# Patent

## Title
Therameutin modulators

## Abstract

This invention relates to agents that are inhibitors or activators of variant forms of endogenous proteins and novel methods of identifying such variants. Of particular interest arc inhibitors and activators of endogenous protein variants, encoded by genes which have mutated, which variants often arise or are at least first identified as having arisen following exposure to a chemical agent which is known to be an inhibitor or activator of the corresponding unmutated endogenous protein.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This is the national phase application of PCT Application No. PCT/US2005/018412, filed May 23, 2005, and claims priority to U.S. Ser. No. 60/633,013, filed Dec. 3, 2004, and U.S. Ser. No. 60/573,962, filed May 23, 2004.


BACKGROUND OF THE INVENTION
The progressive development of drug resistance in a patient is the hallmark of chronic treatment with many classes of drugs, especially in the therapeutic areas of cancer and infectious diseases. Molecular mechanisms have been identified which mediate certain types of drug resistance phenomena, whereas in other cases the mechanisms of acquired as well as de novo resistance remain unknown today.
One mechanism of induced (acquired) drug resistance originally thought to be relevant in the area of cancer therapy involves increased expression of a protein known as P-glycoprotein (P-gp). P-gp is located in the cell membrane and functions as a drug efflux pump. The protein is capable of pumping toxic chemical agents, including many classical anti-cancer drugs, out of the cell. Consequently, upregulation of P-glycoprotein usually results in resistance to multiple drugs. Upregulation of P-glycoprotein in tumor cells may represent a defense mechanism which has evolved in mammalian cells to prevent damage from toxic chemical agents. Other related drug resistance proteins have now been identified with similar functions to P-gp, including multidrug-resistance-associated protein family members such as MRP1 and ABCG2. In any event, with the advent of the development of compounds that are specific for a given target protein, and less toxic, the importance of P-glycoprotein and related ATP-binding cassette (ABC) transporter proteins in clinically significant drug resistance has lessened.
Another possible molecular mechanism of acquired drug resistance is that alternative signal pathways are responsible for continued survival and metabolism of cells, even though the original drug is still effective against its target. Furthermore, alterations in intracellular metabolism of the drug can lead to loss of therapeutic efficacy as well. In addition, changes in gene expression as well as gene amplification events can occur, resulting in increased or decreased expression of a given target protein, and frequently requiring increasing dosages of the drug to maintain the same effects. (Adcock and Lane, 2003)
Mutation induced drug resistance is a frequently occurring event in the infectious disease area. For example, several drugs have been developed that inhibit either the viral reverse transcriptase or the viral protease encoded in the human immunodeficiency (HIV) viral genome. It is well established in the literature that repeated treatment of HIV-infected AIDS patients using, for example, a reverse transcriptase inhibitor eventually gives rise to mutant forms of the virus that have reduced sensitivity to the drug which resulted from mutations that have occurred in the gene encoding reverse transcriptase that render the mutant form of the enzyme less affected by the drug.
The appearance of drug resistance during the course of HIV treatment is not surprising considering the rate at which errors are introduced into the HIV genome. The HIV reverse transcriptase enzyme is known to be particularly error prone, with a forward mutation rate of about 3.4×10−5 mutations per base pair per replication cycle (Mansky et al., J. Virol. 69:5087-94 (1995)). However, analogous mutation rates for endogenous genes encoded in mammalian cells are more than an order of magnitude lower.
New evidence shows that drug resistance can also arise from a mutational event involving the gene encoding the drug target (Gorre et al., Science, 2001; PCT/US02/18729). In this case, exposure of the patient to a specific therapeutic substance such as a given cancer drug that targets a specific protein-of-interest (POI, or “target” protein) may be followed by the outgrowth of a group of cells harboring a mutation occurring in the gene encoding the protein that is the target of the therapeutic substance. Whether the outgrowth of this population of cells results from a small percentage of pre-existing cells in the patient which already harbor a mutation which gives rise to a drug-resistant POI, or whether such mutations arise de novo during or following exposure of the animal or human being to a therapeutic agent capable of activating or inhibiting said POI, is presently unknown. In either case, such mutation events may result in a mutated protein (defined below as a theramutein) which is less affected, or perhaps completely unaffected, by said therapeutic substance.
Chronic myelogenous leukemia (CML) is characterized by excess proliferation of myeloid progenitors that retain the capacity for differentiation during the stable or chronic phase of the disease. Multiple lines of evidence have established deregulation of the Abl tyrosine kinase as the causative oncogene in certain forms of CML. The deregulation is commonly associated with a chromosomal translocation known as the Philadelphia chromosome (Ph), which results in expression of a fusion protein comprised of the BCR gene product fused to the Abelson tyrosine kinase, thus forming p210Bcr-Abl which has tyrosine kinase activity. A related fusion protein, termed p190Bcr-Abl, that arises from a different breakpoint in the BCR gene, and has been shown to occur in patients with Philadelphia chromosome positive (Ph+) Acute Lymphoblastic Leukemia (ALL) (Melo, 1994; Ravandi et al., 1999). Transformation appears to result from activation of multiple signal pathways including those involving RAS, MYC, and JUN. Imatinib mesylate (“STI-571” or “Gleevec®”) is a 2-phenylamino pyrimidine that targets the ATP binding site of the kinase domain of Abl (Druker et al, NEJM 2001, p. 1038). Subsequently it has also been found by other methods to be an inhibitor of platelet-derived growth factor (PDGF) β receptor, and the Kit tyrosine kinase, the latter of which is involved in the development of gastrointestinal stromal tumors (see below).
