutic effect refers to one or more of the following: (a) a reduction in tumor size; (b) inhibition (i.e., slowing or stopping) tumor metastasis; (c) inhibition of tumor growth; and (d) relieving to some extent one or more of the symptoms associated with the abnormal condition. Compounds demonstrating efficacy against leukemias can be identified as described herein, except that rather than inhibiting metastasis, the compounds may instead slow 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 their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival. Aberrant cell proliferative conditions include cancers such as fibrotic and 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 rates, and tissue grafting techniques. Aberrant cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated. A number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death. Cell proliferation, differentiation, and survival are phenomena simply measured by methods in the art. These methods can involve observing the number of cells or the appearance of cells under a microscope with respect to time (for example, days). The term "administering" relates broadly to the provision to an organism and more specifically to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications. For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques. In a preferred embodiment, the invention relates to a method of preventing or treating an abnormal condition in an organism, where the azabenzimidazole-based compound has a structure set forth in formula I, II, or III as defined herein or any of the subgroups thereof set forth herein. In another preferred embodiment, the invention relates to a method of preventing or treating an abnormal condition in an organism, where the azabenzimidazole compound is selected from the group consisting of SABI compounds. In another preferred embodiment, the invention relates to a method of preventing or treating an abnormal condition in an organism, where the organism is a mammal. The term "mammal" refers preferably to such organisms as mice, rats, rabbits, guinea pigs, and goats, more preferably to monkeys and apes, and most preferably to humans. In another preferred embodiment, the invention relates to a method of preventing or treating an abnormal condition in an organism, where the abnormal condition is cancer or a fibrotic disorder. In yet another preferred embodiment, the invention relates to a method of preventing or treating an abnormal condition in an organism, where the cancer is selected from the group consisting of lung cancer, ovarian cancer, breast cancer, brain cancer, intra-axial brain cancer, colon cancer, prostate cancer, sarcoma, Kaposi's sarcoma, melanoma, and glioma. In still another preferred embodiment, the invention relates to a method of preventing or treating an abnormal condition in an organism, where the method applies to an abnormal condition associated with an aberration in a signal transduction pathway characterized by an interaction between a serine/threonine protein kinase and a natural binding partner. The term "signal transduction pathway" 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 encompases signals that are propagated entirely within a cell. The polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein kinases, receptor and non-receptor protein phosphatases, nucleotide exchange factors, and transcription factors. The term "aberration", in conjunction with a signal transduction process, refers to a protein kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner, The term "promoting or disrupting the abnormal interaction" refers to a method that can be accomplished 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 natural binding partners by forming favorable interactions with multiple atoms at the complex interface. Alternatively, a compound can inhibit an interaction between a protein kinase and natural binding partners by compromising favorable interactions formed between atoms at the complex interface. In another preferred embodiment, the invention relates to a method of preventing or treating an abnormal condition in an organism, where the serine/threonine protein kinase is RAF. III. Compounds and Pharmaceutical Compositions of the Invention In another aspect, the invention features azabenzimidazole compounds having structures set forth in formula I, II, or III: where (a) R1, R2, R3, and R4 are independently selected from the group consisting of (i) hydrogen; (ii) saturated or unsaturated alkyl; (iii) NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and homocyclic or heterocyclic ring moieties; (iv) halogen or trihalomethyl; (v) a ketone of formula -CO-X4, where X4 is selected from the group consisting of hydrogen, alkyl, and homocyclic or heterocyclic ring moieties; (vi) a carboxylic acid of formula -(X5)n-COOH or ester of formula - (X6) n-COO-X7, where X5, X6, and X7 are independently selected from the group consisting of alkyl and homocyclic or heterocyclic ring moieties and where n is 0 or 1; (vii) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula -(X8)n-O-X9, where X8 and X9 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and homocyclic or heterocyclic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisitng of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester and where n is 0 or 1; (viii) an amide of formula -NHCOX10, where X10 is selected from the group consisting of alkyl, hydroxyl, and homocyclic or heterocyclic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisitng of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (ix) -SO2NX11X12, where X11 and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic or heterocyclic ring moieties; (x) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisitng of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties; (xi) an aldehyde of formula -CO-H; and (xii) a sulfone of formula -SO2-X13, where X13 is selected from the group consisting of saturated or unsaturated alkyl and homocyclic or heterocyclic ring moieties; (b) Z1 and Z2 are independently selected from the group consisting of nitrogen, sulfur, oxygen, NH and NR4, provided that if one of Z1 and Z2 is nitrogen, NH, or NR4 then the other of Z1 and Z2 is nitrogen, sulfur, oxygen, NH, or NR4; and (c) Z3 and X1 are independently selected from the group consisting of nitrogen, sulfur, and oxygen. The term "saturated alkyl" refers to an alkyl moiety that does not contain any alkene or alkyne moieties. The alkyl moiety may be branched or non-branched. The term "unsaturated alkyl" refers to an alkyl moiety that contains at least one alkene or alkyne moiety. The alkyl moiety may be branched or non-branched. The term "amine" refers to a chemical moiety of formula NR1R2 where R1 and R2 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and homocyclic or heterocyclic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties. The term "aryl" refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl (e.g. phenyl) and heterocyclic aryl groups (e.g. pyridine). The term "carbocyclic" refers to a compound which contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom which is different from carbon. The term "heteroarly" refers to an aryl group which contains at least one heterocyclic ring. The term "halogen" refers to an atom selected from the group consisting of fluorine, chlorine, bromine, and iodine. The term "ketone" refers to a chemical moiety with formula -(R)n-CO-R', where R and R' are selected from the group consisting of saturated or unsaturated alkyl and homocyclic or heterocyclic ring moieties and where n is 0 or 1. The term "carboxylic acid" refers to a chemical moiety with formula -(R)n-COOH, where R is selected from the group consisting of saturated or unsaturated alkyl and homocyclic or heterocyclic ring moieties, and where n is 0 or 1. The term "ester" refers to a chemical moiety with formula -(R)n-COOR', where R and R' are independently selected from the group consisting of saturated or unsaturated alkyl and homocyclic or heterocyclic ring moieties and where n is 0 or 1. The term "alcohol" refers to a chemical substituent of formula -ROH, where R is selected from the group consisting of hydrogen, saturated or unsaturated alkyl, and homocyclic or heterocyclic ring moieties, where the ring moiety is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, and ester moieties. The term "amide" refers to a chemical substituent of formula -NHCOR, where R is selected from the group consisting of hydrogen, alkyl, hydroxyl, and homocyclic or heterocyclic ring moieties, where the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, halogen, trihalomethyl, carboxylate, nitro, or ester. The term "alkoxy moiety" refers to a chemical substituent of formula -OR, where R is hydrogen or a saturated or unsaturated alkyl moiety. The term "aldehyde" refers to a chemical moiety with formula -(R)n-CHO, where R is selected from the group consisting of saturated or unsaturated alkyl and homocyclic or heterocyclic ring moieties and where n is 0 or 1. The term "sulfone" refers to a chemical moiety with formula -SO2-R, where R is selected from the group consisting of saturated or unsaturated-alkyl and homocyclic or heterocyclic ring moieties. In another preferred embodiment, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where Z1 and Z2 are independently selected from the group consisting of nitrogen and NH. In yet another preferred embodiment, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, saturated or unsaturated alkyl optionally substituted with a homocyclic or heterocyclic ring moieties, where the ring moiety is optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, and ester moieties, and a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, hydroxy, alkoxy, carboxylate, nitro, and ester moieties. In other preferred embodiments, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where R2 and R3 are hydrogen. In another preferred embodiment, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where R1 is phenyl optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, or ester moieties. In yet another preferred embodiment, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where R1 selected from the group consisting of SABI substituents. The term "SABI substituents" refers to the group of substituents consisting of phenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 4-(trifluoromethyl)phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-carboxyphenyl, 3-carboxyphenyl, and 4-carboxyphenyl. In other preferred embodiments, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where X1 is sulfur. In another preferred embodiment, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where X1 is oxygen. In yet another preferred embodiment, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where Z3 is oxygen. In other preferred embodiments, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where R4 is selected from the group consisting of methyl and ethyl. In another preferred embodiment, the invention relates to an azabenzimidazole-based compound having a structure set forth in formula I, II, or III, where the azabenzimidazole compound is selected from the group consisting of SABI compounds. In another aspect, the invention features a pharmaceutical composition comprising a compound of the invention, as specified herein, or its salt, and a physiologically acceptable carrier or diluent. In another aspect, the invention relates to a pharmaceutical composition comprising a compound having a structure of formula I, II, or III as defined herein or any of the subgroups thereof set forth herein. In another preferred embodiment, the invention relates to a pharmaceutical composition, where the azabenzimidazole compound is selected from the group consisting of SABI compounds. The term "pharmaceutical composition" refers to a mixture of an azabenzimidazole compound of the invention with other chemical components, such as diluents or parriers . The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but 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 the incorporation of a compound into cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it 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 buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt 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. IV. Synthetic Methods of the Invention In another aspect, the invention relates to a method for synthesizing an azabenzimidazole compound of formula I, II, or III, comprising the steps of: (a) reacting 2-amino- 6-chloro-3-nitropyridine with a second reactant in a solvent to yield the first intermediate, where the second reactant is a substituted aryl ring; (b) reducing the first intermediate in the presence of a catalyst and a reducing agent to yield the second intermediate; (c) reacting the second intermediate with a third reactant; and (d) purifying the compound of invention. In a preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the substituted aryl ring is a substituted phenol, substituted thiophenol, and substituted aniline. In another preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the substituted phenol, substituted thiophenol, and substituted aniline are selected from the group consisting of SABI reactants. The term "SABI reactants" refers to the group of reactants consisting of the sodium salt of phenol, 2- nitrophenol, 3-nitrophenol, 4-nitrophenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2-cresol, 3-cresol, 4- cresol, 2-fluorophenol, 3-fluorophenol, 4-fluorophenol, 2- (trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4- (trifluoromethyl)phenol, 2-methoxyphenol, 3-methoxyphenol, 4-methoxyphenol, 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, thiophenol, 2-nitrothiophenol, 3-nitrothiophenol, 4-nitrothiophenol, 2-chlorothiophenol, 3-chlorothiophenol, 4-chlorothiophenol, 2-thiocresol, 3- thiocresol, 4-thiocresol, 2-fluorothiophenol, 3- fluorothiophenol, 4-fluorothiophenol, 2- (trifluoromethyl)thiophenol, 3-(trifluoromethyl)thiophenol, 4-(trifluoromethyl)thiophenol, 2-methoxybenzenethiol, 3- methoxybenzenethiol, 4-methoxybenzenethiol, 2- mercaptobenzoic acid, 3-mercaptobenzoic acid, 4- mercaptobenzoic acid, aniline, 2-nitroaniline, 3- nitroaniline, 4-nitroaniline, 2-chloroaniline, 3- chloroaniline, 4-chloroaniline, 2-toluidine, 3-toluidine, 4-toluidine, 2-fluoroaniline, 3-fluoroaniline, 4- fluoroaniline, 2-(trifluoromethyl)aniline, 3- (trifluoromethyl)aniline, 4-(trifluoromethyl)aniline, 2- anisidine, 3-anisidine, 4-anisidine, 2-aminobenzoic acid, 3-aminobenzoic acid, and 4-aminobenzoic acid. In a preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the solvent is n-propanol. In another preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the reducing agent is hydrogen. In yet another preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the catalyst is Raney nickel. In still another preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the third reactant is O-methylisourea. In another preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the third reactant is the product of the reaction of S-methylisothiouronium sulphate and alkyl chloroformate. In yet another preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the alkyl chloroformate is methyl chloroformate. In still another preferred embodiment, the invention relates to the method of synthesizing a compound of the invention where the alkyl chloroformate is ethyl chloroformate. 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 from the claims. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed in part towards methods of modulating the function of serine/threpnine protein kinases with azabenzimidazole-based compounds. In addition, the invention relates in part to methods for identifying compounds that modulate the function of serine/threonine protein kinases. The methods incorporate cells that express a serine/threonine protein kinase, such as RAF. RAF is a non-receptor protein kinase that is recruited to the cell membrane when it binds to activated RAS, a guanine triphosphate hydrolyzing enzyme. RAS is activated when an activated receptor protein tyrosine kinase, such as EGFR or PDGFR, bind to an adaptor protein, GRB2, and a guanine nucleotide exchange factor, SOS. SOS removes guanine diphosphate from RAS, replaces it with guanine triphosphate, and thereby activates RAS. RAS then binds RAF and consequently activates RAF. RAF may then phosphorylate other protein targets on serine and threonine residues, such as the kinase (MEK) that phosphorylates and consequently activates mitogen-activated protein kinase (MAPK). Thus, RAF serves as an intermediary controlling factor in mitogen-activated signal transduction. 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. RAF's role in cell proliferation is underscored by the observation that mutations to RAF's amino acid sequence have been associated with tumors and cancers. Because the mutations to RAF that give rise to cancer in cells lead to RAF molecules that display unregulated catalytic activity, inhibitors of RAF may alleviate or even abrogate the cell proliferation that leads to cancer in these cells. Methods of the present invention can detect compounds that modulate the function of the protein kinase RAF in cells. RAF phosphorylates a protein kinase (MEK) which in turn phosphorylates mitogen-activated protein kinase (MAPK). Assays that monitor only the phosphorylation of MEK by RAF are not sensitive because the phosphorylation levels of MEK are not significant. To overcome this sensitivity dilemma, the phosphorylation of both MEK and MAPK are 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 immunosorbant assays. In addition, the assay of the invention is preferrably performed in a high throughput format such that many compounds can be rapidly monitored in a short period of time. The methods of the present invention have identified compounds that inhibit the function of the RAF protein kinase. These compounds are azabenzimidazole-based derivatives. Although azabenzimidazole-based derivatives have been tested for their ability to inhibit enzymes involved with nucleotide synthesis in bacteria, many of these compounds have not yet been significantly explored with respect to protein kinase inhibition. Because RAF exhibits significant amino acid homology to other serine/threonine protein kinases, the azabenzimidazole-based compounds of the invention may likely inhibit serine/threonine protein kinases other than RAF. Thus, the methods of the invention also relate to serine/threonine protein kinases other than RAF, including receptor and non-receptor serine/threonine protein kinases. The methods of the invention also pertain to other compounds that modulate RAF function in cells as the high throughput aspect of the methods allows a wide array of molecules to be tested in a short period of time. Therefore, the methods of the invention can identify existing molecules not disclosed in the present invention that modulate STK function. I. Biological Activity of Azabenzimidazole—Based Compounds Azabenzimidazole-based compounds of the present invention were tested for their ability to inhibit RAF protein kinase function. The biological assays and results of these inhibition studies are reported herein. The methods used to measure azabenzimidazole-based compound modulation of protein kinase function are similar to those described in 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 Docket No. 221/187), filed August 23, 1996, with respect to the high throughput aspect of the method. The 08/702,232 application is incorporated herein by reference in its entirety, including any drawings. II. Target Diseases to be Treated by Azabenzimidazole- Based Compounds 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 harm 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 may be a result of RAF molecules that function inappropriately or a result of RAF-related protein kinase molecules that function inappropriately. Alterations in the function of the RAF protein kinase or protein kinases related to RAF can lead to enhanced or decreased cell proliferative conditions evident 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 relate to the abnormal formation of the cellular extracellular matrix. An example of a fibrotic disorder is hepatic cirrhosis. Hepatic cirrhosis is characterized by an increased concentration of extracellular matrix constituents resulting in the formation of a hepatic scar. Hepatic 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 renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. Preferred types of cancers that may be treated by the methods and compounds of the invention are lung cancer, ovarian cancer, breast cancer, brain cancer, intra-axial brain cancer, colon cancer, prostate cancer, Kaposi's sarcoma, melanoma, and glioma. Evidence that the compounds and methods of the invention can effectively be utilized to stem and reverse the proliferation of cancer cells is provided herein by reference. Angiogenic and vasculogenic disorders result from excess proliferation of blood vessels. Blood vessel proliferation is necessary in a variety of normal physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. However, blood vessel proliferation is also essential in cancer tumor development. Other examples of blood vessel proliferative disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage. In addition, blood vessel proliferative diseases include ocular diseases, such as diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, are also implicated in adverse regulation of protein kinases. Moreover, vasculogenesis and angiogenesis are associated with the growth of malignant solid tumors and metastasis. A vigorously growing cancer tumor requires a nutrient and oxygen rich blood supply to continue growing. As a consequence, an abnormally large number of capillary blood vessels often grow in concert with the tumor and act as supply lines to the tumor. In addition to supplying nutrients to the tumor, the new blood vessels embedded in a tumor provide a gateway for tumor cells to enter the circulation and metastasize to distant sites in the organism. Folkman, 1990, J. Natl. Cancer Inst. 82:4-6. Inappropriate RAF activity can stimulate cell proliferative disorders. Molecules specifically designed to modulate the function of the RAF protein kinase have been shown to inhibit cellular proliferation. Specifically, antisense nucleic acid molecules, which are designed to both bind to message RNA encoding the RAF protein kinase and block translation from that message, effectively reversed transformation of A54 9 cells in vitro. Monia et al., 1996, Nature Medicine 2: 688, incorporated herein by reference in its entirety including all figures and tables. A54 9 cells are human malignant cells. These RAF-targeted antisense studies provide evidence that the azabenzimidazole molecules of the invention, which modulate the function of the RAF protein kinase, can stem, and likely reverse, the proliferation of malignant cells in an organism. These azabenzimidazole compounds can be tested in the in vitro methods provided herein by example. Furthermore, the azabenzimidazole compounds may be tested for their effect upon tumor cells in vivo by the xenograft methods also provided herein by example. There exist at least two ways in which inappropriate RAF activity can stimulate unwanted cell proliferation of a particular type of cells: (1) directly stimulating growth of the particular cell, or (2) increasing vascularization of a particular area, such as tumor tissue, thereby facilitating growth of the tissue. The use of the present invention is facilitated by first identifying whether the cell proliferation disorder is RAF driven. Once such disorders are identified, patients suffering from such a disorder can be identified by analysis of their symptoms using procedures well known to physicians or veterinarians of ordinary skill in the art. Such patients can then be treated as described herein. Determining whether the cell proliferation disorder is RAF driven may be accomplished by first determining the level of RAF activity occurring in the cell or in a particular location in a patient's body. For example, in the case of cancer cells the level of one or more RAF activities may be compared for non-RAF driven cancers and RAF driven cancers. If the cancer cells have a higher level of RAF activity than RAF driven cancers, preferably equal to or greater than RAF driven cancers, then they are candidates for treatment using the described RAF-modulating methods and compounds of the invention. In the case of cell proliferative disorders arising due to unwanted proliferation of non-cancer cells, the level of RAF activity is compared to that level occurring in the general population (e.g., the average level occurring in the general population of people or animals excluding those people or animals suffering from a cell proliferative disorder). If the unwanted cell proliferation disorder is characterized by a higher RAF level than occurring in the general population then the disorder is a candidate for treatment using the described RAF modulating methods and compounds of the invention. III. Pharmaceutical Compositions and Administration of Azabenzimidazole-Based Compounds Methods of preparing pharmaceutical formulations of the compounds, methods of determining the amounts of compounds to be administered to a patient, and modes of administering compounds to an organism are disclosed in 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 Docket No. 221/187), filed August 23, 1996, and International patent publication number 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 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 merely representative of 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 commercially available and the sequences of genes for the various protein kinases are readily accessible in sequence data banks. Thus, a person of ordinary skill in the art can readily recreate the cell lines in a timely • manner by combining the commercially available cells, the commercially available nucleic acid vectors, and the protein kinase genes using techniques readily available to persons of ordinary skill in the art. EXAMPLE 1: PROCEDURES FOR SYNTHESIZING AZABENZIMIDAZOLE COMPOUNDS OF THE INVENTION The invention will now be illustrated in the following non-limiting examples in which, unless otherwise stated: (i) evaporations were carried out by rotary evaporation under vacuum; (ii) operations were carried out under an atmosphere of an inert gas such as nitrogen; (iii) high performance liquid chromatography (HPLC) was performed on Merck LiChrosorb RP-18 reversed-phase silica obtained from E. Merck, Darmstadt, Germany; (iv) yields are given for illustration only and are not necessarily the maximum attainable; (v) melting points are uncorrected and were determined using a HWS Mainz SG 2000 digital melting point apparatus; (vi) the structures of all compounds of the formula I, II, and III of this invention were confirmed by proton magnetic resonance spectroscopy on a Bruker AMX500-NMR spectrophotometer, by elemental microanalysis and, in certain cases, by mass spectroscopy; (vii) the purity of the structures were performed by thin layer chromatography (TLC) using silica gel (Merck Silica Gel 60 F254) or by HPLC; and (vii) intermediates were not generally fully characterized and purity was assessed by thin layer chromatography (TLC) or by HPLC. SYNTHETIC PROCEDURES Compound A-90: 2-Methoxycarbonylamino- (6-phenylmercapto.