Until recently, it had not been observed that during the course of treatment with a specific inhibitor of a given endogenous cellular protein that a mutation in its corresponding endogenous gene could lead to the expression of protein variants whose cellular functioning was resistant to the inhibitor. Work by Charles Sawyers and colleagues (Gorre et al., Science 293:876-80 (2001); PCT/US02/18729) demonstrated for the first time that treatment of a patient with a drug capable of inhibiting the p210Bcr-Abl tyrosine kinase (i.e., STI-571) could be followed by the emergence of a clinically significant population of cells within said patient harboring a mutation in the gene encoding the p210Bcr-Abl cancer causing target protein which contains the Abelson tyrosine kinase domain. Various such mutations gave rise to mutant forms of p210Bcr-Abl which were less responsive to Gleevec treatment than was the original cancer causing version. Notably, the mutations that emerged conferred upon the mutant protein a relative resistance to the effects of the protein kinase inhibitor drug, while maintaining a certain degree of the original substrate specificity of the mutant protein kinase. Prior to Gorre et al.'s work, it was generally believed by those skilled in the art that the types of resistance that would be observed in patients exposed to a compound which inhibited the Abelson protein kinase, such as STI-571, would have resulted from one or more of the other mechanisms of drug resistance listed above, or by some other as yet unknown mechanism, but that in any event said resistance would involve a target (protein or otherwise) which was distinct from the drug's target POI.
Accordingly, the ability to treat clinically relevant resistant mutant forms of proteins that are otherwise the targets of an existing therapy would be extremely useful. Such mutated proteins (theramuteins as defined below) are beginning to be recognized and understood to be important targets in recurring cancers, and will become important in other diseases as well. There exists a need for therapeutic agents that are active against such drug resistant variant forms of cellular proteins that may arise before, during or following normally effective drug therapies. A key purpose of this invention is to provide compounds that may serve as potential therapeutic agents useful in overcoming mutation-induced drug resistance in endogenously occurring proteins.
BRIEF SUMMARY OF THE INVENTION
This invention relates to agents that are inhibitors or activators of variant forms of endogenous proteins and novel methods of identifying such variants. Of particular interest are inhibitors and activators of endogenous protein variants, encoded by genes which have mutated, which variants often arise or are at least first identified as having arisen following exposure to a chemical agent which is known to be an inhibitor or activator of the corresponding unmutated endogenous protein. Such protein variants (mutant proteins) are herein termed “theramuteins,” may occur either spontaneously in an organism (and be pre-existing mutations in some cases), or said mutants may arise as a result of selective pressure which results when the organism is treated with a given chemical agent capable of inhibiting the non-mutated form of said theramutein (herein termed a “prototheramutein”). It will be understood that in some cases a prototheramutein may be a “wild type” form of a POI (e.g., a protein that gives rise to a disease due to disregulation). In other cases, the prototheramutein will be a disease causing variant of a “wild type” protein, having already mutated and thereby contributing to the development of the diseased state as a result of said prior mutation. One example of the latter type of prototheramutein is the P210BCR-ABL oncoprotein, and a mutant form of this protein harboring a threonine (T) to isoleucine (I) mutation at position 315 is termed P210BCR-ABL-T315I and is one example of a theramutein. As used herein, the designation “P210BCR-ABL” is synonymous with the term “p210Bcr-Abl”, the “wild-type Bcr-Abl protein”, and the like.
Theramuteins are a rare class of endogenous proteins that harbor mutations that render said proteins resistant to drugs that are known to inhibit or activate in a therapeutically effective manner their non-mutated counterparts. The endogenous genes encoding a few such proteins are presently known to exhibit such mutations under certain circumstances. This Invention is directed toward compositions that inhibit certain drug-resistant mutants (theramuteins) of the Abelson tyrosine kinase protein, originally termed P210-Bcr-Abl in the literature, that is involved in the development of chronic myelogenous leukemia. The invention is also directed toward general methods of identifying compounds that inhibit or activate any theramutein.
The present method is particularly directed toward the identification of specific inhibitors or specific activators of theramuteins. Use of the term “specific” in the context of the terms “inhibitor” or “activator” (see definitions below) means that said inhibitor or activator binds to the theramutein and inhibits or activates the cellular functioning of the theramutein without also binding to and activating or inhibiting a wide variety of other proteins or non-protein targets in the cell. The skilled investigator is well aware that there is a certain degree of variability in the medical literature with respect to the concept of a specific inhibitor or a specific activator, and of the related concept of target protein “specificity” when discussing the actions of inhibitors or activators of a protein. Accordingly, for the purposes of this Invention, a substance is a specific inhibitor or a specific activator of a given theramutein if said substance is capable of inhibiting or activating said theramutein at a given concentration such that a corresponding phenoresponse is modulated in the appropriate manner, without having an appreciable effect at the same given concentration upon the phenoresponse of a corresponding control cell that essentially does not express either the theramutein or its corresponding prototheramutein.
In certain embodiments, a substance may be a modulator of the prototheramutein as well as the theramutein. In other embodiments, in addition to being a modulator of the prototheramutein and theramutein, a substance may also modulate the activities of proteins that have similar functions. As discussed above, in addition to inhibiting the p210Bcr-Abl tyrosine kinase, imatinib mesylate is also capable of inhibiting the c-kit oncogene product (also a tyrosine kinase) which is overexpressed in certain gastrointestinal stromal tumors, as well as the PDGF β receptor (also a tyrosine kinase), which is expressed in certain chronic myelomonocytic leukemias (CMML). Such a compound is sometimes termed a “moderately specific” inhibitor.
The invention also provides a general method that can be used to identify substances that will activate or inhibit a theramutein, to the same extent, and preferably to an even greater extent than a known drug substance is capable of inhibiting the corresponding “wild type” form of that protein. (The skilled artisan is well aware, however, that said “wild type” forms of such proteins may have already mutated in the course of giving rise to the corresponding disease in which said protein participates.)
In a preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula I