-3H- imidazo [4,5—b] pyridine 2-amino-3-nitro-6-(phenylmercapto)pyridine was prepared by heating 2-amino-6-chloro-3-nitropyridine (84.0 g, 0.484 mol) and sodium thiophenolate (Fluka) (72.0 g, 0.545 mol) in 2-propanol (1500 mL) under reflux for 2 hours. After cooling to room temperature the suspension was diluted with water (100 ml), the solid was collected by vacuum filtration, washed with water and 2-propanol, and dried at 50°C under vacuum to give 109.1 g (95 % yield) of 2-amino-3-nitro-6-(phenylmercapto)pyridine, m.p. 148-152 °C). 2,3-Diamino-6-(phenylmercapto)pyridine was prepared by hydrogenating 2-amino-3-nitro-6-(phenylmercapto)pyridine (107.1 g, 0.433 mol) under 5 atm of H2 in the presence of 30 g of Raney-Ni in 1200 mL of 2-propanol at 70°C. After 4 hours (29.1 L of hydrogen) the reaction mixture was cooled to 4°C while stirring continuously. The precipitate was collected by vacuum filtration, washed with 2-propanol and dried at 50°C under vacuum. The combined filtrates were concentrated under reduced pressure and recrystallized from 2-propanol. After washing the hydrogenation apparatus twice with 1000 mL of THF, evaporation under reduced pressure and recrystallization from 2-propanol, the precipitate was collected and dried at 50°C under vacuum to give 80.4 g (87.1 % yield) of 2,3-diamino-6- (phenylmercapto)pyridine, m.p. 119-122°C). 2-Methoxycarbonylamino-6-phenylmercapto-3H- imidazo [4, 5-b]pyridine was prepared by adding methyl chloroformate (34 mL, 0.44 mol) dropwise to a cold (5-15°C) solution of S-methylisothiouronium sulphate (53 g, 0.19 mol) (Aldrich) in 68 mL of water while the temperature was kept below 20°C. Afterwards, aqueous sodium hydroxide (116 g, 25% NaOH) was added carefully and a white precipitate occured. After 20 minutes, water (210 mL) was added and the pH adjusted to 4.0 with acetic acid (glacial, 34 mL) . To this mixture a solution of 2,3-diamino-6- (phenylmercapto)pyridine (37.8 g, 0.174 mol) in 210 mL of ethanol was added dropwise and heated to 85-90°C for 2 hours. After cooling the reaction mixture overnight, the precipitate was isolated by filtration, washed with warm water (1000 mL), dried and recrystallized from acetic acid and ethanol at 4 °C. The precipitate was collected by filtration, washed with methanol and dried at 50°C under vacuum to give 30 g (57.4 % yield) of 2- methoxycarbonylamino-6-phenylmercapto-3H-imidazo[4,5- b]pyridine, m.p. 269-274 °C (dec). Compound A-3: 2-Methoxycarbonylamino- (6-phenoxy-3H- imidazo [4,5-b]pyridine By substituting sodium phenolate in place of sodium thiophenolate in Example A-90, the identical process gives 2-methoxycarbonylamino-(6-phenoxy-3H-imidazo[4,5- b]pyridine, m.p. >280 °C (dec). Compound A-4: 2-Ethoxycarbonylamino—6-phenoxy-3H- imidazo [4,5-b]pyridine By substituting ethyl chloroformate in place of methyl chloroformate in Example A-3, the identical process gives 2-ethoxycarbonylamino- (6-phenoxy-3H-imidazo [4,5-b] pyridine, m.p. >280 °C (dec.). Compound A-l: 2-Oxo-6-phenoxy-3H-imidazo [4,5-b] pyridine By reacting O-methylisourea directly with 2,3-diamino- 6-(phenylmercapto)pyridine in place of the product of the reaction of S-methylisothiouronium sulfate and methyl chloroformate in Example A-3, the identical process gives 2-oxo-(6-phenoxy-3H-imidazo[4,5-b]pyridine, m.p. 277-278 °C. Compound A—2: 2-Oxo-6-phenylmercapto-3H-imidazo[4,5- b] pyridine By substituting sodium thiophenolate in place of sodium phenolate in Example A-l, the identical process gives 2-oxo- (6-phenylmercapto-3H-imidazb [4, 5-b] pyridine, m.p. 253-254 °C. Compounds A-5 - A-25: By substituting the appropriate phenolate for sodium phenolate in Example A-l, the identical process gives the following examples. For examples A-23, A-24, and A-25, the carboxy groups are protected by a methyl, ethyl, benzyl, t- butyl or other suitable ester and then deprotected in the last step to give the compounds. A-5 2-Oxo-6- (2-nitrophenoxy) -3H-imidazo [4, 5-b] pyridine A-6 2-Oxo-6- (3-nitrophenoxy) -3H-imidazo [4, 5-b] pyridine A-7 2-0x0-6-(4-nitrophenoxy)-3H-imidazo [4, 5-b] pyridine A-8 2-Oxo-6- (2-chlorophenoxy) -3H-imidazo [4, 5-b] pyridine A-9 2-0xo-6- (3-chlorophenoxy) -3H-imidazo [4, 5-b] pyridine A-10 2-Oxo-6- (4-chlorophenoxy) -3H-imidazo [4, 5-b] pyridine A-ll 2-Oxo-6- (2-methylphenoxy) -3H-imidazo [4, 5-b] pyridine A-12 2-Oxo-6- (3-methylphenoxy) -3H-imidazo [4, 5-b] pyridine A-13 2-Oxo-6-(4-methylphenoxy)-3H-imidazo[4,5-b]pyridine A-14 2-Oxo-6- (2-fluorophenoxy) -3H-imidazo [4, 5-b] pyridine A-15 2-Oxo-6- (3-fluorophenoxy) -3H-imidazo [4, 5-b] pyridine A-16 2-Oxo-6- (4-fluorophenoxy) -3H-imidazo [4, 5-b] pyridine A-17 2-0xo-6-[2-(trifluoromethyl)phenoxy]-3H-imidazo[4,5- b]pyridine A-18 2-Oxo-6-[3-(trifluoromethyl)phenoxy]-3H-imidazo[4,5- b] pyridine A-19 2-0x0-6-[4-(trifluoromethyl)phenoxy]-3H-imidazo[4,5- b] pyridine A-20 2-OXo-6- (2-methoxyphenoxy) -3H-imidazo [4, 5-b] pyridine A-21 2-Oxo-6-(3-methoxyphenoxy)-3H-imidazo [4, 5-b] pyridine A-22 2-Oxo-6-(4-methoxyphenoxy)-3H-imidazo [4, 5-b] pyridine A-23 2-Oxo-6- (2-carboxyphenoxy) -3H-imidazo [4, 5-b] pyridine A-24 2-Oxo-6- (3-carboxyphenoxy) -3H-imidazo [4, 5-b] pyridine A-25 2-Oxo-6- (4-carboxyphenoxy) -3H-imidazo [4, 5-b] pyridine Compounds A—26 — A—46. By substituting the appropriate thiophenolate for sodium thiophenolate in Example A-2, the identical process gives the following examples. For examples A-44, A-45, an A-4 6, the carboxy groups are protected by a methyl, ethyl, benzyl, t-butyl or other suitable ester and then deprotected in the last step to give the compounds. A-26 2-Oxo-6- (2-nitrophenylmerapto) -3H-imidazo [4,5- b] pyridine A-27 2-Oxo-6-(3-nitrophenylmercapto)-3H-imidazo[4,5- b]pyridine A-28 2-Oxo-6-(4-nitrophenylmercapto)-3H-imidazo[4,5- b] pyridine A-29 2-Oxo-6-(2-chlorophenylmercapto)-3H-imidazo[4,5- b] pyridine A-30 2-Oxo-6-(3-chlorophenylmercapto)-3H-imidazo[4,5- b]pyridine A-31 2-Oxo-6-(4-chlorophenylmercapto)-3H-imidazo[4,5- b]pyridine A-32 2-Oxo-6-(2-methylphenylmercapto)-3H-imidazo[4,5- b] pyridine A-33 2-Oxo-6- (3-methylphenylmercapto) -3H-imidazo [4,5- b]pyridine A-34 2-Oxo-6- (4-methylphenylmercapto) -3H-imidazo [4,5- b] pyridine A-35 2-Oxo-6-(2-fluorophenylmercapto)-3H-imidazo[4,5- b]pyridine A-36 2-Oxo-6- (3-f luorophenylmercapto) -3H-imidazo [4,5- b] pyridine A-37 2-Oxo-6-(4-fluorophenylmercapto)-3H-imidazo[4,5- b]pyridine A-38 2-Oxo-6-[2-(trifluoromethyl)phenylmercapto]-3H- imidazo [ 4, 5-b] pyridine A-39 2-Oxo-6-[3-(trifluoromethyl)phenylmercapto]-3H- imidazo [4, 5-b] pyridine A-40 2-Oxo-6- [4- (trifluoromethyl) phenylmercapto] -3H- imidazo[4,5-b]pyridine A-41 2-Oxo-6-(2-methoxyphenylmercapto)-3H-imidazo[4,5- b] pyridine A-42 2-Oxo-6-(3-methoxyphenylmercapto)-3H-imidazo[4,5- b]pyridine A-43 2-Oxo-6-(4-methoxyphenylmercapto)-3H-imidazo[4,5- b]pyridine A-44 2-Oxo-6-(2-carboxyphenylmercapto)-3H-imidazo [4,5- b]pyridine A-45 2-Oxo-6-(3-carboxyphenylmercapto)-3H-imidazo [4,5- b] pyridine A-4 6 2-Oxo-6-(4-carboxyphenylmercapto)-3H-imidazo[4,5- b] pyridine Compounds A-47 - A-68. By substituting the appropriate aniline salt for sodium phenolate in Example A-l, the identical process gives the following examples. For examples A-66, A-67, and A-68, the carboxy groups are protected by a methyl, ethyl, benzyl, t-butyl or other suitable ester and then deprotected in the last step to give the compounds. A-47 2-Oxo-6-phenylamino-3H-imidazo [4, 5-b]pyridine A-48 2-Oxo-6- (2-nitrophenylamino) -3H-imidazo [4,5-b]pyridine A-49 2-Oxo-6- (3-nitrophenylamino) -3H-imidazo [4, 5-b] pyridine A-50 2-Oxo-6- (4-nitrophenylamino) -3H-imidazo [4, 5-b] pyridine A-51 2-Oxo-6-(2-chlorophenylamino)-3H-imidazo[4,5- b] pyridine A-52 2-Oxo-6-(3-chlorophenylamino)-3H-imidazo[4,5- b] pyridine A-53 2-Oxo-6-(4-chlorophenylamino)-3H-imidazo[4,5- b] pyridine A-54 2-0xo-6- (2-methylphenylamino) -3H-imidazo [4,5- b]pyridine A-55 2-Oxo-6-(3-methylphenylamino)-3H-imidazo[4, 5- b] pyridine A-56 2-Oxo-6-(4-methylphenylamino)-3H-imidazo[4,5- b]pyridine A-57 2-Oxo-6-(2-fluorophenylamino)-3H-imidazo[4,5- b] pyridine A-58 2-Oxo-6-(3-fluorophenylamino)-3H-imidazo[4,5- b] pyridine A-59 2-Oxo-6- (4-f luorophenylamino) -3H-imidazo [4,5- b] pyridine A-60 2-0xo-6-[2-(trifluoromethyl)phenylamino]-3H- imidazo [ 4, 5-b] pyridine A-61 2-Oxo-6-[3-(trifluoromethyl)phenylamino]-3H- imidazo [ 4, 5-b] pyridine A-62 2-Oxo-6-[4-(trifluoromethyl)phenylamino]-3H- imidazo [ 4, 5-b] pyridine A-63 2-Oxo-6-(2-methoxyphenylamino)-3H-imidazo[4,5- b] pyridine A-64 2-Oxo-6-(3-methoxyphenylamino)-3H-imidazo[4,5- b] pyridine A-65 2-Oxo-6-(4-methoxyphenylamino)-3H-imidazo[4,5- b]pyridine A-66 2-Oxo-6-(2-carboxyphenylamino) -3H-imidazo[4,5- b] pyridine A-67 2-Oxo-6-(3-carboxyphenylamino)-3H-imidazo [4,5- b] pyridine A-68 2-Oxo-6-(4-carboxyphenylamino)-3H-imidazo[4,5- b] pyridine Compounds A-69 - A-8 9. By substituting the appropriate phenolate for sodium phenolate in Example A-3, the identical process gives the following examples. For examples A-87, A-88, and A-89, the carboxy groups are protected by a methyl, ethyl, benzyl, t- butyl or other suitable ester and then deprotected in the last step to give the compounds. A-69 2-Methoxycarbonylamino-6-(2-nitrophenoxy)-3H- imidazo [ 4 , 5-b] pyridine A-70 2-Methoxycarbonylamino-6-(3-nitrophenoxy)-3H- imidazo [ 4, 5-b] pyridine A-71 2-Methoxycarbonylamino-6-(4-nitrophenoxy)-3H- imidazo[4,5-b]pyridine A-72 2-Methoxycarbonylamino-6-(2-chlorophenoxy)-3H- imidazo[4, 5-b]pyridine A-73 2-Methoxycarbonylamino-6-(3-chlorophenoxy)-3H- imidazo[4,5-b]pyridine A-74 2-Methoxycarbonylamino-6-(4-chlorophenoxy)-3H- imidazo[4,5-b]pyridine A-75 2-Methoxycarbonylamino-6-(2-methylphenoxy)-3H- imidazo [4 , 5-Jb] pyridine A-76 2-Methoxycarbonylamino-6-(3-methylphenoxy)-3H- imidazo [4, 5-b] pyridine A-77 2-Methoxycarbonylamino-6-(4-methylphenoxy)-3H- imidazo[4, 5-b]pyridine A-78 2-Methoxycarbonylamino-6-(2-fluorophenoxy)-3H- imidazo [4,5-b]pyridine A-7 9 2-Methoxycarbonylamino-6- (3-fluorophenoxy) -3H- imidazo [ 4 , 5-b] pyridine A-80 2-Methoxycarbonylamino-6-(4-fluorophenoxy)-3H- imidazo[4,5-b]pyridine A-81 2-Methoxycarbonylamino-6-[2-(trifluoromethyl)phenoxy]- 3H-imidazo [4, 5-b] pyridine A-82 2-Methoxycarbonylamino-6-[3-(trifluoromethyl)phenoxy]- 3H-imidazo [ 4, 5-b] pyridine A-83 2-Methoxycarbonylamino-6-[4-(trifluoromethyl)phenoxy]- 3H-imidazo[4,5-b]pyridine A-8 4 2-Methoxycarbonylamino-6-(2-methoxyphenoxy)-3H- imidazo [ 4, 5-b] pyridine A-85 2-Methoxycarbonylamino-6-(3-methoxyphenoxy)-3H- imidazo [ 4, 5-b] pyridine A-86 2-Methoxycarbonylamino-6-(4-methoxyphenoxy)-3H- imidazo [ 4, 5-b] pyridine A-87 2-Methoxycarbonylamino-6-(2-carboxyphenoxy)-3H- imidazo [ 4, 5-b] pyridine A-88 2-Methoxycarbonylamino-6-(3-carboxyphenoxy)-3H- imidazo[4,5-b]pyridine A-89 2-Methoxycarbonylamino-6-(4-carboxyphenoxy)-3H- imidazo[4,5-b]pyridine Compounds A-91 - A-111. By substituting the appropriate thiophenolate for sodium thiophenolate in Example A-90, the identical process gives the following examples. For examples A-109, A-110, and A-lll, the carboxy groups are protected by a methyl, ethyl, benzyl, t-butyl or other suitable ester and then deprotected in the last step to give the compounds. A-91 2-Methoxycarbonylamino-6-(2-nitrophenylmercapto)- 3H-imidazo [ 4, 5-b] pyridine A-92 2-Methox,ycarbonylamino-6- (3-nitrophenylmercapto) - 3H-imidazo [ 4 , 5-b] pyridine A-93 2-Methoxycarbonylamino-6-(4-nitrophenylmercapto)- 3H-imidazo [ 4, 5-b] pyridine A-94 2-Methoxycarbonylamino-6-(2- chlorophenylmercapto) -3H-imidazo [4 , 5-b] pyridine A-95 2-Methoxycarbonylamino-6-(3- chlorophenylmercapto) -3H-imidazo [ 4 , 5-b] pyridine A-96 2-Methoxycarbonylamino-6-(4- chlorophenylmercapto) -3H-imidazo [4,5-b]pyridine A-97 2-Methoxycarbonylamino-6-(2- methylphenylmercapto) -3H-imidazo [4,5-b]pyridine A-98 2-Methoxycarbonylamino-6-(3- methylphenylmercapto) -3H-imidazo [4,5-b]pyridine A-99 2-Methoxycarbonylamino-6-(4- methylphenylmercapto) -3H-imidazo [4,5-b]pyridine A-100 2-Methoxycarbonylamino-6-(2- fluorophenylmercapto)-3H-imidazo [4,5-b]pyridine A-101 2-Methoxycarbonylamino-6-(3- fluorophenylmercapto) -3H-imidazo [4,5-b]pyridine A-102 2-Methoxycarbonylamino-6-(4- fluorophenylmercapto) -3H-imidazo [4,5-b]pyridine A-103 2-Methoxycarbonylamino-6-[2- (trifluoromethyl)phenylmercapto]-3H-imidazo [4,5-b]pyridine A-104 2-Methoxycarbonylamino-6-[3- (trifluoromethyl) phenylmercapto] -3H-imidazo [4,5-b]pyridine A-105 2-Methoxycarbonylamino-6-[4- (trifluoromethyl)phenylmercapto]-3H-imidazo[4,5-b]pyridine A-106 2-Methoxycarbonylamino-6-(2- methoxyphenylmercapto) -3H-imidazo [4,5-b]pyridine A-107 2-Methoxycarbonylamino-6-(3- methoxyphenylmercapto) -3H-imidazo [4,5-b]pyridine A-108 2-Methoxycarbonylamino-6-(4- methoxyphenylmercapto) -3H-imidazo [4,5-b]pyridine A-109 2-Methoxycarbonylamino-6-(2- carboxyphenylmercapto) -3H-imidazo [4,5-b]pyridine A-110 2-Methoxycarbonylamino-6-(3- carboxyphenylmercapto) -3H-imidazo [4,5-b]pyridine A-111 2-Methoxycarbonylamino-6-(4- carboxyphenylmercapto) -3H-imidazo [4,5-b]pyridine Compounds A-112 - A-133. By substituting the appropriate aniline salt for sodium phenolate in Example A-3, the identical process gives the following examples. For examples A-131, A-132, and A-133, the carboxy groups are protected by a methyl, ethyl, benzyl, t-butyl or other suitable ester and then deprotected in the last step to give the compounds. A-112 2-Methoxycarbonylamino-6-phenylamino-3H- imidazo[4,5-A-112 A-113 2-Methoxycarbonylamino-6-(2-nitrophenylamino)-3H- imidazo [4,5-b]pyridine A-114 2-Methoxycarbonylamino-6-(3-nitrophenylamino)-3H- imidazo[4, 5-b]pyridine A-115 2-Methoxycarbonylamino-6-(4-nitrophenylamino)-3H- imidazo[4,5-b]pyridine A-116 2-Methoxycarbonylamino-6-(2-chlorophenylamino)- 3H- imidazo[4,5-b]pyridine A-117 2-Methoxycarbonylamino-6-(3-chlorophenylamino)- 3H-imidazo [4,5-b]pyridine A-118 2-Methoxycarbonylamino-6-(4-chlorophenylamino)- 3H-imidazo [4,5-b]pyridine A-119 2-Methoxycarbonylamino-6-(2-methylphenylamino)- 3H-imidazo [4,5-b]pyridine A-120 2-Methoxycarbonylamino-6-(3-methylphenylamino)- 3H-imidazo [4,5-b]pyridine A-121 2-Methoxycarbonylamino-6-(4-methylphenylamino)- 3H-imidazo[4,5-b]pyridine A-122 2-Methoxycarbonylamino-6- (2-fluorophenylamino) - 3H-imidazo[4,5-b]pyridine A-123 2-Methoxycarbonylamino-6-(3-fluorophenylamino)- 3H-imidazo[4,5-b]pyridine A-124 2-Methoxycarbonylamino-6-(4-fluorophenylamino)- 3H-imidazo[4,5-b]pyridine A-125 2-Methoxycarbonylamino-6-[2- (trifluoromethyl) phenylamino] -3H-imidazo[4,5-b]pyridine A-126 2-Methoxycarbonylamino-6-[3- (trifluoromethyl) phenylamino] -3H-imidazo[4,5-b]pyridine A-127 2-Methoxycarbonylamino-6-[4- (trifluoromethyl) phenylamino] -3H-imidazo[4,5-b]pyridine A-128 2-Methoxycarbonylamino-6-(2-methoxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-129 2-Methoxycarbonylamino-6- (3-methoxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-130 2-Methoxycarbonylamino-6-(4-methoxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-131 2-Methoxycarbonylamino-6-(2-carboxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-132 2-Methoxycarbonylamino-6-(3-carboxyphenylamino)- 3H-imidazo [ 4, 5-b] pyridine A-133 2-Methoxycarbonylamino-6-(4-carboxyphenylamino)- 3H-imidazo [ 4, 5-b] pyridine Compounds A-134 - A-154. By substituting the appropriate phenolate for sodium phenolate in Example A-4, the identical process gives the following examples. For examples A-152, A-153, and A-154, the carboxy groups are protected by a methyl, ethyl, benzyl, t-butyl ester or other suitable ester and then deprotected in the last step to give the compounds. A-134 2-Ethoxycarbonylamino-6-(2-nitrophenoxy)-3H- imidazo[4,5-b]pyridine A-135 2-Ethoxycarbonylamino-6-(3-nitrophenoxy)-3H- imidazo[4,5-b]pyridine A-136 2-Ethoxycarbonylamino-6-(4-nitrophenoxy)-3H- imidazo[4,5-b]pyridine A-137 2-Ethoxycarbonylamino-6-(2-chlorophenoxy)-3H- imidazo[4,5-b]pyridine A-138 2-Ethoxycarbonylamino-6-(3-chlorophenoxy)-3H- imidazo[4,5-b]pyridine A-139 2-Ethoxycarbonylamino-6-(4-chlorophenoxy)-3H- imidazo[4,5-b]pyridine A-14 0 2-Ethoxycarbonylamino-6-(2-methylphenoxy)-3H- imidazo[4,5-b]pyridine A-141 2-Ethoxycarbonylamino-6-(3-methylphenoxy)-3H- imidazo[4,5-b]pyridine A-142 2-Ethoxycarbonylamino-6-(4-methylphMenoxy)-3H imidazo[4,5-b]pyridine A-143 2-Ethoxycarbonylamino-6-(2-fluorophenoxy)-3H- imidazo[4,5-b]pyridine A-144 2-Ethoxycarbonylamino-6-(3-fluorophenoxy)-3H- imidazo[4,5-b]pyridine A-145 2-Ethoxycarbonylamino-6-(4-fluorophenoxy)-3H- imidazo[4,5-b]pyridine A-146 2-Ethoxycarbonylamino-6-[2- (trifluoromethyl) phenoxy] -3H-imidazo[4,5-b]pyridine A-147 2-Ethoxycarbonylamino-6-[3- (trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine A-148 2-Ethoxycarbonylamino-6-[4- (trifluoromethyl) phenoxy] -3H-imidazo[4,5-b]pyridine A-149 2-Ethoxycarbonylamino-6-(2-methoxyphenoxy)-3H- imidazo[4,5-b]pyridine A-150 2-Ethoxycarbonylamino-6-(3-methoxyphenoxy)-3H- imidazo[4,5-b]pyridine A-151 2-Ethoxycarbonylamino-6-(4-methoxyphenoxy)-3H- imidazo[4,5-b]pyridine A-152 2-Ethoxycarbonylamino-6-(2-carboxyphenoxy)-3H- imidazo[4,5-b]pyridine A-153 2-Ethoxycarbonylamino-6- (3-carboxyphenoxy) -3H- imidazo[4,5-b]pyridine A-154 2-Ethoxycarbonylamino-6-(4-carboxyphenoxy)-3H- imidazo[4,5-b]pyridine Compounds A-155 - A-176. By substituting the appropriate thiophenolate for sodium phenolate in Example A-4, the identical process gives the following examples. For examples A-174, A-175, and A-176, the carboxy groups are protected by a methyl, ethyl, benzyl, t-butyl or other suitable ester and then deprotected in the last step to give the compounds. A-155 2-Ethoxycarbonylamino-6-phenylmercapto-3H- imidazo[4,5-b]pyridine A-156 2-Ethoxycarbonylamino-6-(2-nitrophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-157 2-Ethoxycarbonylamino-6-(3-nitrophenylmercapto) - 3H-imidazo[4,5-b]pyridine A-158 2-Ethoxycarbonylamino-6-(4-nitrophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-159 2-Ethoxycarbonylamino-6-(2-chlorophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-160 2-Ethoxycarbonylamino-6-(3-chlorophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-161 2-Ethoxycarbonylamino-6-(4-chlorophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-162 2-Ethoxycarbonylamino-6-(2-methylphenylmercapto)- 3H-imidazo[4,5-b]pyridine A-163 2-Ethoxycarbonylamino-6-(3-methylphenylmercapto)- 3H-imidazo[4,5-b]pyridine A-164 2-Ethoxycarbonylamino-6-(4-methylphMenoxy)-3H- imidazo[4,5-b]pyridine A-165 2-Ethoxycarbonylamino-6-(2-fluorophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-166 2-Ethoxycarbonylamino-6-(3-fluorophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-167 2-Ethoxycarbonylamino-6-(4-fluorophenylmercapto)- 3H-imidazo[4,5-b]pyridine A-168 2-Ethoxycarbonylamino-6-[2- (trifluoromethyl)phenylmercapto] 3H-imidazo[4,5-b]pyridine A-169 2-Ethoxycarbonylamino-6- [3- (trifluoromethyl) phenylmercapto] 3H-imidazo[4,5-b]pyridine A-170 2-Ethoxycarbonylamino-6-[4- (trif luoromethyl) phenylmercapto] 3H-imidazo[4,5-b]pyridine A-171 2-Ethoxycarbonylamino-6-(2- methoxyphenylmercapto) -3H-imidazo[4,5-b]pyridine A-172 2-Ethoxycarbonylamino-6-(3- methoxyphenylmercapto) -3H-imidazo[4,5-b]pyridine A-173 2-Ethoxycarbonylamino-6-(4- methoxyphenylmercapto) -3H-imidazo[4,5-b]pyridine A-174 2-Ethoxycarbonylamino-6-(2- carboxyphenylmercapto) -3H-imidazo[4,5-b]pyridine A-175 2-Ethoxycarbonylamino-6-(3- carboxyphenylmercapto) -3H-imidazo[4,5-b]pyridine A-17 6 2-Ethoxycarbonylamino-6-(4- carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine Compounds A-177 - A-198. By substituting the appropriate aniline salt for sodium phenolate in Example A-4, the identical process gives the following examples. For examples A-196, A-197, and A-198, the carboxy groups are protected by a methyl, ethyl, benzyl, t-butyl or other suitable ester and then deprotected in the last step to give the compounds. A-177 2-Ethoxycarbonylamino-6-phenylamino-3H- imidazo[4,5-b]pyridine A-178 2-Ethoxycarbonylamino-6-(2-nitrophenylamino)-3H- imidazo[4,5-b]pyridine A-179 2-Ethoxycarbonylamino-6-(3-nitrophenylamino)-3H- imidazo[4,5-b]pyridine A-180 2-Ethoxycarbonylamino-6-(4-nitrophenylamino)-3H- imidazo [4 , 5-Jb] pyridine A-181 2-Ethoxycarbonylamino-6-(2-chlorophenylamino)-3H- imidazo[4,5-b]pyridine A-182 2-Ethoxycarbonylamino-6-(3-chlorophenylamino)-3H- imidazo[4,5-b]pyridine A-183 2-Ethoxycarbonylamino-6-(4-chlorophenylamino)-3H- imidazo[4,5-b]pyridine A-184 2-Ethoxycarbonylamino-6-(2-methylphenylamino)-3H- imidazo[4,5-b]pyridine A-185 2-Ethoxycarbonylamino-6-(3-methylphenylamino)-3H- imidazo[4,5-b]pyridine A-186 2-Ethoxycarbonylamino-6-(4-methylphMenoxy)-3H- imidazo[4,5-b]pyridine A-187 2-Ethoxycarbonylamino-6-(2-fluorophenylamino)-3H- imidazo[4,5-b]pyridine A-188 2-Ethoxycarbonylamino-6-(3-fluorophenylamino)-3H- imidazo[4,5-b]pyridine A-189 2-Ethoxycarbonylamino-6-(4-fluorophenylamino)-3H- imidazo[4,5-b]pyridine A-190 2-Ethoxycarbonylamino-6-[2- (trif luoromethyl) phenylamino] -imidazo[4,5-b]pyridine A-191 2-Ethoxycarbonylamino-6-[3- (trif luoromethyl) phenylamino] 3H-imidazo[4,5-b]pyridine A-192 2-Ethoxycarbonylamino-6-[4- (trif luoromethyl) phenylamino] 3H-imidazo[4,5-b]pyridine A-193 2-Ethoxycarbonylamino-6-(2-methoxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-194 2-Ethoxycarbonylamino-6-(3-methoxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-195 2-Ethoxycarbonylamino-6-(4-methoxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-196 2-Ethoxycarbonylamino-6-(2-carboxyphenylamino)- 3H-imidazo[4,5-b]pyridine A-197 2-Ethoxycarbonylamino-6-(3-carboxypheriylamino)- 3H-imidazo[4,5-b]pyridine A-198 2-Ethoxycarbonylamino-6-(4-carboxyphenylamino)- 3H-imidazo[4,5-b]pyridine EXAMPLE 2: ASSAY MEASURING PHOSPHORYLATING FUNCTION OF RAF The following assay reports the amount of RAF- catalyzed phosphorylation of its target protein MEK as well as MEK's target MAPK. The RAF gene sequence is described in Bonner et al., 1985, Molec. Cell. Biol. 5: 1400-1407, and is readily accessible in multiple gene sequence data banks. Construction of the nucleic acid vector and cell lines utilized for this portion of the .invention are fully described in Morrison et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8855-8859. Materials and Reagents 1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, MD. 2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10 % glycerol, 1 mM PMSF, 5 mg/L Aprotenin, 0.5 % Triton X- 100; 3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression and purification by affinity chromatography were performed 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 was expressed in XL1 Blue cells transformed with pUC18 vector encoding His-MAPK. His-MAPK was purified by Ni-affinity chromatography. Cat# 27-4949-01, Pharmacia, Alameda, CA, as described herein. 5. Sheep anti mouse IgG: Jackson laboratories, West Grove, PA. Catalog, # 515-006-008, Lot# 28563 6. RAF-1 protein kinase specflc antibody: URP2653 from UBI. 7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg, MD 8. Wash buffer: TBST - 50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1 % Triton X-100 9. Block buffer: TBST, 0.1 % ethanolamine pH 7.4 10. DMSO, Sigma, St. Louis, MO 11. Kinase buffer (KB): 20 mM Hepes/HCl pH 7.2, 150 mM NaCl, 0.1 % Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 µM sodium ortho vanadate, 0.5 MM DTT and 10 mM MgCl2. 12. ATP mix: 100 mM MgCl2, 300 µM ATP, 10 µCi ?-33P ATP (Dupont-NEN)/mL. 13 Stop solution: 1 % phosphoric acid; Fisher, Pittsburgh, PA. 14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku, Finnland. 15. Filter wash solution: 1 % phosphoric acid, Fisher, Pittsburgh, PA. 16. Tomtec plate harvester, Wallac, Turku, Finnland. 17. Wallac beta plate reader # 1205, Wallac, Turku, Finnland. 18. NUNC 96-well V bottom polypropylene plates for compounds Applied Scientific Catalog # AS-72092. Procedure All of the following steps were conducted at room temperature unless specifically indicated. 1. ELISA plate coating: ELISA wells are coated with 100 (aLof Sheep anti mouse affinity purified antiserum (1 µg/100 µL coating buffer) over night at 4 °C. ELISA plates . can be used for two weeks when stored at 4 °C. 2. Invert the plate and remove liquid. Add 100 µL of blocking solution and incubate for 30 min. 3. Remove blocking solution and wash four times with wash buffer. Pat the plate on a paper towel to remove excess liquid. 4. Add 1 µg of antibody specific, for RAF-1 to each well and incubate for 1 hour. Wash as described in step 3. 5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute with TBST to 10 µg/100 µL. Add 10 µg of diluted lysate to the wells and incubate for 1 hour. Shake the plate during incubation. Negative controls receive no lysate. Lysates from RAS/RAF infected Sf9 insect cells are prepared after cells are infected with recombinant baculoviruses at a MOI of 5 for each virus, and harvested 48 hours later. The cells are washed once with PBS and lysed in RIPA buffer. Insoluble material is removed by centrifugation (5 min at 10 000 x g). Aliquots of lysates are frozen in dry ice/ethanol and stored at - 80 °C until use. 6. Remove non-bound material and wash as outlined 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 kinase buffer. Methods for purifying T-MEK and MAPK from cell extracts are provided herein by example. 8. Predilute compounds (stock solution 10 mg/mL DMSO) or extracts 20 fold in TBST plus 1% DMSO. Add 5 µL of the prediluted compounds/extracts to the wells described in step 6. Incubate for 20 min. Controls receive no drug. 9. Start the kinase reaction by addition of 5 µL ATP mix; Shake the plates on an ELISA "plate shaker during incubation. 10. Stop the kinase reaction after 60 min by addition of 30 µL stop solution to each well. 11. Place the phosphocellulose mat and the ELISA plate in the Tomtec plate harvester. Harvest and wash the filter with the filter wash solution according to the manufacturers recommendation. Dry the filter mats. Seal the filter mats and place them in the holder. Insert the holder into radioactive detection apparatus and quantify the radioactive phosphorous on the filter mats. Alternatively, 40 µL aliquots from individual wells of the assay plate can be transferred to the corresponding Positions on the phosphocellulose filter mat. After air- drying the filters, put the filters in a tray. Gently rock the tray, changing the wash solution at 15 min intervals for 1 hour. Air-dry the filter mats. Seal the filter mats and place them in a holder suitable for measuring the radioactive phosphorous in the samples. Insert the holder into a detection device and quantify the radioactive phosphorous on the filter mats. IC50 values were measured according to the protocol for the following azabenzimidazole-based compounds in the RAF-1 ELISA assay: An IC50 value is the concentration of the azabenzimidazole- based inhibitor required to decrease the maximum amount of phosphorylated target protein or cell growth by 50%. The IC50 values measured in the RAF-1 phosphorylation assay are depicted in Table 1: EXAMPLE 3: PURIFICATION OF MAPK AND MEK The MAPK and MEK proteins are readily expressed in cells by sublconing a gene encoding these proteins into a commercially available vector that expresses the proteins with a poly-Histidine tag. Genes encoding these proteins are readily available from laboratories' that normally work with these proteins or by cloning these genes from cells containing cDNA libraries. The libraries are readily commercially available and a person skilled in the art can readily design nucleic acid probes homologous to cDNA molecules encoding MEK or MAPK from the nucleic acid sequences of MEK and MAPK, available in multiple gene data bases such as Genbank. The cloning of a gene can be accomplished in a short time period using techniques currently available to persons skilled in the art. Purification of the MEK and MAPK proteins from cell extracts can be accomplished using the following protocol, which is adapted from Robbins et al., 1993, J. Biol. Chem. 268: 5097-5106: 1. Lyse cells by sonication, osmotic stress, or French Press techniques readily available to persons skilled in the art. An appropriate sonication buffer is provided below. 2. Equilibrate a solid support which is conjugated with nickel or cobalt with the equilibration buffer disclosed below. The poly-histidine tag specifically binds to the nickel and cobalt atoms on the solid support. Equilibration can be achieved by washing the resin three times with a volume of the equilibration buffer equal to ten times the volume of the solid support. The solid support is readily available to persons of ordinary skill in the art. 3. Add the cell lysate to the solid support and equilibrate in a vessel for a period of time. Alternatively, the solid support can be packed within a protein chromatography column and the lysate may be flowed through the solid support. 4. Wash the solid support with the wash buffer disclosed below. 5. Elute the MEK or MAPK protein from the solid support with an amount of elution buffer (provided below) that removes a significant portion of the protein from the solid support. EXAMPLE 4: ASSAY MEASURING PHOSPHORYLATING FUNCTION OF EGF RECEPTOR EGF Receptor kinase activity (EGFR-NIH3T3 assay) in whole cells was measured as described in detail in PCT Publication WO9640116, filed 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. The IC50 values measured in the EGF receptor phosphorylation assay are depicted in Table 2: EXAMPLE 5: ASSAY MEASURING THE EFFECT OF AZABENZIMIDAZQLE-BASED COMPOUNDS ON THE GROWTH OF CELLS EXPRESSING RAS The following assay measures growth rates fo'r NIH-3T3 cells expressing RAS. The purpose of the assay is to determine the effects of compounds on the growth of NIH 3T3 cells over expressing H-Ras. Cell line: 3T3/H-Ras (NIH 3T3 clone 7 cells expressing genomic fragment of oncogenic H-Ras). The cells can be constructed using the following protocol: 1. Subclone a gene fragment encoding Ras into a commercially available vector that will stably transfect NIH-3T3 cells. The fragment is from the genomic transforming allele of cHa-ras. 2. Transfect NIH-3T3 cells with the subcloned vecto by a calcium phosphate method. Select cells expressing th Ras construct in 2% serum in DMEM. Visible foci are observed after 2 weeks. Pool the transformed cells to generate a stably transformed cell line. Growth medium: 2% calf serum/DMEM + 2 mM glutamine, Pen/Strep Protocol: Day 0: Cell Plating: This part of assay is carried out in a laminar flow hood. 1. Trypsinize cells. Transfer 200 µL of cell suspension to 10 mL of isotone. Count cells with a Coulte: Counter. 2. Dilute cells in growth medium to 60,000 cell/mL. Transfer 100 \|iL of cells to each well in a 96-well flat bottom plate to give 6000 cells/well. 3. Use half of plate (4 rows) for each comound and quadruplicate wells for each comound concentration, and a set of 4 wells for medium control. 4. Gently shake plates to allow for uniform attachment of the cells. 5. Incubate the plates at 37 °C in a 10% CO2 incubator. Day 1: Addition of Compound: This part of assay is carried out in a laminar flow hood. 1. In a 96-well round bottom plate, add 120 µL of growth medium containing 2X final % DMSO found in highest screening concentration of compound to columns 1 to 11. For example, if the highest concentration is 100 µL, and this is made from a 100 mM stock, 1X DMSO is 0.1%, so 2X DMSO is 0.2%. This plate is used to titrate out the compound, 4 rows per compound. 2. In a sterile 15 mL tube, make a 2X solution of the highest screening concentration of compound in growth medium plus 2X DMSO. 1 mL per cell line is needed. The starting concentration of the compound is usually 100 µM but this concentration may vary depending upon the solubility of the compound. 3. Transfer 240 µL of the 2X starting compound solution to qudruplicate wells in column 12 of the 96-well round bottom plate. Do 1:2 serial dilutions across the plate from right to left by transferring 12 µL from column 12 to column 11, column 11 to 10 and so on through column 2. Transfer 100 µL of compound dilutions, and 100 µL of medium in column 1, onto 100 µL medium on cells in corresponding wells of 96-well flat bottom plate. Total volume per well should be 200 µL. 4. Return the plate to the incubattor and incubate for 3 days. Day 4: Development of Assay This party of assay is carried out on the bench. 1. Aspirate or pour off medium. Add 200 µL cold 10% TCA to each well to fix cells. Incubate plate for at least 60 min. at 4 °C. 2. Discard TCA and rinse wells 5 times with tap water. Dry plates upside down on paper towels. 3. Stain cells with 100 µL/well 0.4% SRB for 10 min. 4. Pour of SRB and rinse wells 5 times with 1% acetic acid. Dry plates completely upside down on paper towels. 5. Solubilize dye with 100 µL/well 10 mM Tris base for 5-10 min. on shaker. 6. Read plates on Dynatech ELISA Plate REader at 570 nm with reference at 630 nm. Select compounds inhibited the growth rate of cells over-expressing RAS as illustrated in Table 3. EXAMPLE 6: ASSAY MEASURING EFFECT OF AZABENZIMIDAZOLE- BASED COMPOUNDS ON GROWTH OF A54 9 CELLS The following assay measures growth rates for A549 cells. The purpose of the assay is to determine the effects of compounds on the growth of A54 9 human lung carcinoma cells. A54 9 cells are readily-accessible from commercial sources, such as ATCC (CCL185)• Materials: 96-well flat bottom sterile plates 96-well round bottom sterile plates sterile 25 mL or 100 mL reservoir pipets, multi-channel pipetman sterile pipet tips sterile 15 mL and 50 mL tubes Reagents: 0.4% SRB in 1% acetic acid l0mM Tris base 10% TCA 1% acetic acid sterile DMSO (Sigma) compound in DMSO (100 mM or less stock solution) Trypsin-EDTA (GIBCO BRL) Cell line and growth medium: A549 human lung carcinoma cells (ATCC CCL185) 10% fetal calf serum in Ham's F12-K Protocol: Day 0: Cell Plating: This part of assay is carried out in a laminar flow hood. 1. Trypsinize cells. Transfer 200 uL of cell suspension to 10 mL of isotone. Count cells with a Coulter Counter. 2. Dilute cells in growth medium to 20,000 cell/mL. Transfer 100 uL of cells to each well in a 96-well flat bottom plate to give 2000 cells/well. 3. Use half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, and a set of 4 wells for medium control. 4. Gently shake plates to allow for uniform attachment of the cells. 5. Incubate the plates at 37°C in a 10% CO2 incubator. Day 1: Addition of Compound: This part of assay is carried out in a laminar flow hood. 1. In a 96 well-round bottom plate, add 120 uL of growth medium containing 2X final %DMSO found in highest screening concentration of compound to columns 1 to 11. For example, if the highest screening concentration is 100 µM, and this is made from a lOOmM stock, IX DMSO is 0.1%, so 2X DMSO is 0.2%. This plate is used to titrate out the compound, 4 rows per compound. 2. In a sterile 15 mL tube, make a 2X solution of the highest screening concentration of compound in growth medium plus 2X DMSO. 1 mL per cell line is needed. The starting concentration of the compound is usually 100 pM but this concentration may vary depending upon the solubility of the compound. 3. Transfer 240 uL of the 2X starting compound solution to quadruplicate wells in column 12 of the 96-well round bottom plate. Do 1:2 serial dilutions across the plate from right to left by transferring 120 uL from column 12 to column 11, column 11 to 10 and so on through column 2. Transfer 100 uL of compound dilutions, and 100 uL of medium in column 1, onto 100 uL medium on cells in corresponding wells of 96-well flat bottom plate. Total volume per well should be 200 uL. 4. Return the plate to the incubator and incubate for 3 days. Day 5: Development of Assay This part of assay is carried out on the bench. 1. Aspirate or pour off medium. Add 200 uL cold 10% TCA to each well to fix cells. Incubate plate for at least 60 min. at 4°C. 2. Discard TCA and rinse wells 5 times with tap water. Dry plates upside down on paper towels. 3. Stain cells with 100 uL/well 0.4% SRB for 10 min. 4. Pour off SRB and rinse wells 5 times with 1% acetic acid. Dry plates completely upside down on paper towels. 5. Solubilize dye with lOOµL/well 10 mM Tris base for 5-10 min. on shaker. 6. Read plates on Dynatech ELISA Plate Reader at 570 nm with reference at 630 nm. Select compounds inhibited the growth rates of A54 9 cells, as illstrated in Table 4. EXAMPLE 7: METHOD FOR DETERMINING THE BIOLOGICAL ACTIVITY OF RAF MODULATORS IN VTVO Xenograft studies can be utilized to monitor the effect of compounds of the invention upon the inhibition of ovarian, melanoma, prostate, lung and mammary tumor cells. The protocol for the assay is described in detail in PCT Publication WO9640116, filed 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. The invention illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The molecular complexes and the methods, procedures, treatments, molecules, specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. Those references not previously incorporated herein by reference, including both patent and non-patent references, are expressly incorporated herein by reference for all purposes. Other embodiments are within the following claims. WE CLAIM : 1. Azabenzimidazole compound having the formula set forth in formula I: wherein, (a) R1, R2, and R3 are independently selected from the group consisting of: (i) unsaturated alkyl; (ii) —NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula -CO-X4. where X4, is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X5)n- COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and heter'ocyclic ring moieties, and wherein n. is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O- X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or' more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii) an amide of formula —NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyJ, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro. and ester; (viii) —SO2NX11X12, wherein Xn and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or' three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester' moieties; (x) an aldehyde of formula —CO-H; and (xi) a sulfone of formula --SO2-X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring, moieties; (b) Z1 and Z2 are NH; and (c) X1 is independently selected fl'om the group consisting of NH, sulfur, and oxygen. 2. A pharmaceutical composition comprising an azabenzimidazole-based compound as claimed in claim 1, or salt thereof, and a physiologically acceptable carrier or' diluent. 3. A pharmaceutical composition for preventing or treating a cell proliferative disorder in an organism, comprising the azabenzimidazole compound as claimed in claim 1, or a salt thereof, and a physiologically acceptable carrier or diluent,wherein said cell proliferative is associated with an aberration in a signal transduction pathway characterized by an interaction between the setine/threonine protein kinase RAF and a natural binding partner. 4. The pharmaceutical composition as claimed in claim 3, wherein said organism is a mammal. 5. The pharmaceutical composition as claimed in claim 4, wherein said cell proliferative disorder is selected from the group consisting of cancer, fibrotic disorder, mcsangial disorders, abnormal angiogenesis, abnormal vasculogenesis, wound healing, psoriasis, restenosis. and inflammation. 6. The pharmaceutical composition as claimed in claim 5, wherein said cancer is selected from the group consisting of lung cancer, ovarian cancer, breast cancer, brain cancer, intra-axial brain cancer, colon cancer, prostate cancer, sarcoma, Kaposi 's sarcoma, melanoma, and glioma. 7. The pharmaceutical composition as claimed in claim 6, wherein said cell proliferative disorder is associated with an aberration in a signal transduction pathway characterized by an interaction between serine/threonine protein kinase and a natural binding protein. 8. An azabenzimidazole compound having a structure as set forth in formula II or III: wherein (a) R1, R2, R3, and R4 are independently selected from the group consisting of: (i) hydrogen; (ii) saturated or unsaturated alkyl; (ii) NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic or heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula -CO—X4, where X4 selected from the group consisting of hydrogen, alky], homocyclic ring moieties and. heterocyclic ring moieties; (v) a caxboxylic acid of formula —(X5)n-COOH or ester of formula --(X6)n- COO-X7, wherein X5, X6, and. X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O- X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, txihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii) an amide of formula —NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro. and ester; (viii) -SO2NX1 1X12, wherein X11 and X12 are selected from the group consisting of hydrogen, alkyl. and Homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, car'boxylate. nitro, and ester moieties; (x) an aldehyde of formula —CO-H; and (xi) a sulfone of formula —SO2,X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring, moieties; (b) Z1 and Z2 are independently selected from the group consisting of nitrogen, NH, and NR4; and (c) Z3 and X1 arc independently selected from the group consisting of NH, sulfur, and oxygen. 9. A pharmaceutical composition comprising an azabenzimidazole-based compound as claimed in claim 8, or salt thereof, and a physiologically acceptable carrier or diluent. 10. A pharmaceutical composition for preventing or treating a cell prolifcrative disorder in an organism, comprising the azabenzimidazole compound as claimed in claim 8, or a salt thereof, and a physiologically acceptable carrier or diluent, wherein said cell proliferative is associated with an aberration in a signal transduction pathway characterized by an interaction between the serine/threonine protein kinase RAF and a natural binding partner. 11. The pharmaceutical composition as claimed in claim 10, wherein said organism is a mammal. 12. The pharmaceutical composition as claimed in claim 11, wherein said cell proliferative disorder is selected from, the group consisting of cancer, fibrotic disorder, mesangial disorders, abnormal angiogenesis, abnormal vasculogenesis, wound, healing, psoriasis, restenosis, and inflammation. 13. The pharmaceutical composition as claimed in claim 12, wherein said cancer is selected flom the group consisting of lung cancer, ovarian cancer, breast cancer, brain cancer, intra-axial brain cancer, colon cancer, prostate cancer, sarcoma, Kaposi's sarcoma, melanoma, and glioma. 14. The pharmaceutical composition as claimed in claim 13, wherein said cell proliferative disorder is associated with an aberration in a signal transduction pathway characterized by an interaction between serine/threonine protein kinase and a natural binding protein. 15. A method of modulating the function of a serine/threonine protein kinase with an azabenzimidazole based compound in vitro said compound having the formula set forth in formula I: wherein, (a) R1, R2, and R3 are independently selected from the group consisting of: (i) unsaturated alkyl; (ii) —NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv)- a ketone of formula -CO-X4. where X4, is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (v) a carboxylic acid of formula —(X5)nCOOH or ester of formula —(X6)n- COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and hetero cyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula -(X8)n-O- X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl. alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii) an amide of formula —NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein (he ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii) —SO2NX11X12, wherein X11 and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro. and ester moieties; (x) an aldehyde of formula —CO-H; and (xi) a sulfone of formula -SO2-X13, where X13 is selected, from the group consisting of saturated alkyl, unsaturated alkyl. homocyclic ring moieties, arid heterocyclic ring moieties; (b) Z1 and Z2 are NH; and (c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen, comprising the steps of contacting the cells expressing said serme/threonine protein kinase with said azabenzimida.zole based compound. 16. The method as claimed in claim 15, wherein said serine/threonine protein is 17. A method of modulating the function of a serine/threonine protein kinase with an azabenzimidazole-based compound in vitro, said compound having the formula as set forth in formula II or III: wherein Ri, R2, R3, and R4 are independently selected from the group consisting of: (i) hydrogen; (ii) saturated or unsaturated alkyl; (ii) NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic or heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula —CO—X4, where X4 is selected from the group consisting of hydrogen., alkyl, homocyclic ring moieties and heterocyclic ring moieties; (v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X6)n- COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, hornocyciic ring moieties, and heterocyclic ring moieties, and wherein, n. is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O- X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii) an amide of formula —NHCOX10, wherein X10 is selected from the group consisting of alkyl hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy. halogen, tribalomethyl, carboxylate, nitro, and ester; (viii) -SO2NX11X12, wherein X11 and X12 are selected from the group consisting of hydrogen, alkyl, and honiocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester moieties; (x) an aldehyde of formula --CO-H; and (xi) a sulfone of formula -SO2 -X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (b) Z1 and 4 arc independently selected from the group consisting of nitrogen, NH. and NR4; and (c) Z3 and X1 are independently selected from the group consisting of NH, sulfur, and oxygen, comprising the steps of contacting the cells expressing said serine/threonine protein kinase with said azabenzimidazole based compound. 18. The method as claimed in claim 1 7, wherein said serine/threonine protein is RAF. 19. A method for synthesizing a compound having a structure as set forth in formula 'I, II or III, and having been defined in claim 1 or 8, said method comprising the steps of: (a) reacting 2-amino-6-chloro-3-nittOpyridine with a second reactant in a solvent, yielding a first intermediate, wherein said second reactant is selected from the group consisting of a substituted phenol, substituted thiophenol, and substituted aniline; (b) reducing the said first intermediate in the presence of a catalyst and a reducing agent, yielding a second intermediate: (c) reacting the second intermediate with a third reactant; and (d) purifying the compound, so obtained. 20. The method as claimed in claim 19. wherein said second reactant is selected from the group consisting of phenol, 2-nitrophenol. 3-nitrophenol, 4-nitrophenol, 2- chlor'ophenol, 3 -chlorophenol, 4-chlorophenol, 2-cresol, 3-cresol, 4-cresol, 2- nuor'ophenol, 3-fluorophenol, 4-fluorophenol, 2.-(trifluoromethyl) phenol, 3- (trifiuoromethyl) phenol, 4-(trifluoromethyl) phenol, 2-methoxyphenol, 3- methoxyphenol, 4-.methoxyphenol, thiophenol, 2-hydroxybenzoic acid, 3- hydroxybenzoic acid, 4-hydroxybenzoic acid, thiophenol, 2-nitrothiophenol, 3- nitrothiophenol, 4-nitrothiophenol, 2-chlorothiophenol, 3 -chlorothiophenol, 4- chlorothiophenol, 2-thiocresol, 3 -thiocresol, 4-thiocresol, 2-fluorothiophenol, 3- fluorothiophenol, 4-fluorothiophenol 2-(1rifluoromethyl)thiophenol, 3.-(trifluor'omethyl) thiophenol, 4-(trifluoromethyl) thiophenol, 2-methoxybenzenethiol, 3- methoxybenzenethiol, 4-methoxybenzene1hiol, 2-met captobenzoic acid, 3- mercaptobenzoic acid, 4-mercaptobenzoic acid, aniline, 2-nitroaniline, 3»-nitroaniline, 4- nitroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, 2-toluidine, 3-toluidine, 4- toluidine. 2-fluoroaniline, 3-fluoronaniline, 4-fluoronaniline, 2-(trifluorornethyl) aniline, 3 (trifluoromethyl) aniline, 4-(trilfl.uoroinethyl) aniline, 2-anisidine, 3-amisidine, 4- anisidine, 2-aminobenzoic acid, 3-aminobenzoic acid, and 4-aminobenzoic acid. 21. The method as claimed in claim 19, wherein said reducing agent is hydrogen. 22. The method as claimed in claim 19. wherein said catalyst is Raney nickel. 23. A pharmaceutical composition, having a compound as defined in claim 1 or 8, substantially as herein described, particularly with reference to the foregoing examples. 24. A method for synthesising a compound, as defined in claim 1 or 8, substantially as herein described, particularly with reference lo the foregoing examples.. 25. A method of modulating the function of serine/threonine protein kinase, substantially as herein described, particularly with reference to the foregoing examples. The present invention provides an azabenzimidazole compound having the formula set forth in formula I: wherein, (a) R1, R2, and R3 are independently selected from the group consisting of: (i) unsaturated alkyl; (ii) —NX2X3, where X2 and X3 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (iii) halogen or trihalomethyl; (iv) a ketone of formula -CO-X4, where X4, is selected from the group consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X6)n- COO-X7, wherein X5, X6, and X7, and are independently selected from the group consisting of alkyl, homocyclic ring moieties, and heter'ocyclic ring moieties, and wherein n is 0 or 1; (vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O- X9, wherein X8 and X9 are independently selected from the group consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or' more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester, and wherein n is 0 or 1; (vii) an amide of formula —NHCOX10, wherein X10 is selected from the group consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic ring moieties, and wherein the ring is optionally substituted with one or more substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester; (viii) —SO2NX11X12, wherein Xn and X12 are selected from the group consisting of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring moieties; (ix) a homocyclic or heterocyclic ring moiety optionally substituted with one, two, or' three substituents independently selected from the group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester' moieties; (x) an aldehyde of formula —CO-H; and (xi) a sulfone of formula --SO2-X13, where X13 is selected from the group consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and heterocyclic ring moieties; (b) Z1 and Z2 are NH; and (c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen.

Full Text

DESCRIPTION
BACKGROUND OF THE INVENTION
The following description of the background of the
invention is provided to aid in understanding the invention
but is not admitted to be prior art to the invention.
Cellular signal transduction is a fundamental
mechanism whereby external stimuli regulating diverse
cellular processes are relayed to the interior of cells.
One of the key biochemical mechanisms of signal
transduction involves the reversible phosp.horylation of
proteins, which enables regulation of the activity of
mature proteins by altering their structure, and function.
The best characterized protein kinases in eukaryotes
phosphorylate proteins on the alcohol moiety of serine,
threonine, and tyrosine residues. These kinases largely
fall into two groups, those specific for phosphorylating
serine and threonine, and those specific for
phosphorylating tyrosine. Some kinases, referred to as
"dual specificity" kinases, are able to phosphorylate on
tyrosine as well as serine/threonine residues.
Protein kinases can also be characterized by their
location within the cell. Some kinases are transmembrane
receptor proteins capable of binding ligands external to
the cell membrane. Binding the ligands alters the receptor
protein kinase's catalytic activity. Others are non-
receptor proteins lacking a transmembrane domain. Non-
receptor protein kinases can be found in a variety of
cellular 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 state. Ultimately
the activity of a downstream effector is modulated by
phosphorylation resulting from activation of such a
pathway.
The serine/threonine kinase family includes members
that regulate many steps of signaling cascades, including
cascades controlling cell growth, migration,
differentiation, gene expression, muscle contraction,
glucose metabolism, cellular protein synthesis, and
regulation of the cell cycle.
An example of a non-receptor protein kinase that
phosphorylates protein targets on serine and threonine
residues is RAF. RAF modulates the catalytic activity of
other protein kinases, such as the protein kinase that
phosphorylates and thereby activates mitogen activated
protein kinase (MAPK). RAF itself is activated by the
membrane anchored protein RAS, which in turn is activated
in response to ligand activated tyrosine receptor protein
kinases such as epidermal growth factor receptor (EGFR) and
platelet-derived growth factor receptor (PDGFR). The
biological importance of RAF in controlling cellular events
is underscored by the finding that altered forms of RAF can
cause cancer in organisms. Evidence for importance of RAF
in malignancies is provided in Monia et al., 1996, Nature
Medicine 2: 668, incorporated herein by reference in its
entirety including all figures and tables.
In an effort to discover novel treatments for cancer
and other diseases, biomedical researchers and chemists
have designed, synthesized, and tested molecules that
inhibit the function of protein kinases. Some small
organic molecules form 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-azaindole derivatives
(PCT WO 94/14808), 1-cyclopropyl-4-pyridyl-quinolones (U.S.
Patent No. 5,330,992), styryl compounds (U.S. Patent No.
5,217,999), styryl-substituted pyridyl compounds (U.S.
Patent No. 5,302,606), certain quinazoline derivatives (EP
Application No. 0 566 266 A1) , seleoindoles and selenides
(PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT
WO 92/21660), and benzylphosphonic acid compounds (PCT WO
91/15495).
The compounds that can traverse cell membranes and are
resistant to acid hydrolysis are potentially advantageous
therapeutics as 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, many inhibit a
variety of protein kinases and will therefore cause
multiple side-effects as therapeutics for diseases.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides an
azabenzimidazole compound having the formula set forth in
formula I:
wherein,
(a) R1, R2, and R3 are independently selected from the
group consisting of:
(i) unsaturated alkyl;
(ii) -NX2X3, where X2 and X3 are independently
selected from the group consisting of hydrogen,
saturated alkyl, unsaturated alkyl, homocyclic
ring moieties, and heterocyclic ring moieties;
(iii)halogen or trihalomethyl;
(iv) a ketone of formula -CO-X4, where X4, is
selected from the group consisting of hydrogen,
alkyl, homocyclic ring moieties, and
heterocyclic ring moieties;
(v) a carboxylic acid of formula -(X5)n-COOH or
ester of formula - (X6) n-COO-X7, wherein X5, X6,
and X7, and are independently selected from the
group consisting of alkyl, homocyclic ring
moieties, and heter'ocyclic ring moieties, and
wherein n is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy
moiety of formula -(X8)n-O-X9, wherein X8 and X9
are independently selected from the group
consisting of hydrogen, saturated alkyl,
unsaturated alkyl,,. homocyclic ring moieties,
and heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or'
more substituents independently selected from
the group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester,
and wherein n is 0 or 1;
(vii)an amide of formula -NHCOX10, wherein X10 is
selected from the group consisting of alkyl,
hydroxyl, homocyclic ring moieties, and
heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or more
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester;
(viii) -SO2NX1 1X12, wherein Xn and X12 are selected from
the group consisting of hydrogen, alkyl, and
homocyclic ring moieties, and heterocyclic ring
moieties;
(ix) a homocyclic or heterocyclic ring moiety
optionally substituted with one, two, or' three
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester'
moieties;
(x) an aldehyde of formula -CO-H; and
(xi) a sulfone of formula -SO2-X13, where X13 is
selected from the group consisting of saturated
alkyl, unsaturated alkyl, homocyclic ring
moieties, and heterocyclic ring moieties;
(b) Z1 and Z2 are NH; and
(c) X1 is independently selected from the group
consisting of NH, sulfur, and oxygen.
The present invention also provides an azabenzimida-
zole compound having a structure set forth in formulas II
or III:
wherein
(a) Rl, R2, R3, and R4 are independently selected from
the group consisting of:
(i) hydrogen;
(ii) saturated or unsaturated alkyl;
(ii) NX2X3, where X2 and X3 are independently selected
from the group consisting of hydrogen,
saturated alkyl, unsaturated alkyl, homocyclic
or heterocyclic ring moieties;
(iii)halogen or trihalomethyl;
(iv) a ketone of formula -CO-X4, where X4 is selected
from the group consisting of hydrogen, alkyl,
homocyclic ring moieties and heterocyclic ring
moieties;
(v) a caxboxylic acid of formula -(X5)n-COOH or
ester of formula - (X6) n-COO-X7, wherein X5, X6,
and X7, and are independently selected from the
group consisting of alkyl, homocyclic ring
moieties, and heterocyclic ring moieties, and
wherein n is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy
moiety of formula -(X8)n-O-X9, wherein X8 and X9
are independently selected from the group
consisting of hydrogen, saturated alkyl,
unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or more
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
txihalomethyl, carboxylate, nitro, and ester,
and wherein n is 0 or 1;
(vii)an amide of formula -NHCOX10, wherein X10 is
selected from the group consisting of alkyl,
hydroxyl, homocyclic ring moieties, and
heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or more
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester;
(viii)-SO2NX1 1X12, wherein X11 and X12 are selected from
the group consisting of hydrogen, alkyl, and
liomocyclic ring moieties, and beterocyclic
ring moieties;
(ix) a homocyclic or heterocyclic ring moiety
optionally substituted with one, two, or three
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, car'boxylate, nitro, and ester
moieties;
(x) an aldehyde of formula -CO-H; and
(xi) a sulfone of formula -SO2-X13, where X13 is
selected from the group consisting of saturated
alkyl, unsaturated alkyl, homocyclic ring
moieties, and heterocyclic ring moieties;
(b) Z1 and Z2 are independently selected from the group
. consisting of nitrogen,, NH, and NR4; and
(c) Z3 and X1 are independently selected from the group
consisting of NH, sulfur, and oxygen.
The present invention further provides a method of
modulating the function of a serine/threonine protein
kina.se with an azabenzimidazole based compound in vitro ,
said compound having the formula set forth in formula I:
wherein,
(a) R1, R2, and R3 are independently selected from the
group consisting of:
(i) unsaturated alkyl;
(ii)-NX2X3, where X2 and X3 are independently selected
from the group consisting of hydrogen,
saturated alkyl, unsaturated alkyl, homocyclic
ring moieties, and heterocyclic ring moieties;
(iii) halogen or trihalomethyl;
(iv) a ketone of formula -CO-X4 where X4, is selected
from the group consisting of hydrogen, alkyl,
homocyclic ring moieties, and heterocyclic ring
moieties;
(v) a carboxylic acid of formula (X5)n-COOH or ester
of formula (X6) n-COO-X7, wherein X5, X6, and X7,
and are independently selected from the group
consisting of alkyl, homocyclic ring moieties,
and hetero cyclic ring moieties, and wherein n
is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy
moiety of formula -(X8)n-O-X9, wherein X8 and X9
are independently selected from the group
consisting of hydrogen, saturated alkyl,
unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or more
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester,
and wherein n is 0 or 1;
(vii)an amide of formula -NHCOX10, wherein X10 is
selected from the group consisting of alkyl,
hydroxyl, homocyclic ring moieties, and
heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or more
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester;
(viii) -SO2NX1 1X12, wherein X11 and X12 are selected
from the group consisting of hydrogen, alkyl,
and homocyclic ring moieties, and heterocyclic
ring moieties;
(ix) a homocyclic or heterocyclic ring moiety
optionally substituted with one, two, or three
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester
moieties;
(x) an aldehyde of formula -CO-H; and
(xi) a sulfone of formula -SO2-X13, where X13 is
selected from the group consisting of saturated
alkyl, unsaturated alkyl, homocyclic ring
moieties, arid heterocyclic ring moieties;
(b) Z1 and Z2 are NH; and
(c) X1 is independently selected from the group
consisting of NH, sulfur, and oxygen, comprising the
steps of contacting the cells expressing said serine/
threonine protein kinase with said azabenzimidazole based
compound.
The present invention also provides a method of
modulating the function of a serine/threonine protein
kinase with an azabenzimidazole-based compound in vitro,
said compound having the formula as set forth in formula
II or III:
wherein
(a) R1, R2, R3, and R4 are independently selected from
the group consisting of:
(i) hydrogen;
(ii) saturated or unsaturated alkyl;
(ii) NX2X3, where X2 and X3 are independently selected
from the group consisting of hydrogen,
saturated alkyl, unsaturated alkyl, homocyclic
or heterocyclic ring moieties;
(iii) halogen or trihalomethyl;
(iv) a ketone of formula -CO-X4, where X4 is selected
from the group consisting of hydrogen, alkyl,
homocyclic ring moieties and heterocyclic ring
moieties;
(v) a carboxylic acid of formula -(X5)n-COOH or
ester of formula - (X6) n-COO-X7, wherein X5, X6,
and X7, and are independently selected from the
group consisting of alkyl, homocyciic ring
moieties, and heterocyclic ring moieties, and
wherein n is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy
moiety of formula -(X8)n-O-X9, wherein X8 and X9
are independently selected from the group
consisting of hydrogen, saturated alkyl,
unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or more
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester,
and wherein n is 0 or 1;
(vii)an amide of formula -NHCOX10, wherein X10 is
selected from the group consisting of alkyl,
hydroxyl, homocyclic ring moieties, and
heterocyclic ring moieties, and wherein the
ring is optionally substituted with one or more
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester;
(viii)-SO2NX11X12, wherein X11 and X12 are selected from
the group consisting of hydrogen, alkyl, and
honiocyclic ring moieties, and heterocyclic
ring moieties;
(ix) a homocyclic or heterocyclic ring moiety
optionally substituted with one, two, or three
substituents independently selected from the
group consisting of alkyl, alkoxy, halogen,
trihalomethyl, carboxylate, nitro, and ester
moieties;
(x) an aldehyde of formula -CO-H; and
(xi) a sulfone of formula -SO2 -X13, where X13 is
selected from the group consisting of saturated
alkyl, unsaturated alkyl, homocyclic ring
moieties, and heterocyclic ring moieties;
(b) Z, and 4 are independently selected from the group
consisting of nitrogen, NH, and NR,,; and
(c) Z3 and X1 are independently selected from the group
consisting of NH, sulfur, and oxygen, comprising the steps
of contacting the cells expressing said serine/ threonine
protein kinase with said azabenzimidazole based compound.
The present invention further provides a method for
synthesizing a compound having a structure set forth in
formula I, II or III, as defined hereinbefore, said method
comprising the steps of:
(a) reacting 2-amino-6-chloro-3-nittOpyridine with a
second reactant in a solvent, yielding a first
intermediate, wherein said second reactant is selected from
the group consisting of a substituted phenol, substituted
thiophenol, and substituted aniline;
(b) reducing the said first intermediate in the
presence of a catalyst and a reducing agent, yielding a
second intermediate;
(c) reacting the second intermediate with a third
reactant; and
(d) purifying the compound, so obtained.
I. Methods for Screening Compounds that Modulate
Serine/Threonine Protein Kinase Function
The methods of the present invention provide means for
modulating the function of both receptor and cytosolic
serine/threonine protein kinases. These methods provide
umeans of modulating the enzymes both in vitro and in vivo.
For in vitro applications, the methods of the invention
relate in part to method of identifying compounds that
modulate the function of serine/threonine protein kinases.
Thus, in a first aspect, the invention features a
method of modulating the function of a serine/threonine
protein kinase with an azabenzimidazole-based compound.
The azabenzimidazole compound is optionally substituted
with organic groups. The method comprises comprises
contacting cells expressing 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
steps in signaling cascades, including cascades controlling
cell growth, migration, differentiation, gene expression,
muscle contraction, glucose metabolism, cellular protein
synthesis, and regulation of the cell cycle.
The term "modulates" refers to the ability of, a
compound to alter the function of a protein kinase. 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 most preferably inhibits the catalytic activity
of a protein kinase.

The term "catalytic activity", in the context of the
invention, defines the rate at which a protein kinase
phosphorylates a substrate. 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 normally 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. The substrate is preferably a peptide and more
preferably a protein. In relation to the protein kinase
RAF, preferred substrates are MEK and the MEK substrate
MAPK.
The term "activates" refers to increasing the cellular
function of a protein kinase. The protein kinase function
is preferably the interaction with a natural binding
partner and most preferably catalytic activity.
The term "inhibit" refers to decreasing the cellular
function of a protein kinase. The protein kinase function
is preferably the interaction with a natural binding
partner and most preferably catalytic activity.
The term "modulates" also refers to altering the
function of a protein kinase by increasing or decreasing
the probability that a complex forms between a protein
kinase and a natural binding partner. A modulator
preferably increases the probability that such a complex
forms between the protein kinase and the natural binding
partner, more preferably increases or decreases the
probability that a complex forms between the protein kinase
and the natural binding partner depending on the
concentration of the compound exposed to the protein
kinase, and most preferably decreases the probability that
a complex forms between the protein kinase and the natural
binding partner.
The term "complex" refers to an assembly of at least
two molecules bound to one another. Signal transduction
complexes often contain at least two protein molecules
bound to one another. For instance, a protein tyrosine
receptor protein kinase, GRB2, SOS, RAF, and RAS assemble
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 propagating a
signal in a protein kinase signal transduction process. A
change in the interaction between a protein kinase and a
natural binding partner can manifest itself as an increased
or decreased probability that the interaction forms, or an
increased or decreased concentration of the protein
kinase/natural binding partner complex.
A protein kinase natural binding partner can bind to a
protein kinase's intracellular region with high affinity.
High affinity represents an equilibrium binding constant on
the order of 10"6 M or less. In addition, a natural
binding partner can also transiently interact with a
protein kinase intracellular region and chemically modify
it. Protein kinase natural binding partners are chosen
from a group that includes, but is not limited to, SRC
homology 2 (SH2) or 3 (SH3) domains, other phosphoryl
tyrosine binding (PTB) domains, guanine nucleotide exchange
factors, protein phosphatases, and other protein kinases.
Methods of 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 with at least 10%
amino acid identity to other enzymes that phosphorylate
proteins on serine and threonine residues. A
serine/threonine protein kinase catalyzes the addition of
phosphate onto proteins on serine and threonine residues.
Serine/threonine protein kinases can exist as membrane
bound proteins or cytosolic protins.
The term "contacting" as used herein refers to mixing
a solution comprising an azabenzimidazole compound of the
invention with a liquid medium bathing the cells of the
methods. The solution comprising the compound may also
comprise another component, such as dimethylsulfoxide
(DMSO), which facilitates the uptake of the
azabenzimidazole compound or compounds into the cells of
the methods. The solution comprising the azabenzimidazole
compound may be added to the medium bathing the cells by
utilizing a delivery apparatus, such as a pipet-based
device or syringe-based device.
The term "azabenzimidazole-based compound" refers to
an azabenzimidazole organic compound substituted with
chemical substituents. Azabenzimidazole compounds are of
the general structure:
The term "substituted", in reference to the invention,
refers to an azabenzimidazole compound that is derivatized
with any number of chemical substituents.
In a preferred embodiment, the invention relates to
the method of modulating the function of a serine/threonine
protein kinase, where the protein kinase is RAF.
The RAF protein kinase phosphorylates protein targets
on serine or threonine residues. One such protein target
is the protein kinase (MEK) that phosphorylates and
consequently activates mitogen activated protein kinase
(MAPK). RAF itself is activated by the membrane-bound
guanine triphosphate hydrolyzing enzyme RAS in response to
mitogen-stimulated receptor protein tyrosine kinases, such
as epidermal growth factor receptor (EGFR) and platelet-
derived growth factor receptor (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 mitogen-activated protein
kinase (MAPK). Assays that monitor only the
phosphorylation of MEK by RAF are not sensitive because the
phosphorylation levels of MEK are not significant. To
overcome this sensitivity dilemma, the phosphorylation of
both MEK and MAPK are 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 immunosorbant assays. In addition, the assay of the
invention is performed in a high throughput format such
that many compounds can be rapidly monitored in a short
period of time.
In another aspect, the invention describes a method of
identifying compounds that modulate the function of
serine/threonine protein kinase, comprising the steps of
contacting cells expressing the serine/threonine protein
kinase with the compound, and monitoring an effect upon the
cells.
The term "monitoring" refers to observing the effect
of adding the compound to the cells of the method. The
effect can be manifested in a change in cell 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
a change in cell phenotype or cell proliferation. "Effect"
can also describe a change or an absence of a change in the
catalytic activity of the protein kinase. "Effect" can
also describe a change or an 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 of identifying compounds that modulate the function
of serine/threonine protein kinase, where the effect is a
change or an absence of a change in cell 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 numbers of cells), cell differentiation (a
change or absence of a change in cell shape), cell
survival, apoptosis (cell death), or the utilization of a
metabolic nutrient (e.g., glucose uptake). Changes or the
absence of changes in cell phenotype are readily measured
by techniques known in the art.
In another preferred embodiment, the invention relates
to the method of identifying compounds that modulate the
function of serine/threonine protein kinase, where the
effect is a change or an absence of a change in cell
proliferation.
The term "cell proliferation" refers to the rate at
which a group of cells divides. The number of cells
growing in a vessel can be quantified by a person skilled
in the art when that person visually counts the number of
cells in a defined volume using a common light microscope.
Alternatively, cell proliferation rates can be quantified
by laboratory apparatae that optically or conductively
measure the density of cells in an appropriate medium.
In another preferred embodiment, the invention relates
to the method of identifying compounds that modulate the
function of serine/threonine protein kinase, where the
effect is a change or an absence of a change in the
interaction between the serine/threonine protein kinase
with a natural binding partner.
The term "interaction", in the context of the
invention, describes a complex formed between a protein
kinase's intracellular region and a natural binding partner
or compound. The term "interaction" can also extend 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 receptor
protein kinase will harbor both an extracellular and an
intracellular region.
The term "intracellular region" as used herein refers
to the portion of a protein kinase which exists inside a
cell. The term "extracellular region" as used herein
refers to a portion of a protein kinase which exists
outside of the cell.
In a preferred embodiment, the invention relates to
the method of identifying compounds that modulate the
function of serine/threonine protein kinase that further
comprises the following steps:(a) lysing the cells to
render a lysate comprising serine/threonine protein kinase;
(b) adsorbing the serine/threonine protein kinase to an
antibody; (c) incubating the adsorbed serine/threonine
protein kinase 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 "lysing" as used herein refers to a method of
disrupting the integrity of a cell such that its interior
contents are liberated. Cell lysis is accomplished by many
techniques known to persons skilled in the art. The method
is accomplished preferably by sonication or cell sheering
techniques and more preferably by detergent techniques.
The term "antibody" as used herein refers to a protein
molecule that specifically binds a protein kinase. An
antibody preferably binds to one class of protein kinase
and more preferably specifically binds to the RAF protein
kinase.
The term "specifically binds" as used herein refers to
an antibody that binds a protein kinase with higher
affinity than another protein kinase or cellular protein.
An antibody that specifically binds to a protein kinase
will bind a higher concentration of the specific protein
kinase than any other protein kinase or cellular protein.
The term "adsorbing" as used herein refers to the
binding 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.
Antibodies can be linked to solid supports using techniques
well known to individuals of ordinary skill in the art.
See, e.g., Harlo & Lane, Antibodies, A Laboratory Manual,
1989, Cold Spring Harbor Laboratories.
The term "measuring the phosphate concentration" as
used herein refers to techniques commonly known to persons
of ordinary skill in the art. These techniques can involve
quantifying the concentration of phosphate concentrations
within a substrate or determining relative amounts of
phosphate within a substrate. These techniques can 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 the method of identifying compounds that modulate the
function of serine/threonine protein kinase that further
comprises the following steps: (a) lysing the cells to
render a lysate comprising RAF; (b) adsorbing the RAF to an
antibody; (c) incubating the adsorbed RAF with MEK and
MAPK; and (d) adsorbing the MEK and MAPK to a solid support
or antibody or antibodies. The step of measuring the
effect on the cells comprises monitoring the phosphate
concentration of said MEK and MAPK.
In a preferred embodiment, the invention relates to
the method of identifying compounds that modulate the
function of serine/threonine protein kinase, where the
azabenzimidazole-based compound has a structure set forth
in formula I, II, or III as defined herein or any of the
subgroups thereof set forth herein.
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-toluenesulfonic acid, salicylic acid and the like.
The term "prodrug" refers to an agent that is
converted into the parent drug in vivo. Prodrugs may be
easier to administer than the parent drug in some
situations. For example, the prodrug may be bioavailable
by oral administration but the parent is not, or the
prodrug may improve solubility to allow for intravenous
administration.
In another preferred embodiment, the invention relates
to the method of identifying compounds that modulate the
function of serine/threonine protein kinase, where the
azabenzimidazole-based compound has a structure set forth
in formula I, II, or III, where the azabenzimidazole
compound is selected from the group consisting of SABI
compounds.
The term "SABI compounds" refers to the group of
azabenzimidazole-based compounds having a structure set
forth in formula A or B, and numbered A-l through A-198 in
the following table:
In another aspect, the invention features a method of
preventing or treating an abnormal condition in an organism
by administering a compound of the invention, as specified
herein by formula I, II, or III, with any of the
constraints provided herein, to an organism.
The term "organism" relates to any living entity
comprising at least one cell. An organism can be as simple
as one eukaryotic cell or as complex as a mammal. In
preferred embodiments, an organism refers to humans or
mamals.
The term "preventing" refers to the method of the
invention decreasing the probability, or eliminating the
possibility, that an organism contracts or develops the
abnormal condition.
The term "treating" refers to the method of the
invention having a therapeutic effect and at least
partially alleviating or abrogating the abnormal condition
in the organism.
The term "therapeutic effect" refers to the inhibition
of cell growth causing or contributing to an abnormal
condition. The term "therapeutic effect" also refers to
the inhibition of growth factors causing or contributing to
the abnormal condition (e.g. cancer). A therapeutic effect
relieves to some extent one or more of the symptoms of the
abnormal condition. In reference to the treatment of a
cancer, a therapeutic effect refers to one or more of the
following: (a) a reduction in tumor size; (b) inhibition
(i.e., slowing or stopping) tumor metastasis; (c)
inhibition of tumor growth; and (d) relieving to some
extent one or more of the symptoms associated with the
abnormal condition. Compounds demonstrating efficacy
against leukemias can be identified as described herein,
except that rather than inhibiting metastasis, the
compounds may instead slow 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
their normal functions in that organism. An abnormal
condition can relate to cell proliferation, cell
differentiation, or cell survival.
Aberrant cell proliferative conditions include cancers
such as fibrotic and 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 rates, and tissue grafting techniques.
Aberrant cell survival conditions relate to conditions
in which programmed cell death (apoptosis) pathways are
activated or abrogated. A number of protein kinases are
associated with the apoptosis pathways. Aberrations in the
function of any one of the protein kinases could lead to
cell immortality or premature cell death.
Cell proliferation, differentiation, and survival are
phenomena simply measured by methods in the art. These
methods can involve observing the number of cells or the
appearance of cells under a microscope with respect to time
(for example, days).
The term "administering" relates broadly to the
provision to an organism and more specifically to a method
of incorporating a compound into cells or tissues of an
organism. The abnormal condition can be prevented or
treated when the cells or tissues of the organism exist
within the organism or outside of the organism. Cells
existing outside the organism can be maintained or grown in
cell culture dishes. For cells harbored within the
organism, many techniques exist in the art to administer
compounds, including (but not limited to) oral, parenteral,
dermal, injection, and aerosol applications. For cells
outside of the organism, multiple techniques exist in the
art to administer the compounds, including (but not limited
to) cell microinjection techniques, transformation
techniques, and carrier techniques.
In a preferred embodiment, the invention relates to a
method of preventing or treating an abnormal condition in
an organism, where the azabenzimidazole-based compound has
a structure set forth in formula I, II, or III as defined
herein or any of the subgroups thereof set forth herein.
In another preferred embodiment, the invention relates
to a method of preventing or treating an abnormal condition
in an organism, where the azabenzimidazole compound is
selected from the group consisting of SABI compounds.
In another preferred embodiment, the invention relates
to a method of preventing or treating an abnormal condition
in an organism, where the organism is a mammal.
The term "mammal" refers preferably to such organisms
as mice, rats, rabbits, guinea pigs, and goats, more
preferably to monkeys and apes, and most preferably to
humans.
In another preferred embodiment, the invention relates
to a method of preventing or treating an abnormal condition
in an organism, where the abnormal condition is cancer or a
fibrotic disorder.
In yet another preferred embodiment, the invention
relates to a method of preventing or treating an abnormal
condition in an organism, where the cancer is selected from
the group consisting of lung cancer, ovarian cancer, breast
cancer, brain cancer, intra-axial brain cancer, colon
cancer, prostate cancer, sarcoma, Kaposi's sarcoma,
melanoma, and glioma.
In still another preferred embodiment, the invention
relates to a method of preventing or treating an abnormal
condition in an organism, where the method applies to an
abnormal condition associated with an aberration in a
signal transduction pathway characterized by an interaction
between a serine/threonine protein kinase and a natural
binding partner.
The term "signal transduction pathway" 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 encompases signals that
are propagated entirely within a cell. The polypeptide
molecules involved in signal transduction processes are
typically receptor and non-receptor protein kinases,
receptor and non-receptor protein phosphatases, nucleotide
exchange factors, and transcription factors.
The term "aberration", in conjunction with a signal
transduction process, refers to a protein kinase that is
over- or under-expressed in an organism, mutated such that
its catalytic activity is lower or higher than wild-type
protein kinase activity, mutated such that it can no longer
interact with a natural binding partner, is no longer
modified by another protein kinase or protein phosphatase,
or no longer interacts with a natural binding partner,
The term "promoting or disrupting the abnormal
interaction" refers to a method that can be accomplished 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 natural binding
partners by forming favorable interactions with multiple
atoms at the complex interface. Alternatively, a compound
can inhibit an interaction between a protein kinase and
natural binding partners by compromising favorable
interactions formed between atoms at the complex interface.
In another preferred embodiment, the invention relates
to a method of preventing or treating an abnormal condition
in an organism, where the serine/threonine protein kinase
is RAF.
III. Compounds and Pharmaceutical Compositions of the
Invention
In another aspect, the invention features
azabenzimidazole compounds having structures set forth in
formula I, II, or III:
where
(a) R1, R2, R3, and R4 are independently selected
from the group consisting of
(i) hydrogen;
(ii) saturated or unsaturated alkyl;
(iii) NX2X3, where X2 and X3 are
independently selected from the group consisting of
hydrogen, saturated or unsaturated alkyl, and homocyclic or
heterocyclic ring moieties;
(iv) halogen or trihalomethyl;
(v) a ketone of formula -CO-X4, where
X4 is selected from the group consisting of hydrogen,
alkyl, and homocyclic or heterocyclic ring moieties;
(vi) a carboxylic acid of formula
-(X5)n-COOH or ester of formula - (X6) n-COO-X7, where X5, X6,
and X7 are independently selected from the group consisting
of alkyl and homocyclic or heterocyclic ring moieties and
where n is 0 or 1;
(vii) an alcohol of formula (X8)n-OH or
an alkoxy moiety of formula -(X8)n-O-X9, where X8 and X9 are
independently selected from the group consisting of
hydrogen, saturated or unsaturated alkyl, and homocyclic or
heterocyclic ring moieties, where the ring is optionally
substituted with one or more substituents independently
selected from the group consisitng of alkyl, alkoxy,
halogen, trihalomethyl, carboxylate, nitro, and ester and
where n is 0 or 1;
(viii) an amide of formula -NHCOX10,
where X10 is selected from the group consisting of alkyl,
hydroxyl, and homocyclic or heterocyclic ring moieties,
where the ring is optionally substituted with one or more
substituents independently selected from the group
consisitng of alkyl, alkoxy, halogen, trihalomethyl,
carboxylate, nitro, and ester;
(ix) -SO2NX11X12, where X11 and X12 are
selected from the group consisting of hydrogen, alkyl, and
homocyclic or heterocyclic ring moieties;
(x) a homocyclic or heterocyclic ring
moiety optionally substituted with one, two, or three
substituents independently selected from the group
consisitng of alkyl, alkoxy, halogen, trihalomethyl,
carboxylate, nitro, and ester moieties;
(xi) an aldehyde of formula -CO-H; and
(xii) a sulfone of formula -SO2-X13,
where X13 is selected from the group consisting of
saturated or unsaturated alkyl and homocyclic or
heterocyclic ring moieties;
(b) Z1 and Z2 are independently selected from
the group consisting of nitrogen, sulfur, oxygen, NH and
NR4, provided that if one of Z1 and Z2 is nitrogen, NH, or
NR4 then the other of Z1 and Z2 is nitrogen, sulfur, oxygen,
NH, or NR4; and
(c) Z3 and X1 are independently selected from
the group consisting of nitrogen, sulfur, and oxygen.
The term "saturated alkyl" refers to an alkyl moiety
that does not contain any alkene or alkyne moieties. The
alkyl moiety may be branched or non-branched.
The term "unsaturated alkyl" refers to an alkyl moiety
that contains at least one alkene or alkyne moiety. The
alkyl moiety may be branched or non-branched.
The term "amine" refers to a chemical moiety of
formula NR1R2 where R1 and R2 are independently selected
from the group consisting of hydrogen, saturated or
unsaturated alkyl, and homocyclic or heterocyclic ring
moieties, where the ring is optionally substituted with one
or more substituents independently selected from the group
consisting of alkyl, halogen, trihalomethyl, carboxylate,
nitro, and ester moieties.
The term "aryl" refers to an aromatic group which has
at least one ring having a conjugated pi electron system
and includes both carbocyclic aryl (e.g. phenyl) and
heterocyclic aryl groups (e.g. pyridine). The term
"carbocyclic" refers to a compound which contains one or
more covalently closed ring structures, and that the atoms
forming the backbone of the ring are all carbon atoms. The
term thus distinguishes carbocyclic from heterocyclic rings
in which the ring backbone contains at least one atom which
is different from carbon. The term "heteroarly" refers to
an aryl group which contains at least one heterocyclic
ring.
The term "halogen" refers to an atom selected from the
group consisting of fluorine, chlorine, bromine, and
iodine.
The term "ketone" refers to a chemical moiety with
formula -(R)n-CO-R', where R and R' are selected from the
group consisting of saturated or unsaturated alkyl and
homocyclic or heterocyclic ring moieties and where n is 0
or 1.
The term "carboxylic acid" refers to a chemical moiety
with formula -(R)n-COOH, where R is selected from the group
consisting of saturated or unsaturated alkyl and homocyclic
or heterocyclic ring moieties, and where n is 0 or 1.
The term "ester" refers to a chemical moiety with
formula -(R)n-COOR', where R and R' are independently
selected from the group consisting of saturated or
unsaturated alkyl and homocyclic or heterocyclic ring
moieties and where n is 0 or 1.
The term "alcohol" refers to a chemical substituent of
formula -ROH, where R is selected from the group consisting
of hydrogen, saturated or unsaturated alkyl, and homocyclic
or heterocyclic ring moieties, where the ring moiety is
optionally substituted with one or more substituents
independently selected from the group consisting of alkyl,
halogen, trihalomethyl, carboxylate, nitro, and ester
moieties.
The term "amide" refers to a chemical substituent of
formula -NHCOR, where R is selected from the group
consisting of hydrogen, alkyl, hydroxyl, and homocyclic or
heterocyclic ring moieties, where the ring is optionally
substituted with one or more substituents independently
selected from the group consisting of alkyl, halogen,
trihalomethyl, carboxylate, nitro, or ester.
The term "alkoxy moiety" refers to a chemical
substituent of formula -OR, where R is hydrogen or a
saturated or unsaturated alkyl moiety.
The term "aldehyde" refers to a chemical moiety with
formula -(R)n-CHO, where R is selected from the group
consisting of saturated or unsaturated alkyl and homocyclic
or heterocyclic ring moieties and where n is 0 or 1.
The term "sulfone" refers to a chemical moiety with
formula -SO2-R, where R is selected from the group
consisting of saturated or unsaturated-alkyl and homocyclic
or heterocyclic ring moieties.
In another preferred embodiment, the invention relates
to an azabenzimidazole-based compound having a structure
set forth in formula I, II, or III, where Z1 and Z2 are
independently selected from the group consisting of
nitrogen and NH.
In yet another preferred embodiment, the invention
relates to an azabenzimidazole-based compound having a
structure set forth in formula I, II, or III, where R1, R2,
R3, and R4 are independently selected from the group
consisting of hydrogen, saturated or unsaturated alkyl
optionally substituted with a homocyclic or heterocyclic
ring moieties, where the ring moiety is optionally
substituted with one, two, or three substituents
independently selected from the group consisting of alkyl,
alkoxy, halogen, trihalomethyl, hydroxy, alkoxy,
carboxylate, nitro, and ester moieties, and a homocyclic or
heterocyclic ring moiety optionally substituted with one,
two, or three substituents independently selected from the
group consisting of alkyl, alkoxy, halogen, trihalomethyl,
hydroxy, alkoxy, carboxylate, nitro, and ester moieties.
In other preferred embodiments, the invention relates
to an azabenzimidazole-based compound having a structure
set forth in formula I, II, or III, where R2 and R3 are
hydrogen.
In another preferred embodiment, the invention relates
to an azabenzimidazole-based compound having a structure
set forth in formula I, II, or III, where R1 is phenyl
optionally substituted with one, two, or three substituents
independently selected from the group consisting of alkyl,
alkoxy, halogen, trihalomethyl, carboxylate, nitro, or
ester moieties.
In yet another preferred embodiment, the invention
relates to an azabenzimidazole-based compound having a
structure set forth in formula I, II, or III, where R1
selected from the group consisting of SABI substituents.
The term "SABI substituents" refers to the group of
substituents consisting of phenyl, 2-nitrophenyl,
3-nitrophenyl, 4-nitrophenyl, 2-chlorophenyl,
3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl,
3-methylphenyl, 4-methylphenyl, 2-fluorophenyl,
3-fluorophenyl, 4-fluorophenyl, 2-(trifluoromethyl)phenyl,
3-(trifluoromethyl)phenyl, 4-(trifluoromethyl)phenyl,
2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,
2-carboxyphenyl, 3-carboxyphenyl, and 4-carboxyphenyl.
In other preferred embodiments, the invention relates
to an azabenzimidazole-based compound having a structure
set forth in formula I, II, or III, where X1 is sulfur.
In another preferred embodiment, the invention relates
to an azabenzimidazole-based compound having a structure
set forth in formula I, II, or III, where X1 is oxygen.
In yet another preferred embodiment, the invention
relates to an azabenzimidazole-based compound having a
structure set forth in formula I, II, or III, where Z3 is
oxygen.
In other preferred embodiments, the invention relates
to an azabenzimidazole-based compound having a structure
set forth in formula I, II, or III, where R4 is selected
from the group consisting of methyl and ethyl.
In another preferred embodiment, the invention relates
to an azabenzimidazole-based compound having a structure
set forth in formula I, II, or III, where the
azabenzimidazole compound is selected from the group
consisting of SABI compounds.
In another aspect, the invention features a
pharmaceutical composition comprising a compound of the
invention, as specified herein, or its salt, and a
physiologically acceptable carrier or diluent.
In another aspect, the invention relates to a
pharmaceutical composition comprising a compound having a
structure of formula I, II, or III as defined herein or any
of the subgroups thereof set forth herein.
In another preferred embodiment, the invention relates
to a pharmaceutical composition, where the azabenzimidazole
compound is selected from the group consisting of SABI
compounds.
The term "pharmaceutical composition" refers to a
mixture of an azabenzimidazole compound of the invention
with other chemical components, such as diluents or
parriers . The pharmaceutical composition facilitates
administration of the compound to an organism. Multiple
techniques of administering a compound exist in the art
including, but 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 the incorporation of a compound into cells or
tissues. For example dimethyl sulfoxide (DMSO) is a
commonly utilized carrier as it 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 buffered solutions are
utilized as diluents in the art. One commonly used
buffered solution is phosphate buffered saline because it
mimics the salt 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.
IV. Synthetic Methods of the Invention
In another aspect, the invention relates to a method
for synthesizing an azabenzimidazole compound of formula I,
II, or III, comprising the steps of: (a) reacting 2-amino-
6-chloro-3-nitropyridine with a second reactant in a
solvent to yield the first intermediate, where the second
reactant is a substituted aryl ring; (b) reducing the first
intermediate in the presence of a catalyst and a reducing
agent to yield the second intermediate; (c) reacting the
second intermediate with a third reactant; and (d)
purifying the compound of invention.
In a preferred embodiment, the invention relates to
the method of synthesizing a compound of the invention
where the substituted aryl ring is a substituted phenol,
substituted thiophenol, and substituted aniline.
In another preferred embodiment, the invention relates
to the method of synthesizing a compound of the invention
where the substituted phenol, substituted thiophenol, and
substituted aniline are selected from the group consisting
of SABI reactants.
The term "SABI reactants" refers to the group of
reactants consisting of the sodium salt of phenol, 2-
nitrophenol, 3-nitrophenol, 4-nitrophenol, 2-chlorophenol,
3-chlorophenol, 4-chlorophenol, 2-cresol, 3-cresol, 4-
cresol, 2-fluorophenol, 3-fluorophenol, 4-fluorophenol, 2-
(trifluoromethyl)phenol, 3-(trifluoromethyl)phenol, 4-
(trifluoromethyl)phenol, 2-methoxyphenol, 3-methoxyphenol,
4-methoxyphenol, 2-hydroxybenzoic acid, 3-hydroxybenzoic
acid, 4-hydroxybenzoic acid, thiophenol, 2-nitrothiophenol,
3-nitrothiophenol, 4-nitrothiophenol, 2-chlorothiophenol,
3-chlorothiophenol, 4-chlorothiophenol, 2-thiocresol, 3-
thiocresol, 4-thiocresol, 2-fluorothiophenol, 3-
fluorothiophenol, 4-fluorothiophenol, 2-
(trifluoromethyl)thiophenol, 3-(trifluoromethyl)thiophenol,
4-(trifluoromethyl)thiophenol, 2-methoxybenzenethiol, 3-
methoxybenzenethiol, 4-methoxybenzenethiol, 2-
mercaptobenzoic acid, 3-mercaptobenzoic acid, 4-
mercaptobenzoic acid, aniline, 2-nitroaniline, 3-
nitroaniline, 4-nitroaniline, 2-chloroaniline, 3-
chloroaniline, 4-chloroaniline, 2-toluidine, 3-toluidine,
4-toluidine, 2-fluoroaniline, 3-fluoroaniline, 4-
fluoroaniline, 2-(trifluoromethyl)aniline, 3-
(trifluoromethyl)aniline, 4-(trifluoromethyl)aniline, 2-
anisidine, 3-anisidine, 4-anisidine, 2-aminobenzoic acid,
3-aminobenzoic acid, and 4-aminobenzoic acid.
In a preferred embodiment, the invention relates to
the method of synthesizing a compound of the invention
where the solvent is n-propanol.
In another preferred embodiment, the invention relates
to the method of synthesizing a compound of the invention
where the reducing agent is hydrogen.
In yet another preferred embodiment, the invention
relates to the method of synthesizing a compound of the
invention where the catalyst is Raney nickel.
In still another preferred embodiment, the invention
relates to the method of synthesizing a compound of the
invention where the third reactant is O-methylisourea.
In another preferred embodiment, the invention relates
to the method of synthesizing a compound of the invention
where the third reactant is the product of the reaction of
S-methylisothiouronium sulphate and alkyl chloroformate.
In yet another preferred embodiment, the invention
relates to the method of synthesizing a compound of the
invention where the alkyl chloroformate is methyl
chloroformate.
In still another preferred embodiment, the invention
relates to the method of synthesizing a compound of the
invention where the alkyl chloroformate is ethyl
chloroformate.
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 from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed in part towards
methods of modulating the function of serine/threpnine
protein kinases with azabenzimidazole-based compounds. In
addition, the invention relates in part to methods for
identifying compounds that modulate the function of
serine/threonine protein kinases. The methods incorporate
cells that express a serine/threonine protein kinase, such
as RAF.
RAF is a non-receptor protein kinase that is recruited
to the cell membrane when it binds to activated RAS, a
guanine triphosphate hydrolyzing enzyme. RAS is activated
when an activated receptor protein tyrosine kinase, such as
EGFR or PDGFR, bind to an adaptor protein, GRB2, and a
guanine nucleotide exchange factor, SOS. SOS removes
guanine diphosphate from RAS, replaces it with guanine
triphosphate, and thereby activates RAS. RAS then binds
RAF and consequently activates RAF. RAF may then
phosphorylate other protein targets on serine and threonine
residues, such as the kinase (MEK) that phosphorylates and
consequently activates mitogen-activated protein kinase
(MAPK). Thus, RAF serves as an intermediary controlling
factor in mitogen-activated signal transduction.
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.
RAF's role in cell proliferation is underscored by the
observation that mutations to RAF's amino acid sequence
have been associated with tumors and cancers. Because the
mutations to RAF that give rise to cancer in cells lead to
RAF molecules that display unregulated catalytic activity,
inhibitors of RAF may alleviate or even abrogate the cell
proliferation that leads to cancer in these cells.
Methods of the present invention can detect compounds
that modulate the function of the protein kinase RAF in
cells. RAF phosphorylates a protein kinase (MEK) which in
turn phosphorylates mitogen-activated protein kinase
(MAPK). Assays that monitor only the phosphorylation of
MEK by RAF are not sensitive because the phosphorylation
levels of MEK are not significant. To overcome this
sensitivity dilemma, the phosphorylation of both MEK and
MAPK are 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
immunosorbant assays. In addition, the assay of the
invention is preferrably performed in a high throughput
format such that many compounds can be rapidly monitored in
a short period of time.
The methods of the present invention have identified
compounds that inhibit the function of the RAF protein
kinase. These compounds are azabenzimidazole-based
derivatives. Although azabenzimidazole-based derivatives
have been tested for their ability to inhibit enzymes
involved with nucleotide synthesis in bacteria, many of
these compounds have not yet been significantly explored
with respect to protein kinase inhibition.
Because RAF exhibits significant amino acid homology
to other serine/threonine protein kinases, the
azabenzimidazole-based compounds of the invention may
likely inhibit serine/threonine protein kinases other than
RAF. Thus, the methods of the invention also relate to
serine/threonine protein kinases other than RAF, including
receptor and non-receptor serine/threonine protein kinases.
The methods of the invention also pertain to other
compounds that modulate RAF function in cells as the high
throughput aspect of the methods allows a wide array of
molecules to be tested in a short period of time.
Therefore, the methods of the invention can identify
existing molecules not disclosed in the present invention
that modulate STK function.
I. Biological Activity of Azabenzimidazole—Based
Compounds
Azabenzimidazole-based compounds of the present
invention were tested for their ability to inhibit RAF
protein kinase function. The biological assays and results
of these inhibition studies are reported herein. The
methods used to measure azabenzimidazole-based compound
modulation of protein kinase function are similar to those
described in 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 Docket No. 221/187),
filed August 23, 1996, with respect to the high throughput
aspect of the method. The 08/702,232 application is
incorporated herein by reference in its entirety, including
any drawings.
II. Target Diseases to be Treated by Azabenzimidazole-
Based Compounds
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 harm 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
may be a result of RAF molecules that function
inappropriately or a result of RAF-related protein kinase
molecules that function inappropriately.
Alterations in the function of the RAF protein kinase
or protein kinases related to RAF can lead to enhanced or
decreased cell proliferative conditions evident 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 relate to the abnormal formation of
the cellular extracellular matrix. An example of a
fibrotic disorder is hepatic cirrhosis. Hepatic cirrhosis
is characterized by an increased concentration of
extracellular matrix constituents resulting in the
formation of a hepatic scar. Hepatic 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 renal
diseases, such as glomerulonephritis, diabetic nephropathy,
malignant nephrosclerosis, thrombotic microangiopathy
syndromes, transplant rejection, and glomerulopathies.
Preferred types of cancers that may be treated by the
methods and compounds of the invention are lung cancer,
ovarian cancer, breast cancer, brain cancer, intra-axial
brain cancer, colon cancer, prostate cancer, Kaposi's
sarcoma, melanoma, and glioma. Evidence that the compounds
and methods of the invention can effectively be utilized to
stem and reverse the proliferation of cancer cells is
provided herein by reference.
Angiogenic and vasculogenic disorders result from
excess proliferation of blood vessels. Blood vessel
proliferation is necessary in a variety of normal
physiological processes such as embryonic development,
corpus luteum formation, wound healing and organ
regeneration. However, blood vessel proliferation is also
essential in cancer tumor development. Other examples of
blood vessel proliferative disorders include arthritis,
where new capillary blood vessels invade the joint and
destroy cartilage. In addition, blood vessel proliferative
diseases include ocular diseases, such as diabetic
retinopathy, where new capillaries in the retina invade the
vitreous, bleed and cause blindness. Conversely, disorders
related to the shrinkage, contraction or closing of blood
vessels, such as restenosis, are also implicated in adverse
regulation of protein kinases.
Moreover, vasculogenesis and angiogenesis are
associated with the growth of malignant solid tumors and
metastasis. A vigorously growing cancer tumor requires a
nutrient and oxygen rich blood supply to continue growing.
As a consequence, an abnormally large number of capillary
blood vessels often grow in concert with the tumor and act
as supply lines to the tumor. In addition to supplying
nutrients to the tumor, the new blood vessels embedded in a
tumor provide a gateway for tumor cells to enter the
circulation and metastasize to distant sites in the
organism. Folkman, 1990, J. Natl. Cancer Inst. 82:4-6.
Inappropriate RAF activity can stimulate cell
proliferative disorders. Molecules specifically designed
to modulate the function of the RAF protein kinase have
been shown to inhibit cellular proliferation.
Specifically, antisense nucleic acid molecules, which are
designed to both bind to message RNA encoding the RAF
protein kinase and block translation from that message,
effectively reversed transformation of A54 9 cells in vitro.
Monia et al., 1996, Nature Medicine 2: 688, incorporated
herein by reference in its entirety including all figures
and tables. A54 9 cells are human malignant cells.
These RAF-targeted antisense studies provide evidence
that the azabenzimidazole molecules of the invention, which
modulate the function of the RAF protein kinase, can stem,
and likely reverse, the proliferation of malignant cells in
an organism. These azabenzimidazole compounds can be
tested in the in vitro methods provided herein by example.
Furthermore, the azabenzimidazole compounds may be tested
for their effect upon tumor cells in vivo by the xenograft
methods also provided herein by example.
There exist at least two ways in which inappropriate
RAF activity can stimulate unwanted cell proliferation of a
particular type of cells: (1) directly stimulating growth
of the particular cell, or (2) increasing vascularization
of a particular area, such as tumor tissue, thereby
facilitating growth of the tissue.
The use of the present invention is facilitated by
first identifying whether the cell proliferation disorder
is RAF driven. Once such disorders are identified,
patients suffering from such a disorder can be identified
by analysis of their symptoms using procedures well known
to physicians or veterinarians of ordinary skill in the
art. Such patients can then be treated as described
herein.
Determining whether the cell proliferation disorder is
RAF driven may be accomplished by first determining the
level of RAF activity occurring in the cell or in a
particular location in a patient's body. For example, in
the case of cancer cells the level of one or more RAF
activities may be compared for non-RAF driven cancers and
RAF driven cancers. If the cancer cells have a higher
level of RAF activity than RAF driven cancers, preferably
equal to or greater than RAF driven cancers, then they are
candidates for treatment using the described RAF-modulating
methods and compounds of the invention.
In the case of cell proliferative disorders arising
due to unwanted proliferation of non-cancer cells, the
level of RAF activity is compared to that level occurring
in the general population (e.g., the average level
occurring in the general population of people or animals
excluding those people or animals suffering from a cell
proliferative disorder). If the unwanted cell
proliferation disorder is characterized by a higher RAF
level than occurring in the general population then the
disorder is a candidate for treatment using the described
RAF modulating methods and compounds of the invention.
III. Pharmaceutical Compositions and Administration of
Azabenzimidazole-Based Compounds
Methods of preparing pharmaceutical formulations of
the compounds, methods of determining the amounts of
compounds to be administered to a patient, and modes of
administering compounds to an organism are disclosed in
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 Docket No. 221/187), filed August 23, 1996, and
International patent publication number 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 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 merely
representative of 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 commercially available and the sequences of genes
for the various protein kinases are readily accessible in
sequence data banks. Thus, a person of ordinary skill in
the art can readily recreate the cell lines in a timely •
manner by combining the commercially available cells, the
commercially available nucleic acid vectors, and the
protein kinase genes using techniques readily available to
persons of ordinary skill in the art.
EXAMPLE 1: PROCEDURES FOR SYNTHESIZING AZABENZIMIDAZOLE
COMPOUNDS OF THE INVENTION
The invention will now be illustrated in the following
non-limiting examples in which, unless otherwise stated:
(i) evaporations were carried out by rotary
evaporation under vacuum;
(ii) operations were carried out under an atmosphere
of an inert gas such as nitrogen;
(iii) high performance liquid chromatography (HPLC)
was performed on Merck LiChrosorb RP-18 reversed-phase
silica obtained from E. Merck, Darmstadt, Germany;
(iv) yields are given for illustration only and are
not necessarily the maximum attainable;
(v) melting points are uncorrected and were determined
using a HWS Mainz SG 2000 digital melting point apparatus;
(vi) the structures of all compounds of the formula I,
II, and III of this invention were confirmed by proton
magnetic resonance spectroscopy on a Bruker AMX500-NMR
spectrophotometer, by elemental microanalysis and, in
certain cases, by mass spectroscopy;
(vii) the purity of the structures were performed by
thin layer chromatography (TLC) using silica gel (Merck
Silica Gel 60 F254) or by HPLC; and
(vii) intermediates were not generally fully
characterized and purity was assessed by thin layer
chromatography (TLC) or by HPLC.
SYNTHETIC PROCEDURES
Compound A-90: 2-Methoxycarbonylamino- (6-phenylmercapto.-3H-
imidazo [4,5—b] pyridine
2-amino-3-nitro-6-(phenylmercapto)pyridine was
prepared by heating 2-amino-6-chloro-3-nitropyridine (84.0
g, 0.484 mol) and sodium thiophenolate (Fluka) (72.0 g,
0.545 mol) in 2-propanol (1500 mL) under reflux for 2
hours. After cooling to room temperature the suspension
was diluted with water (100 ml), the solid was collected by
vacuum filtration, washed with water and 2-propanol, and
dried at 50°C under vacuum to give 109.1 g (95 % yield) of
2-amino-3-nitro-6-(phenylmercapto)pyridine, m.p. 148-152
°C).
2,3-Diamino-6-(phenylmercapto)pyridine was prepared by
hydrogenating 2-amino-3-nitro-6-(phenylmercapto)pyridine
(107.1 g, 0.433 mol) under 5 atm of H2 in the presence of
30 g of Raney-Ni in 1200 mL of 2-propanol at 70°C. After 4
hours (29.1 L of hydrogen) the reaction mixture was cooled
to 4°C while stirring continuously. The precipitate was
collected by vacuum filtration, washed with 2-propanol and
dried at 50°C under vacuum. The combined filtrates were
concentrated under reduced pressure and recrystallized from
2-propanol. After washing the hydrogenation apparatus
twice with 1000 mL of THF, evaporation under reduced
pressure and recrystallization from 2-propanol, the
precipitate was collected and dried at 50°C under vacuum to
give 80.4 g (87.1 % yield) of 2,3-diamino-6-
(phenylmercapto)pyridine, m.p. 119-122°C).
2-Methoxycarbonylamino-6-phenylmercapto-3H-
imidazo [4, 5-b]pyridine was prepared by adding methyl
chloroformate (34 mL, 0.44 mol) dropwise to a cold (5-15°C)
solution of S-methylisothiouronium sulphate (53 g, 0.19
mol) (Aldrich) in 68 mL of water while the temperature was
kept below 20°C. Afterwards, aqueous sodium hydroxide (116
g, 25% NaOH) was added carefully and a white precipitate
occured. After 20 minutes, water (210 mL) was added and
the pH adjusted to 4.0 with acetic acid (glacial, 34 mL) .
To this mixture a solution of 2,3-diamino-6-
(phenylmercapto)pyridine (37.8 g, 0.174 mol) in 210 mL of
ethanol was added dropwise and heated to 85-90°C for 2
hours. After cooling the reaction mixture overnight, the
precipitate was isolated by filtration, washed with warm
water (1000 mL), dried and recrystallized from acetic acid
and ethanol at 4 °C. The precipitate was collected by
filtration, washed with methanol and dried at 50°C under
vacuum to give 30 g (57.4 % yield) of 2-
methoxycarbonylamino-6-phenylmercapto-3H-imidazo[4,5-
b]pyridine, m.p. 269-274 °C (dec).
Compound A-3: 2-Methoxycarbonylamino- (6-phenoxy-3H-
imidazo [4,5-b]pyridine
By substituting sodium phenolate in place of sodium
thiophenolate in Example A-90, the identical process gives
2-methoxycarbonylamino-(6-phenoxy-3H-imidazo[4,5-
b]pyridine, m.p. >280 °C (dec).
Compound A-4: 2-Ethoxycarbonylamino—6-phenoxy-3H-
imidazo [4,5-b]pyridine
By substituting ethyl chloroformate in place of methyl
chloroformate in Example A-3, the identical process gives
2-ethoxycarbonylamino- (6-phenoxy-3H-imidazo [4,5-b] pyridine,
m.p. >280 °C (dec.).
Compound A-l: 2-Oxo-6-phenoxy-3H-imidazo [4,5-b] pyridine
By reacting O-methylisourea directly with 2,3-diamino-
6-(phenylmercapto)pyridine in place of the product of the
reaction of S-methylisothiouronium sulfate and methyl
chloroformate in Example A-3, the identical process gives
2-oxo-(6-phenoxy-3H-imidazo[4,5-b]pyridine, m.p. 277-278
°C.
Compound A—2: 2-Oxo-6-phenylmercapto-3H-imidazo[4,5-
b] pyridine
By substituting sodium thiophenolate in place of
sodium phenolate in Example A-l, the identical process
gives 2-oxo- (6-phenylmercapto-3H-imidazb [4, 5-b] pyridine,
m.p. 253-254 °C.
Compounds A-5 - A-25:
By substituting the appropriate phenolate for sodium
phenolate in Example A-l, the identical process gives the
following examples. For examples A-23, A-24, and A-25, the
carboxy groups are protected by a methyl, ethyl, benzyl, t-
butyl or other suitable ester and then deprotected in the
last step to give the compounds.
A-5 2-Oxo-6- (2-nitrophenoxy) -3H-imidazo [4, 5-b] pyridine
A-6 2-Oxo-6- (3-nitrophenoxy) -3H-imidazo [4, 5-b] pyridine
A-7 2-0x0-6-(4-nitrophenoxy)-3H-imidazo [4, 5-b] pyridine
A-8 2-Oxo-6- (2-chlorophenoxy) -3H-imidazo [4, 5-b] pyridine
A-9 2-0xo-6- (3-chlorophenoxy) -3H-imidazo [4, 5-b] pyridine
A-10 2-Oxo-6- (4-chlorophenoxy) -3H-imidazo [4, 5-b] pyridine
A-ll 2-Oxo-6- (2-methylphenoxy) -3H-imidazo [4, 5-b] pyridine
A-12 2-Oxo-6- (3-methylphenoxy) -3H-imidazo [4, 5-b] pyridine
A-13 2-Oxo-6-(4-methylphenoxy)-3H-imidazo[4,5-b]pyridine
A-14 2-Oxo-6- (2-fluorophenoxy) -3H-imidazo [4, 5-b] pyridine
A-15 2-Oxo-6- (3-fluorophenoxy) -3H-imidazo [4, 5-b] pyridine
A-16 2-Oxo-6- (4-fluorophenoxy) -3H-imidazo [4, 5-b] pyridine
A-17 2-0xo-6-[2-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b]pyridine
A-18 2-Oxo-6-[3-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b] pyridine
A-19 2-0x0-6-[4-(trifluoromethyl)phenoxy]-3H-imidazo[4,5-
b] pyridine
A-20 2-OXo-6- (2-methoxyphenoxy) -3H-imidazo [4, 5-b] pyridine
A-21 2-Oxo-6-(3-methoxyphenoxy)-3H-imidazo [4, 5-b] pyridine
A-22 2-Oxo-6-(4-methoxyphenoxy)-3H-imidazo [4, 5-b] pyridine
A-23 2-Oxo-6- (2-carboxyphenoxy) -3H-imidazo [4, 5-b] pyridine
A-24 2-Oxo-6- (3-carboxyphenoxy) -3H-imidazo [4, 5-b] pyridine
A-25 2-Oxo-6- (4-carboxyphenoxy) -3H-imidazo [4, 5-b] pyridine
Compounds A—26 — A—46.
By substituting the appropriate thiophenolate for
sodium thiophenolate in Example A-2, the identical process
gives the following examples. For examples A-44, A-45, an
A-4 6, the carboxy groups are protected by a methyl, ethyl,
benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
A-26 2-Oxo-6- (2-nitrophenylmerapto) -3H-imidazo [4,5-
b] pyridine
A-27 2-Oxo-6-(3-nitrophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-28 2-Oxo-6-(4-nitrophenylmercapto)-3H-imidazo[4,5-
b] pyridine
A-29 2-Oxo-6-(2-chlorophenylmercapto)-3H-imidazo[4,5-
b] pyridine
A-30 2-Oxo-6-(3-chlorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-31 2-Oxo-6-(4-chlorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-32 2-Oxo-6-(2-methylphenylmercapto)-3H-imidazo[4,5-
b] pyridine
A-33 2-Oxo-6- (3-methylphenylmercapto) -3H-imidazo [4,5-
b]pyridine
A-34 2-Oxo-6- (4-methylphenylmercapto) -3H-imidazo [4,5-
b] pyridine
A-35 2-Oxo-6-(2-fluorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-36 2-Oxo-6- (3-f luorophenylmercapto) -3H-imidazo [4,5-
b] pyridine
A-37 2-Oxo-6-(4-fluorophenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-38 2-Oxo-6-[2-(trifluoromethyl)phenylmercapto]-3H-
imidazo [ 4, 5-b] pyridine
A-39 2-Oxo-6-[3-(trifluoromethyl)phenylmercapto]-3H-
imidazo [4, 5-b] pyridine
A-40 2-Oxo-6- [4- (trifluoromethyl) phenylmercapto] -3H-
imidazo[4,5-b]pyridine
A-41 2-Oxo-6-(2-methoxyphenylmercapto)-3H-imidazo[4,5-
b] pyridine
A-42 2-Oxo-6-(3-methoxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-43 2-Oxo-6-(4-methoxyphenylmercapto)-3H-imidazo[4,5-
b]pyridine
A-44 2-Oxo-6-(2-carboxyphenylmercapto)-3H-imidazo [4,5-
b]pyridine
A-45 2-Oxo-6-(3-carboxyphenylmercapto)-3H-imidazo [4,5-
b] pyridine
A-4 6 2-Oxo-6-(4-carboxyphenylmercapto)-3H-imidazo[4,5-
b] pyridine
Compounds A-47 - A-68.
By substituting the appropriate aniline salt for
sodium phenolate in Example A-l, the identical process
gives the following examples. For examples A-66, A-67, and
A-68, the carboxy groups are protected by a methyl, ethyl,
benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
A-47 2-Oxo-6-phenylamino-3H-imidazo [4, 5-b]pyridine
A-48 2-Oxo-6- (2-nitrophenylamino) -3H-imidazo [4,5-b]pyridine
A-49 2-Oxo-6- (3-nitrophenylamino) -3H-imidazo [4, 5-b] pyridine
A-50 2-Oxo-6- (4-nitrophenylamino) -3H-imidazo [4, 5-b] pyridine
A-51 2-Oxo-6-(2-chlorophenylamino)-3H-imidazo[4,5-
b] pyridine
A-52 2-Oxo-6-(3-chlorophenylamino)-3H-imidazo[4,5-
b] pyridine
A-53 2-Oxo-6-(4-chlorophenylamino)-3H-imidazo[4,5-
b] pyridine
A-54 2-0xo-6- (2-methylphenylamino) -3H-imidazo [4,5-
b]pyridine
A-55 2-Oxo-6-(3-methylphenylamino)-3H-imidazo[4, 5-
b] pyridine
A-56 2-Oxo-6-(4-methylphenylamino)-3H-imidazo[4,5-
b]pyridine
A-57 2-Oxo-6-(2-fluorophenylamino)-3H-imidazo[4,5-
b] pyridine
A-58 2-Oxo-6-(3-fluorophenylamino)-3H-imidazo[4,5-
b] pyridine
A-59 2-Oxo-6- (4-f luorophenylamino) -3H-imidazo [4,5-
b] pyridine
A-60 2-0xo-6-[2-(trifluoromethyl)phenylamino]-3H-
imidazo [ 4, 5-b] pyridine
A-61 2-Oxo-6-[3-(trifluoromethyl)phenylamino]-3H-
imidazo [ 4, 5-b] pyridine
A-62 2-Oxo-6-[4-(trifluoromethyl)phenylamino]-3H-
imidazo [ 4, 5-b] pyridine
A-63 2-Oxo-6-(2-methoxyphenylamino)-3H-imidazo[4,5-
b] pyridine
A-64 2-Oxo-6-(3-methoxyphenylamino)-3H-imidazo[4,5-
b] pyridine
A-65 2-Oxo-6-(4-methoxyphenylamino)-3H-imidazo[4,5-
b]pyridine
A-66 2-Oxo-6-(2-carboxyphenylamino) -3H-imidazo[4,5-
b] pyridine
A-67 2-Oxo-6-(3-carboxyphenylamino)-3H-imidazo [4,5-
b] pyridine
A-68 2-Oxo-6-(4-carboxyphenylamino)-3H-imidazo[4,5-
b] pyridine
Compounds A-69 - A-8 9.
By substituting the appropriate phenolate for sodium
phenolate in Example A-3, the identical process gives the
following examples. For examples A-87, A-88, and A-89, the
carboxy groups are protected by a methyl, ethyl, benzyl, t-
butyl or other suitable ester and then deprotected in the
last step to give the compounds.
A-69 2-Methoxycarbonylamino-6-(2-nitrophenoxy)-3H-
imidazo [ 4 , 5-b] pyridine
A-70 2-Methoxycarbonylamino-6-(3-nitrophenoxy)-3H-
imidazo [ 4, 5-b] pyridine
A-71 2-Methoxycarbonylamino-6-(4-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-72 2-Methoxycarbonylamino-6-(2-chlorophenoxy)-3H-
imidazo[4, 5-b]pyridine
A-73 2-Methoxycarbonylamino-6-(3-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-74 2-Methoxycarbonylamino-6-(4-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-75 2-Methoxycarbonylamino-6-(2-methylphenoxy)-3H-
imidazo [4 , 5-Jb] pyridine
A-76 2-Methoxycarbonylamino-6-(3-methylphenoxy)-3H-
imidazo [4, 5-b] pyridine
A-77 2-Methoxycarbonylamino-6-(4-methylphenoxy)-3H-
imidazo[4, 5-b]pyridine
A-78 2-Methoxycarbonylamino-6-(2-fluorophenoxy)-3H-
imidazo [4,5-b]pyridine
A-7 9 2-Methoxycarbonylamino-6- (3-fluorophenoxy) -3H-
imidazo [ 4 , 5-b] pyridine
A-80 2-Methoxycarbonylamino-6-(4-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-81 2-Methoxycarbonylamino-6-[2-(trifluoromethyl)phenoxy]-
3H-imidazo [4, 5-b] pyridine
A-82 2-Methoxycarbonylamino-6-[3-(trifluoromethyl)phenoxy]-
3H-imidazo [ 4, 5-b] pyridine
A-83 2-Methoxycarbonylamino-6-[4-(trifluoromethyl)phenoxy]-
3H-imidazo[4,5-b]pyridine
A-8 4 2-Methoxycarbonylamino-6-(2-methoxyphenoxy)-3H-
imidazo [ 4, 5-b] pyridine
A-85 2-Methoxycarbonylamino-6-(3-methoxyphenoxy)-3H-
imidazo [ 4, 5-b] pyridine
A-86 2-Methoxycarbonylamino-6-(4-methoxyphenoxy)-3H-
imidazo [ 4, 5-b] pyridine
A-87 2-Methoxycarbonylamino-6-(2-carboxyphenoxy)-3H-
imidazo [ 4, 5-b] pyridine
A-88 2-Methoxycarbonylamino-6-(3-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-89 2-Methoxycarbonylamino-6-(4-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
Compounds A-91 - A-111.
By substituting the appropriate thiophenolate for
sodium thiophenolate in Example A-90, the identical process
gives the following examples. For examples A-109, A-110,
and A-lll, the carboxy groups are protected by a methyl,
ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
A-91 2-Methoxycarbonylamino-6-(2-nitrophenylmercapto)-
3H-imidazo [ 4, 5-b] pyridine
A-92 2-Methox,ycarbonylamino-6- (3-nitrophenylmercapto) -
3H-imidazo [ 4 , 5-b] pyridine
A-93 2-Methoxycarbonylamino-6-(4-nitrophenylmercapto)-
3H-imidazo [ 4, 5-b] pyridine
A-94 2-Methoxycarbonylamino-6-(2-
chlorophenylmercapto) -3H-imidazo [4 , 5-b] pyridine
A-95 2-Methoxycarbonylamino-6-(3-
chlorophenylmercapto) -3H-imidazo [ 4 , 5-b] pyridine
A-96 2-Methoxycarbonylamino-6-(4-
chlorophenylmercapto) -3H-imidazo [4,5-b]pyridine
A-97 2-Methoxycarbonylamino-6-(2-
methylphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-98 2-Methoxycarbonylamino-6-(3-
methylphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-99 2-Methoxycarbonylamino-6-(4-
methylphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-100 2-Methoxycarbonylamino-6-(2-
fluorophenylmercapto)-3H-imidazo [4,5-b]pyridine
A-101 2-Methoxycarbonylamino-6-(3-
fluorophenylmercapto) -3H-imidazo [4,5-b]pyridine
A-102 2-Methoxycarbonylamino-6-(4-
fluorophenylmercapto) -3H-imidazo [4,5-b]pyridine
A-103 2-Methoxycarbonylamino-6-[2-
(trifluoromethyl)phenylmercapto]-3H-imidazo [4,5-b]pyridine
A-104 2-Methoxycarbonylamino-6-[3-
(trifluoromethyl) phenylmercapto] -3H-imidazo [4,5-b]pyridine
A-105 2-Methoxycarbonylamino-6-[4-
(trifluoromethyl)phenylmercapto]-3H-imidazo[4,5-b]pyridine
A-106 2-Methoxycarbonylamino-6-(2-
methoxyphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-107 2-Methoxycarbonylamino-6-(3-
methoxyphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-108 2-Methoxycarbonylamino-6-(4-
methoxyphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-109 2-Methoxycarbonylamino-6-(2-
carboxyphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-110 2-Methoxycarbonylamino-6-(3-
carboxyphenylmercapto) -3H-imidazo [4,5-b]pyridine
A-111 2-Methoxycarbonylamino-6-(4-
carboxyphenylmercapto) -3H-imidazo [4,5-b]pyridine
Compounds A-112 - A-133.
By substituting the appropriate aniline salt for
sodium phenolate in Example A-3, the identical process
gives the following examples. For examples A-131, A-132,
and A-133, the carboxy groups are protected by a methyl,
ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
A-112 2-Methoxycarbonylamino-6-phenylamino-3H-
imidazo[4,5-A-112
A-113 2-Methoxycarbonylamino-6-(2-nitrophenylamino)-3H-
imidazo [4,5-b]pyridine
A-114 2-Methoxycarbonylamino-6-(3-nitrophenylamino)-3H-
imidazo[4, 5-b]pyridine
A-115 2-Methoxycarbonylamino-6-(4-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-116 2-Methoxycarbonylamino-6-(2-chlorophenylamino)-
3H- imidazo[4,5-b]pyridine
A-117 2-Methoxycarbonylamino-6-(3-chlorophenylamino)-
3H-imidazo [4,5-b]pyridine
A-118 2-Methoxycarbonylamino-6-(4-chlorophenylamino)-
3H-imidazo [4,5-b]pyridine
A-119 2-Methoxycarbonylamino-6-(2-methylphenylamino)-
3H-imidazo [4,5-b]pyridine
A-120 2-Methoxycarbonylamino-6-(3-methylphenylamino)-
3H-imidazo [4,5-b]pyridine
A-121 2-Methoxycarbonylamino-6-(4-methylphenylamino)-
3H-imidazo[4,5-b]pyridine
A-122 2-Methoxycarbonylamino-6- (2-fluorophenylamino) -
3H-imidazo[4,5-b]pyridine
A-123 2-Methoxycarbonylamino-6-(3-fluorophenylamino)-
3H-imidazo[4,5-b]pyridine
A-124 2-Methoxycarbonylamino-6-(4-fluorophenylamino)-
3H-imidazo[4,5-b]pyridine
A-125 2-Methoxycarbonylamino-6-[2-
(trifluoromethyl) phenylamino] -3H-imidazo[4,5-b]pyridine
A-126 2-Methoxycarbonylamino-6-[3-
(trifluoromethyl) phenylamino] -3H-imidazo[4,5-b]pyridine
A-127 2-Methoxycarbonylamino-6-[4-
(trifluoromethyl) phenylamino] -3H-imidazo[4,5-b]pyridine
A-128 2-Methoxycarbonylamino-6-(2-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-129 2-Methoxycarbonylamino-6- (3-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-130 2-Methoxycarbonylamino-6-(4-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-131 2-Methoxycarbonylamino-6-(2-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-132 2-Methoxycarbonylamino-6-(3-carboxyphenylamino)-
3H-imidazo [ 4, 5-b] pyridine
A-133 2-Methoxycarbonylamino-6-(4-carboxyphenylamino)-
3H-imidazo [ 4, 5-b] pyridine
Compounds A-134 - A-154.
By substituting the appropriate phenolate for sodium
phenolate in Example A-4, the identical process gives the
following examples. For examples A-152, A-153, and A-154,
the carboxy groups are protected by a methyl, ethyl,
benzyl, t-butyl ester or other suitable ester and then
deprotected in the last step to give the compounds.
A-134 2-Ethoxycarbonylamino-6-(2-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-135 2-Ethoxycarbonylamino-6-(3-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-136 2-Ethoxycarbonylamino-6-(4-nitrophenoxy)-3H-
imidazo[4,5-b]pyridine
A-137 2-Ethoxycarbonylamino-6-(2-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-138 2-Ethoxycarbonylamino-6-(3-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-139 2-Ethoxycarbonylamino-6-(4-chlorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-14 0 2-Ethoxycarbonylamino-6-(2-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-141 2-Ethoxycarbonylamino-6-(3-methylphenoxy)-3H-
imidazo[4,5-b]pyridine
A-142 2-Ethoxycarbonylamino-6-(4-methylphMenoxy)-3H
imidazo[4,5-b]pyridine
A-143 2-Ethoxycarbonylamino-6-(2-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-144 2-Ethoxycarbonylamino-6-(3-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-145 2-Ethoxycarbonylamino-6-(4-fluorophenoxy)-3H-
imidazo[4,5-b]pyridine
A-146 2-Ethoxycarbonylamino-6-[2-
(trifluoromethyl) phenoxy] -3H-imidazo[4,5-b]pyridine
A-147 2-Ethoxycarbonylamino-6-[3-
(trifluoromethyl)phenoxy]-3H-imidazo[4,5-b]pyridine
A-148 2-Ethoxycarbonylamino-6-[4-
(trifluoromethyl) phenoxy] -3H-imidazo[4,5-b]pyridine
A-149 2-Ethoxycarbonylamino-6-(2-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-150 2-Ethoxycarbonylamino-6-(3-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-151 2-Ethoxycarbonylamino-6-(4-methoxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-152 2-Ethoxycarbonylamino-6-(2-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
A-153 2-Ethoxycarbonylamino-6- (3-carboxyphenoxy) -3H-
imidazo[4,5-b]pyridine
A-154 2-Ethoxycarbonylamino-6-(4-carboxyphenoxy)-3H-
imidazo[4,5-b]pyridine
Compounds A-155 - A-176.
By substituting the appropriate thiophenolate for
sodium phenolate in Example A-4, the identical process
gives the following examples. For examples A-174, A-175,
and A-176, the carboxy groups are protected by a methyl,
ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
A-155 2-Ethoxycarbonylamino-6-phenylmercapto-3H-
imidazo[4,5-b]pyridine
A-156 2-Ethoxycarbonylamino-6-(2-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-157 2-Ethoxycarbonylamino-6-(3-nitrophenylmercapto) -
3H-imidazo[4,5-b]pyridine
A-158 2-Ethoxycarbonylamino-6-(4-nitrophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-159 2-Ethoxycarbonylamino-6-(2-chlorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-160 2-Ethoxycarbonylamino-6-(3-chlorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-161 2-Ethoxycarbonylamino-6-(4-chlorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-162 2-Ethoxycarbonylamino-6-(2-methylphenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-163 2-Ethoxycarbonylamino-6-(3-methylphenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-164 2-Ethoxycarbonylamino-6-(4-methylphMenoxy)-3H-
imidazo[4,5-b]pyridine
A-165 2-Ethoxycarbonylamino-6-(2-fluorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-166 2-Ethoxycarbonylamino-6-(3-fluorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-167 2-Ethoxycarbonylamino-6-(4-fluorophenylmercapto)-
3H-imidazo[4,5-b]pyridine
A-168 2-Ethoxycarbonylamino-6-[2-
(trifluoromethyl)phenylmercapto] 3H-imidazo[4,5-b]pyridine
A-169 2-Ethoxycarbonylamino-6- [3-
(trifluoromethyl) phenylmercapto] 3H-imidazo[4,5-b]pyridine
A-170 2-Ethoxycarbonylamino-6-[4-
(trif luoromethyl) phenylmercapto] 3H-imidazo[4,5-b]pyridine
A-171 2-Ethoxycarbonylamino-6-(2-
methoxyphenylmercapto) -3H-imidazo[4,5-b]pyridine
A-172 2-Ethoxycarbonylamino-6-(3-
methoxyphenylmercapto) -3H-imidazo[4,5-b]pyridine
A-173 2-Ethoxycarbonylamino-6-(4-
methoxyphenylmercapto) -3H-imidazo[4,5-b]pyridine
A-174 2-Ethoxycarbonylamino-6-(2-
carboxyphenylmercapto) -3H-imidazo[4,5-b]pyridine
A-175 2-Ethoxycarbonylamino-6-(3-
carboxyphenylmercapto) -3H-imidazo[4,5-b]pyridine
A-17 6 2-Ethoxycarbonylamino-6-(4-
carboxyphenylmercapto)-3H-imidazo[4,5-b]pyridine
Compounds A-177 - A-198.
By substituting the appropriate aniline salt for
sodium phenolate in Example A-4, the identical process
gives the following examples. For examples A-196, A-197,
and A-198, the carboxy groups are protected by a methyl,
ethyl, benzyl, t-butyl or other suitable ester and then
deprotected in the last step to give the compounds.
A-177 2-Ethoxycarbonylamino-6-phenylamino-3H-
imidazo[4,5-b]pyridine
A-178 2-Ethoxycarbonylamino-6-(2-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-179 2-Ethoxycarbonylamino-6-(3-nitrophenylamino)-3H-
imidazo[4,5-b]pyridine
A-180 2-Ethoxycarbonylamino-6-(4-nitrophenylamino)-3H-
imidazo [4 , 5-Jb] pyridine
A-181 2-Ethoxycarbonylamino-6-(2-chlorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-182 2-Ethoxycarbonylamino-6-(3-chlorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-183 2-Ethoxycarbonylamino-6-(4-chlorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-184 2-Ethoxycarbonylamino-6-(2-methylphenylamino)-3H-
imidazo[4,5-b]pyridine
A-185 2-Ethoxycarbonylamino-6-(3-methylphenylamino)-3H-
imidazo[4,5-b]pyridine
A-186 2-Ethoxycarbonylamino-6-(4-methylphMenoxy)-3H-
imidazo[4,5-b]pyridine
A-187 2-Ethoxycarbonylamino-6-(2-fluorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-188 2-Ethoxycarbonylamino-6-(3-fluorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-189 2-Ethoxycarbonylamino-6-(4-fluorophenylamino)-3H-
imidazo[4,5-b]pyridine
A-190 2-Ethoxycarbonylamino-6-[2-
(trif luoromethyl) phenylamino] -imidazo[4,5-b]pyridine
A-191 2-Ethoxycarbonylamino-6-[3-
(trif luoromethyl) phenylamino] 3H-imidazo[4,5-b]pyridine
A-192 2-Ethoxycarbonylamino-6-[4-
(trif luoromethyl) phenylamino] 3H-imidazo[4,5-b]pyridine
A-193 2-Ethoxycarbonylamino-6-(2-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-194 2-Ethoxycarbonylamino-6-(3-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-195 2-Ethoxycarbonylamino-6-(4-methoxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-196 2-Ethoxycarbonylamino-6-(2-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
A-197 2-Ethoxycarbonylamino-6-(3-carboxypheriylamino)-
3H-imidazo[4,5-b]pyridine
A-198 2-Ethoxycarbonylamino-6-(4-carboxyphenylamino)-
3H-imidazo[4,5-b]pyridine
EXAMPLE 2: ASSAY MEASURING PHOSPHORYLATING FUNCTION OF
RAF
The following assay reports the amount of RAF-
catalyzed phosphorylation of its target protein MEK as well
as MEK's target MAPK. The RAF gene sequence is described
in Bonner et al., 1985, Molec. Cell. Biol. 5: 1400-1407,
and is readily accessible in multiple gene sequence data
banks. Construction of the nucleic acid vector and cell
lines utilized for this portion of the .invention are fully
described in Morrison et al., 1988, Proc. Natl. Acad. Sci.
USA 85: 8855-8859.
Materials and Reagents
1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL,
Gaithersburg, MD.
2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl,
10 % glycerol, 1 mM PMSF, 5 mg/L Aprotenin, 0.5 % Triton X-
100;
3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK
expression and purification by affinity chromatography were
performed 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 was expressed
in XL1 Blue cells transformed with pUC18 vector encoding
His-MAPK. His-MAPK was purified by Ni-affinity
chromatography. Cat# 27-4949-01, Pharmacia, Alameda, CA,
as described herein.
5. Sheep anti mouse IgG: Jackson laboratories, West
Grove, PA. Catalog, # 515-006-008, Lot# 28563
6. RAF-1 protein kinase specflc antibody: URP2653
from UBI.
7. Coating buffer: PBS; phosphate buffered saline,
GIBCO-BRL, Gaithersburg, MD
8. Wash buffer: TBST - 50 mM Tris/HCL pH 7.2, 150 mM
NaCl, 0.1 % Triton X-100
9. Block buffer: TBST, 0.1 % ethanolamine pH 7.4
10. DMSO, Sigma, St. Louis, MO
11. Kinase buffer (KB): 20 mM Hepes/HCl pH 7.2, 150
mM NaCl, 0.1 % Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin,
75 µM sodium ortho vanadate, 0.5 MM DTT and 10 mM MgCl2.
12. ATP mix: 100 mM MgCl2, 300 µM ATP, 10 µCi ?-33P
ATP (Dupont-NEN)/mL.
13 Stop solution: 1 % phosphoric acid; Fisher,
Pittsburgh, PA.
14. Wallac Cellulose Phosphate Filter mats; Wallac,
Turku, Finnland.
15. Filter wash solution: 1 % phosphoric acid,
Fisher, Pittsburgh, PA.
16. Tomtec plate harvester, Wallac, Turku, Finnland.
17. Wallac beta plate reader # 1205, Wallac, Turku,
Finnland.
18. NUNC 96-well V bottom polypropylene plates for
compounds Applied Scientific Catalog # AS-72092.
Procedure
All of the following steps were conducted at room
temperature unless specifically indicated.
1. ELISA plate coating: ELISA wells are coated with
100 (aLof Sheep anti mouse affinity purified antiserum (1
µg/100 µL coating buffer) over night at 4 °C. ELISA plates .
can be used for two weeks when stored at 4 °C.
2. Invert the plate and remove liquid. Add 100 µL
of blocking solution and incubate for 30 min.
3. Remove blocking solution and wash four times with
wash buffer. Pat the plate on a paper towel to remove
excess liquid.
4. Add 1 µg of antibody specific, for RAF-1 to each
well and incubate for 1 hour. Wash as described in step 3.
5. Thaw lysates from RAS/RAF infected Sf9 cells and
dilute with TBST to 10 µg/100 µL. Add 10 µg of diluted
lysate to the wells and incubate for 1 hour. Shake the
plate during incubation. Negative controls receive no
lysate. Lysates from RAS/RAF infected Sf9 insect cells are
prepared after cells are infected with recombinant
baculoviruses at a MOI of 5 for each virus, and harvested
48 hours later. The cells are washed once with PBS and
lysed in RIPA buffer. Insoluble material is removed by
centrifugation (5 min at 10 000 x g). Aliquots of lysates
are frozen in dry ice/ethanol and stored at - 80 °C until
use.
6. Remove non-bound material and wash as outlined
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 kinase buffer. Methods
for purifying T-MEK and MAPK from cell extracts are
provided herein by example.
8. Predilute compounds (stock solution 10 mg/mL
DMSO) or extracts 20 fold in TBST plus 1% DMSO. Add 5 µL
of the prediluted compounds/extracts to the wells described
in step 6. Incubate for 20 min. Controls receive no drug.
9. Start the kinase reaction by addition of 5 µL ATP
mix; Shake the plates on an ELISA "plate shaker during
incubation.
10. Stop the kinase reaction after 60 min by addition
of 30 µL stop solution to each well.
11. Place the phosphocellulose mat and the ELISA
plate in the Tomtec plate harvester. Harvest and wash the
filter with the filter wash solution according to the
manufacturers recommendation. Dry the filter mats. Seal
the filter mats and place them in the holder. Insert the
holder into radioactive detection apparatus and quantify
the radioactive phosphorous on the filter mats.
Alternatively, 40 µL aliquots from individual wells of
the assay plate can be transferred to the corresponding
Positions on the phosphocellulose filter mat. After air-
drying the filters, put the filters in a tray. Gently rock
the tray, changing the wash solution at 15 min intervals
for 1 hour. Air-dry the filter mats. Seal the filter mats
and place them in a holder suitable for measuring the
radioactive phosphorous in the samples. Insert the holder
into a detection device and quantify the radioactive
phosphorous on the filter mats.
IC50 values were measured according to the protocol
for the following azabenzimidazole-based compounds in the
RAF-1 ELISA assay:
An IC50 value is the concentration of the azabenzimidazole-
based inhibitor required to decrease the maximum amount of
phosphorylated target protein or cell growth by 50%. The
IC50 values measured in the RAF-1 phosphorylation assay are
depicted in Table 1:
EXAMPLE 3: PURIFICATION OF MAPK AND MEK
The MAPK and MEK proteins are readily expressed in
cells by sublconing a gene encoding these proteins into a
commercially available vector that expresses the proteins
with a poly-Histidine tag. Genes encoding these proteins
are readily available from laboratories' that normally work
with these proteins or by cloning these genes from cells
containing cDNA libraries. The libraries are readily
commercially available and a person skilled in the art can
readily design nucleic acid probes homologous to cDNA
molecules encoding MEK or MAPK from the nucleic acid
sequences of MEK and MAPK, available in multiple gene data
bases such as Genbank. The cloning of a gene can be
accomplished in a short time period using techniques
currently available to persons skilled in the art.
Purification of the MEK and MAPK proteins from cell
extracts can be accomplished using the following protocol,
which is adapted from Robbins et al., 1993, J. Biol. Chem.
268: 5097-5106:
1. Lyse cells by sonication, osmotic stress, or
French Press techniques readily available to persons
skilled in the art. An appropriate sonication buffer is
provided below.
2. Equilibrate a solid support which is conjugated
with nickel or cobalt with the equilibration buffer
disclosed below. The poly-histidine tag specifically binds
to the nickel and cobalt atoms on the solid support.
Equilibration can be achieved by washing the resin three
times with a volume of the equilibration buffer equal to
ten times the volume of the solid support. The solid
support is readily available to persons of ordinary skill
in the art.
3. Add the cell lysate to the solid support and
equilibrate in a vessel for a period of time.
Alternatively, the solid support can be packed within a
protein chromatography column and the lysate may be flowed
through the solid support.
4. Wash the solid support with the wash buffer
disclosed below.
5. Elute the MEK or MAPK protein from the solid
support with an amount of elution buffer (provided below)
that removes a significant portion of the protein from the
solid support.
EXAMPLE 4: ASSAY MEASURING PHOSPHORYLATING FUNCTION OF
EGF RECEPTOR
EGF Receptor kinase activity (EGFR-NIH3T3 assay) in
whole cells was measured as described in detail in PCT
Publication WO9640116, filed 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.
The IC50 values measured in the EGF receptor
phosphorylation assay are depicted in Table 2:
EXAMPLE 5: ASSAY MEASURING THE EFFECT OF
AZABENZIMIDAZQLE-BASED COMPOUNDS ON THE
GROWTH OF CELLS EXPRESSING RAS
The following assay measures growth rates fo'r NIH-3T3
cells expressing RAS. The purpose of the assay is to
determine the effects of compounds on the growth of NIH 3T3
cells over expressing H-Ras.
Cell line:
3T3/H-Ras (NIH 3T3 clone 7 cells expressing genomic
fragment of oncogenic H-Ras).
The cells can be constructed using the following
protocol:
1. Subclone a gene fragment encoding Ras into a
commercially available vector that will stably transfect
NIH-3T3 cells. The fragment is from the genomic
transforming allele of cHa-ras.
2. Transfect NIH-3T3 cells with the subcloned vecto
by a calcium phosphate method. Select cells expressing th
Ras construct in 2% serum in DMEM. Visible foci are
observed after 2 weeks. Pool the transformed cells to
generate a stably transformed cell line.
Growth medium:
2% calf serum/DMEM + 2 mM glutamine, Pen/Strep
Protocol:
Day 0: Cell Plating:
This part of assay is carried out in a laminar flow hood.
1. Trypsinize cells. Transfer 200 µL of cell
suspension to 10 mL of isotone. Count cells with a Coulte:
Counter.
2. Dilute cells in growth medium to 60,000 cell/mL.
Transfer 100 |iL of cells to each well in a 96-well flat
bottom plate to give 6000 cells/well.
3. Use half of plate (4 rows) for each comound and
quadruplicate wells for each comound concentration, and a
set of 4 wells for medium control.
4. Gently shake plates to allow for uniform
attachment of the cells.
5. Incubate the plates at 37 °C in a 10% CO2
incubator.
Day 1: Addition of Compound:
This part of assay is carried out in a laminar flow hood.
1. In a 96-well round bottom plate, add 120 µL of
growth medium containing 2X final % DMSO found in highest
screening concentration of compound to columns 1 to 11.
For example, if the highest concentration is 100 µL, and
this is made from a 100 mM stock, 1X DMSO is 0.1%, so 2X
DMSO is 0.2%. This plate is used to titrate out the
compound, 4 rows per compound.
2. In a sterile 15 mL tube, make a 2X solution of
the highest screening concentration of compound in growth
medium plus 2X DMSO. 1 mL per cell line is needed. The
starting concentration of the compound is usually 100 µM
but this concentration may vary depending upon the
solubility of the compound.
3. Transfer 240 µL of the 2X starting compound
solution to qudruplicate wells in column 12 of the 96-well
round bottom plate. Do 1:2 serial dilutions across the
plate from right to left by transferring 12 µL from column
12 to column 11, column 11 to 10 and so on through column
2. Transfer 100 µL of compound dilutions, and 100 µL of
medium in column 1, onto 100 µL medium on cells in
corresponding wells of 96-well flat bottom plate. Total
volume per well should be 200 µL.
4. Return the plate to the incubattor and incubate
for 3 days.
Day 4: Development of Assay
This party of assay is carried out on the bench.
1. Aspirate or pour off medium. Add 200 µL cold 10%
TCA to each well to fix cells. Incubate plate for at least
60 min. at 4 °C.
2. Discard TCA and rinse wells 5 times with tap
water. Dry plates upside down on paper towels.
3. Stain cells with 100 µL/well 0.4% SRB for 10 min.
4. Pour of SRB and rinse wells 5 times with 1%
acetic acid. Dry plates completely upside down on paper
towels.
5. Solubilize dye with 100 µL/well 10 mM Tris base
for 5-10 min. on shaker.
6. Read plates on Dynatech ELISA Plate REader at 570
nm with reference at 630 nm.
Select compounds inhibited the growth rate of cells
over-expressing RAS as illustrated in Table 3.
EXAMPLE 6: ASSAY MEASURING EFFECT OF AZABENZIMIDAZOLE-
BASED COMPOUNDS ON GROWTH OF A54 9 CELLS
The following assay measures growth rates for A549
cells. The purpose of the assay is to determine the
effects of compounds on the growth of A54 9 human lung
carcinoma cells. A54 9 cells are readily-accessible from
commercial sources, such as ATCC (CCL185)•
Materials:
96-well flat bottom sterile plates
96-well round bottom sterile plates
sterile 25 mL or 100 mL reservoir
pipets, multi-channel pipetman
sterile pipet tips
sterile 15 mL and 50 mL tubes
Reagents:
0.4% SRB in 1% acetic acid
l0mM Tris base
10% TCA
1% acetic acid
sterile DMSO (Sigma)
compound in DMSO (100 mM or less stock solution)
Trypsin-EDTA (GIBCO BRL)
Cell line and growth medium:
A549 human lung carcinoma cells (ATCC CCL185)
10% fetal calf serum in Ham's F12-K
Protocol:
Day 0: Cell Plating:
This part of assay is carried out in a laminar flow hood.
1. Trypsinize cells. Transfer 200 uL of cell
suspension to 10 mL of isotone. Count cells with a Coulter
Counter.
2. Dilute cells in growth medium to 20,000 cell/mL.
Transfer 100 uL of cells to each well in a 96-well flat
bottom plate to give 2000 cells/well.
3. Use half of plate (4 rows) for each compound and
quadruplicate wells for each compound concentration, and a
set of 4 wells for medium control.
4. Gently shake plates to allow for uniform
attachment of the cells.
5. Incubate the plates at 37°C in a 10% CO2
incubator.
Day 1: Addition of Compound:
This part of assay is carried out in a laminar flow hood.
1. In a 96 well-round bottom plate, add 120 uL of
growth medium containing 2X final %DMSO found in highest
screening concentration of compound to columns 1 to 11.
For example, if the highest screening concentration is 100
µM, and this is made from a lOOmM stock, IX DMSO is 0.1%,
so 2X DMSO is 0.2%. This plate is used to titrate out the
compound, 4 rows per compound.
2. In a sterile 15 mL tube, make a 2X solution of
the highest screening concentration of compound in growth
medium plus 2X DMSO. 1 mL per cell line is needed. The
starting concentration of the compound is usually 100 pM
but this concentration may vary depending upon the
solubility of the compound.
3. Transfer 240 uL of the 2X starting compound
solution to quadruplicate wells in column 12 of the 96-well
round bottom plate. Do 1:2 serial dilutions across the
plate from right to left by transferring 120 uL from column
12 to column 11, column 11 to 10 and so on through column
2. Transfer 100 uL of compound dilutions, and 100 uL of
medium in column 1, onto 100 uL medium on cells in
corresponding wells of 96-well flat bottom plate. Total
volume per well should be 200 uL.
4. Return the plate to the incubator and incubate
for 3 days.
Day 5: Development of Assay
This part of assay is carried out on the bench.
1. Aspirate or pour off medium. Add 200 uL cold 10%
TCA to each well to fix cells. Incubate plate for at least
60 min. at 4°C.
2. Discard TCA and rinse wells 5 times with tap
water. Dry plates upside down on paper towels.
3. Stain cells with 100 uL/well 0.4% SRB for 10 min.
4. Pour off SRB and rinse wells 5 times with 1%
acetic acid. Dry plates completely upside down on paper
towels.
5. Solubilize dye with lOOµL/well 10 mM Tris base
for 5-10 min. on shaker.
6. Read plates on Dynatech ELISA Plate Reader at 570
nm with reference at 630 nm.
Select compounds inhibited the growth rates of A54 9
cells, as illstrated in Table 4.
EXAMPLE 7: METHOD FOR DETERMINING THE BIOLOGICAL
ACTIVITY OF RAF MODULATORS IN VTVO
Xenograft studies can be utilized to monitor the
effect of compounds of the invention upon the inhibition of
ovarian, melanoma, prostate, lung and mammary tumor cells.
The protocol for the assay is described in detail in PCT
Publication WO9640116, filed 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.
The invention illustratively described herein may be
practiced in the absence of any element or elements,
limitation or limitations which is not specifically
disclosed herein. The terms and expressions which have
been employed are used as terms of description and not of
limitation, and there is no intention that in the use of
such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but
it is recognized that various modifications are possible
within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments and
optional features, modification and variation of the
concepts herein disclosed may be resorted to by those
skilled in the art, and that such modifications and
variations are considered to be within the scope of this
invention as defined by the appended claims.
One skilled in the art would readily appreciate that
the present invention is well adapted to carry out the
objects and obtain the ends and advantages mentioned, as
well as those inherent therein. The molecular complexes
and the methods, procedures, treatments, molecules,
specific compounds described herein are presently
representative of preferred embodiments are exemplary and
are not intended as limitations on the scope of the
invention. Changes therein and other uses will occur to
those skilled in the art which are encompassed within the
spirit of the invention are defined by the scope of the
claims.
It will be readily apparent to one skilled in the art
that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the
scope and spirit of the invention.
All patents and publications mentioned in the
specification are indicative of the levels of those skilled
in the art to which the invention pertains. All patents
and publications are herein incorporated by reference to
the same extent as if each individual publication was
specifically and individually indicated to be incorporated
by reference.
The invention illustratively described herein suitably
may be practiced in the absence of any element or elements,
limitation or limitations which is not specifically
disclosed herein. Thus, for example, in each instance
herein any of the terms "comprising", "consisting
essentially of" and "consisting of" may be replaced with
either of the other two terms. The terms and expressions
which have been employed are used as terms of description
and not of limitation, and there is no intention that in
the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed.
Thus, it should be understood that although the present
invention has been specifically disclosed by preferred
embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted
to by those skilled in the art, and that such modifications
and variations are considered to be within the scope of
this invention as defined by the appended claims.
In addition, where features or aspects of the
invention are described in terms of Markush groups, those
skilled in the art will recognize that the invention is
also thereby described in terms of any individual member or
subgroup of members of the Markush group. For example, if
X is described as selected from the group consisting of
bromine, chlorine, and iodine, claims for X being bromine
and claims for X being bromine and chlorine are fully
described.
Those references not previously incorporated herein by
reference, including both patent and non-patent references,
are expressly incorporated herein by reference for all
purposes. Other embodiments are within the following
claims.
WE CLAIM :
1. Azabenzimidazole compound having the formula set forth in formula I:
wherein,
(a) R1, R2, and R3 are independently selected from the group consisting of:
(i) unsaturated alkyl;
(ii) —NX2X3, where X2 and X3 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and
heterocyclic ring moieties;
(iii) halogen or trihalomethyl;
(iv) a ketone of formula -CO-X4. where X4, is selected from the group
consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic
ring moieties;
(v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X5)n-
COO-X7, wherein X5, X6, and X7, and are independently selected from the
group consisting of alkyl, homocyclic ring moieties, and heter'ocyclic ring
moieties, and wherein n. is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O-
X9, wherein X8 and X9 are independently selected from the group consisting
of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring moieties, and wherein the ring is optionally
substituted with one or' more substituents independently selected from the
group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate,
nitro, and ester, and wherein n is 0 or 1;
(vii) an amide of formula —NHCOX10, wherein X10 is selected from the group
consisting of alkyl, hydroxyJ, homocyclic ring moieties, and heterocyclic
ring moieties, and wherein the ring is optionally substituted with one or
more substituents independently selected from the group consisting of
alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro. and ester;
(viii) —SO2NX11X12, wherein Xn and X12 are selected from the group consisting
of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring
moieties;
(ix) a homocyclic or heterocyclic ring moiety optionally substituted with one,
two, or' three substituents independently selected from the group
consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and
ester' moieties;
(x) an aldehyde of formula —CO-H; and
(xi) a sulfone of formula --SO2-X13, where X13 is selected from the group
consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring, moieties;
(b) Z1 and Z2 are NH; and
(c) X1 is independently selected fl'om the group consisting of NH, sulfur, and
oxygen.
2. A pharmaceutical composition comprising an azabenzimidazole-based compound
as claimed in claim 1, or salt thereof, and a physiologically acceptable carrier or' diluent.
3. A pharmaceutical composition for preventing or treating a cell proliferative
disorder in an organism, comprising the azabenzimidazole compound as claimed in claim
1, or a salt thereof, and a physiologically acceptable carrier or diluent,wherein said cell
proliferative is associated with an aberration in a signal transduction pathway
characterized by an interaction between the setine/threonine protein kinase RAF and a
natural binding partner.
4. The pharmaceutical composition as claimed in claim 3, wherein said organism is
a mammal.
5. The pharmaceutical composition as claimed in claim 4, wherein said cell
proliferative disorder is selected from the group consisting of cancer, fibrotic disorder,
mcsangial disorders, abnormal angiogenesis, abnormal vasculogenesis, wound healing,
psoriasis, restenosis. and inflammation.
6. The pharmaceutical composition as claimed in claim 5, wherein said cancer is
selected from the group consisting of lung cancer, ovarian cancer, breast cancer, brain
cancer, intra-axial brain cancer, colon cancer, prostate cancer, sarcoma, Kaposi 's
sarcoma, melanoma, and glioma.
7. The pharmaceutical composition as claimed in claim 6, wherein said cell
proliferative disorder is associated with an aberration in a signal transduction pathway
characterized by an interaction between serine/threonine protein kinase and a natural
binding protein.
8. An azabenzimidazole compound having a structure as set forth in formula II or
III:
wherein
(a) R1, R2, R3, and R4 are independently selected from the group consisting of:
(i) hydrogen;
(ii) saturated or unsaturated alkyl;
(ii) NX2X3, where X2 and X3 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic or
heterocyclic ring moieties;
(iii) halogen or trihalomethyl;
(iv) a ketone of formula -CO—X4, where X4 selected from the group
consisting of hydrogen, alky], homocyclic ring moieties and. heterocyclic
ring moieties;
(v) a caxboxylic acid of formula —(X5)n-COOH or ester of formula --(X6)n-
COO-X7, wherein X5, X6, and. X7, and are independently selected from the
group consisting of alkyl, homocyclic ring moieties, and heterocyclic ring
moieties, and wherein n is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O-
X9, wherein X8 and X9 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring
moieties, and heterocyclic ring moieties, and wherein the ring is optionally
substituted with one or more substituents independently selected from the
group consisting of alkyl, alkoxy, halogen, txihalomethyl, carboxylate,
nitro, and ester, and wherein n is 0 or 1;
(vii) an amide of formula —NHCOX10, wherein X10 is selected from the group
consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic
ring moieties, and wherein the ring is optionally substituted with one or
more substituents independently selected from the group consisting of
alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro. and ester;
(viii) -SO2NX1 1X12, wherein X11 and X12 are selected from the group consisting
of hydrogen, alkyl. and Homocyclic ring moieties, and heterocyclic ring
moieties;
(ix) a homocyclic or heterocyclic ring moiety optionally substituted with one,
two, or three substituents independently selected from the group consisting
of alkyl, alkoxy, halogen, trihalomethyl, car'boxylate. nitro, and ester
moieties;
(x) an aldehyde of formula —CO-H; and
(xi) a sulfone of formula —SO2,X13, where X13 is selected from the group
consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring, moieties;
(b) Z1 and Z2 are independently selected from the group consisting of nitrogen, NH,
and NR4; and
(c) Z3 and X1 arc independently selected from the group consisting of NH, sulfur, and
oxygen.
9. A pharmaceutical composition comprising an azabenzimidazole-based compound
as claimed in claim 8, or salt thereof, and a physiologically acceptable carrier or diluent.
10. A pharmaceutical composition for preventing or treating a cell prolifcrative
disorder in an organism, comprising the azabenzimidazole compound as claimed in claim
8, or a salt thereof, and a physiologically acceptable carrier or diluent, wherein said cell
proliferative is associated with an aberration in a signal transduction pathway
characterized by an interaction between the serine/threonine protein kinase RAF and a
natural binding partner.
11. The pharmaceutical composition as claimed in claim 10, wherein said organism is
a mammal.
12. The pharmaceutical composition as claimed in claim 11, wherein said cell
proliferative disorder is selected from, the group consisting of cancer, fibrotic disorder,
mesangial disorders, abnormal angiogenesis, abnormal vasculogenesis, wound, healing,
psoriasis, restenosis, and inflammation.
13. The pharmaceutical composition as claimed in claim 12, wherein said cancer is
selected flom the group consisting of lung cancer, ovarian cancer, breast cancer, brain
cancer, intra-axial brain cancer, colon cancer, prostate cancer, sarcoma, Kaposi's
sarcoma, melanoma, and glioma.
14. The pharmaceutical composition as claimed in claim 13, wherein said cell
proliferative disorder is associated with an aberration in a signal transduction pathway
characterized by an interaction between serine/threonine protein kinase and a natural
binding protein.
15. A method of modulating the function of a serine/threonine protein kinase with an
azabenzimidazole based compound in vitro said compound having the formula set forth
in formula I:
wherein,
(a) R1, R2, and R3 are independently selected from the group consisting of:
(i) unsaturated alkyl;
(ii) —NX2X3, where X2 and X3 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring
moieties, and heterocyclic ring moieties;
(iii) halogen or trihalomethyl;
(iv)- a ketone of formula -CO-X4. where X4, is selected from the group
consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic
ring moieties;
(v) a carboxylic acid of formula —(X5)nCOOH or ester of formula —(X6)n-
COO-X7, wherein X5, X6, and X7, and are independently selected from the
group consisting of alkyl, homocyclic ring moieties, and hetero cyclic ring
moieties, and wherein n is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula -(X8)n-O-
X9, wherein X8 and X9 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring
moieties, and heterocyclic ring moieties, and wherein the ring is optionally
substituted with one or more substituents independently selected from the
group consisting of alkyl. alkoxy, halogen, trihalomethyl, carboxylate,
nitro, and ester, and wherein n is 0 or 1;
(vii) an amide of formula —NHCOX10, wherein X10 is selected from the group
consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic
ring moieties, and wherein (he ring is optionally substituted with one or
more substituents independently selected from the group consisting of
alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester;
(viii) —SO2NX11X12, wherein X11 and X12 are selected from the group consisting
of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring
moieties;
(ix) a homocyclic or heterocyclic ring moiety optionally substituted with one,
two, or three substituents independently selected from the group consisting
of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro. and ester
moieties;
(x) an aldehyde of formula —CO-H; and
(xi) a sulfone of formula -SO2-X13, where X13 is selected, from the group
consisting of saturated alkyl, unsaturated alkyl. homocyclic ring moieties,
arid heterocyclic ring moieties;
(b) Z1 and Z2 are NH; and
(c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen,
comprising the steps of contacting the cells expressing said serme/threonine protein
kinase with said azabenzimida.zole based compound.
16. The method as claimed in claim 15, wherein said serine/threonine protein is
17. A method of modulating the function of a serine/threonine protein kinase with an
azabenzimidazole-based compound in vitro, said compound having the formula as set
forth in formula II or III:
wherein
Ri, R2, R3, and R4 are independently selected from the group consisting of:
(i) hydrogen;
(ii) saturated or unsaturated alkyl;
(ii) NX2X3, where X2 and X3 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic or
heterocyclic ring moieties;
(iii) halogen or trihalomethyl;
(iv) a ketone of formula —CO—X4, where X4 is selected from the group
consisting of hydrogen., alkyl, homocyclic ring moieties and heterocyclic
ring moieties;
(v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X6)n-
COO-X7, wherein X5, X6, and X7, and are independently selected from the
group consisting of alkyl, hornocyciic ring moieties, and heterocyclic ring
moieties, and wherein, n. is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O-
X9, wherein X8 and X9 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring
moieties, and heterocyclic ring moieties, and wherein the ring is optionally
substituted with one or more substituents independently selected from the
group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate,
nitro, and ester, and wherein n is 0 or 1;
(vii) an amide of formula —NHCOX10, wherein X10 is selected from the group
consisting of alkyl hydroxyl, homocyclic ring moieties, and heterocyclic
ring moieties, and wherein the ring is optionally substituted with one or
more substituents independently selected from the group consisting of
alkyl, alkoxy. halogen, tribalomethyl, carboxylate, nitro, and ester;
(viii) -SO2NX11X12, wherein X11 and X12 are selected from the group consisting
of hydrogen, alkyl, and honiocyclic ring moieties, and heterocyclic ring
moieties;
(ix) a homocyclic or heterocyclic ring moiety optionally substituted with one,
two, or three substituents independently selected from the group consisting
of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester
moieties;
(x) an aldehyde of formula --CO-H; and
(xi) a sulfone of formula -SO2 -X13, where X13 is selected from the group
consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring moieties;
(b) Z1 and 4 arc independently selected from the group consisting of nitrogen, NH.
and NR4; and
(c) Z3 and X1 are independently selected from the group consisting of NH, sulfur, and
oxygen, comprising the steps of contacting the cells expressing said serine/threonine
protein kinase with said azabenzimidazole based compound.
18. The method as claimed in claim 1 7, wherein said serine/threonine protein is RAF.
19. A method for synthesizing a compound having a structure as set forth in formula
'I, II or III, and having been defined in claim 1 or 8, said method comprising the steps of:
(a) reacting 2-amino-6-chloro-3-nittOpyridine with a second reactant in a solvent,
yielding a first intermediate, wherein said second reactant is selected from the group
consisting of a substituted phenol, substituted thiophenol, and substituted aniline;
(b) reducing the said first intermediate in the presence of a catalyst and a reducing
agent, yielding a second intermediate:
(c) reacting the second intermediate with a third reactant; and
(d) purifying the compound, so obtained.
20. The method as claimed in claim 19. wherein said second reactant is selected from
the group consisting of phenol, 2-nitrophenol. 3-nitrophenol, 4-nitrophenol, 2-
chlor'ophenol, 3 -chlorophenol, 4-chlorophenol, 2-cresol, 3-cresol, 4-cresol, 2-
nuor'ophenol, 3-fluorophenol, 4-fluorophenol, 2.-(trifluoromethyl) phenol, 3-
(trifiuoromethyl) phenol, 4-(trifluoromethyl) phenol, 2-methoxyphenol, 3-
methoxyphenol, 4-.methoxyphenol, thiophenol, 2-hydroxybenzoic acid, 3-
hydroxybenzoic acid, 4-hydroxybenzoic acid, thiophenol, 2-nitrothiophenol, 3-
nitrothiophenol, 4-nitrothiophenol, 2-chlorothiophenol, 3 -chlorothiophenol, 4-
chlorothiophenol, 2-thiocresol, 3 -thiocresol, 4-thiocresol, 2-fluorothiophenol, 3-
fluorothiophenol, 4-fluorothiophenol 2-(1rifluoromethyl)thiophenol, 3.-(trifluor'omethyl)
thiophenol, 4-(trifluoromethyl) thiophenol, 2-methoxybenzenethiol, 3-
methoxybenzenethiol, 4-methoxybenzene1hiol, 2-met captobenzoic acid, 3-
mercaptobenzoic acid, 4-mercaptobenzoic acid, aniline, 2-nitroaniline, 3»-nitroaniline, 4-
nitroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, 2-toluidine, 3-toluidine, 4-
toluidine. 2-fluoroaniline, 3-fluoronaniline, 4-fluoronaniline, 2-(trifluorornethyl) aniline,
3 (trifluoromethyl) aniline, 4-(trilfl.uoroinethyl) aniline, 2-anisidine, 3-amisidine, 4-
anisidine, 2-aminobenzoic acid, 3-aminobenzoic acid, and 4-aminobenzoic acid.
21. The method as claimed in claim 19, wherein said reducing agent is hydrogen.
22. The method as claimed in claim 19. wherein said catalyst is Raney nickel.
23. A pharmaceutical composition, having a compound as defined in claim 1 or 8,
substantially as herein described, particularly with reference to the foregoing examples.
24. A method for synthesising a compound, as defined in claim 1 or 8, substantially as
herein described, particularly with reference lo the foregoing examples..
25. A method of modulating the function of serine/threonine protein kinase,
substantially as herein described, particularly with reference to the foregoing examples.
The present invention provides an azabenzimidazole compound having the
formula set forth in formula I:
wherein,
(a) R1, R2, and R3 are independently selected from the group consisting of:
(i) unsaturated alkyl;
(ii) —NX2X3, where X2 and X3 are independently selected from the group
consisting of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties, and
heterocyclic ring moieties;
(iii) halogen or trihalomethyl;
(iv) a ketone of formula -CO-X4, where X4, is selected from the group
consisting of hydrogen, alkyl, homocyclic ring moieties, and heterocyclic
ring moieties;
(v) a carboxylic acid of formula —(X5)n-COOH or ester of formula —(X6)n-
COO-X7, wherein X5, X6, and X7, and are independently selected from the
group consisting of alkyl, homocyclic ring moieties, and heter'ocyclic ring
moieties, and wherein n is 0 or 1;
(vi) an alcohol of formula (X8)n-OH or an alkoxy moiety of formula —(X8)n-O-
X9, wherein X8 and X9 are independently selected from the group consisting
of hydrogen, saturated alkyl, unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring moieties, and wherein the ring is optionally
substituted with one or' more substituents independently selected from the
group consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate,
nitro, and ester, and wherein n is 0 or 1;
(vii) an amide of formula —NHCOX10, wherein X10 is selected from the group
consisting of alkyl, hydroxyl, homocyclic ring moieties, and heterocyclic
ring moieties, and wherein the ring is optionally substituted with one or
more substituents independently selected from the group consisting of
alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and ester;
(viii) —SO2NX11X12, wherein Xn and X12 are selected from the group consisting
of hydrogen, alkyl, and homocyclic ring moieties, and heterocyclic ring
moieties;
(ix) a homocyclic or heterocyclic ring moiety optionally substituted with one,
two, or' three substituents independently selected from the group
consisting of alkyl, alkoxy, halogen, trihalomethyl, carboxylate, nitro, and
ester' moieties;
(x) an aldehyde of formula —CO-H; and
(xi) a sulfone of formula --SO2-X13, where X13 is selected from the group
consisting of saturated alkyl, unsaturated alkyl, homocyclic ring moieties,
and heterocyclic ring moieties;
(b) Z1 and Z2 are NH; and
(c) X1 is independently selected from the group consisting of NH, sulfur, and oxygen.

Documents:

01722-cal-1998-abstract.pdf

01722-cal-1998-assignment.pdf

01722-cal-1998-claims.pdf

01722-cal-1998-correspondence.pdf

01722-cal-1998-description (complete).pdf

01722-cal-1998-form 1.pdf

01722-cal-1998-form 18.pdf

01722-cal-1998-form 2.pdf

01722-cal-1998-form 3.pdf

01722-cal-1998-form 5.pdf

01722-cal-1998-form 6.pdf

01722-cal-1998-letter patent.pdf

01722-cal-1998-pa.pdf

01722-cal-1998-priority document.pdf

01722-cal-1998-reply f.e.r.pdf

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Patent Number

211434

Indian Patent Application Number

1722/CAL/1998

PG Journal Number

44/2007

Publication Date

02-Nov-2007

Grant Date

29-Oct-2007

Date of Filing

24-Sep-1998

Name of Patentee

ZENTARIS GMBH

Applicant Address

WEISMULLERSTRASSE 50, 60314 FRANKFURT/MAIN

Inventors:

#	Inventor's Name	Inventor's Address
1	GERALD MCMAHON	1414, GREENWICH STREET, SAN FRANCISCO, CALIFORNIA 94109
2	HEINZ WEINBERGER	AM HOLZWEG 3, D-65843, SULZBACH, TS
3	BERNHARD KUTSCHER	STRESENMANNSTRASSE 9, D-63477, MAINTAL
4	HARALD APP	630 27TH STREET, SAN FRANCISCO, CALIFORNIA 94131

PCT International Classification Number

A 61 K 7/4745

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/060,145	1997-09-26	U.S.A.