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pR12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R2 is selected from —CR21a—, —NR22b—, and —(C═R23)—;
    
        each R21 is independently selected from H, halo, —NH2, —N(H)(C1-3 alkyl), —N(C1-3 alkyl)2, —O—(C1-3 alkyl), OH and C1-3 alkyl;
        each R22 is independently selected from H and C1-3 alkyl;
        R23 is selected from O, S, N—R0, and N—OR0;
    
    
    R3 is selected from —CR31c—, —NR32d—, and —(C═R33)—;
    each R31 group is selected from H, halo, —NH2, —N(H)(R0), —N(R0)2, —O—R0, OH and C1-3 alkyl;
    
        each R32 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, aryl, and a heterocyclic ring;
        R33 is selected from O, S, N—R34, and N—OR0;
        R34 is selected from H, NO2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring;
    
    
    R4 is selected from —CR41e—, —NR42f—, —(C═R43)—, and —O—;
    
        each R41 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO2R0, C(O)R0, aralkyl, aryl, and a heterocyclic ring;
        each R42 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, aryl, and a heterocyclic ring;
        each R43 is selected from O, S, N—R0, and N—OR0;

with the provisos that when R2 is —NR22b— and R4 is —NR42f—, then R3 is not —NR32d—; and that both R3 and R4 are not simultaneously selected from —(C═R33)— and —(C═R43)—, respectively;

    
    
    R5 is selected from —Y—R6 and —Z—R7;
    
        Y is selected from a chemical bond, O, NR0,
        R6 is selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
        R7 is H or is selected from aryl and a heterocyclic ring;
    
    
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring;
    a is 1 or 2;
    b is 0 or 1;
    c is 1 or 2;
    d is 0 or 1;
    e is 1 or 2; and
    f is 0 or 1.


The invention provides for a fundamentally new way of treating cancer and other diseases where treatment with an existing drug compound, by whatever mechanism, is followed by identifiable (clinically significant) theramutein-mediated drug resistance, by providing alternative drugs that can be administered as theramuteins arise and are identified as such (Wakai et al., 2004, reports an example wherein a theramutein may arise during the course of an on-going treatment regimen), or preemptively before the outgrowth of clinically significant populations of theramutein expressing cells. Further, where a drug treatment for a particular disease is less effective in a subset of individuals that express a certain theramutein of a protein that the drug targets, the invention enables the tailoring of treatments for those subjects by providing alternative drug substances that will be effective against said theramutein.
1. The invention provides a method of determining whether a chemical agent is at least as effective a modulator of a theramutein in a cell as a known substance is a modulator of a corresponding prototheramutein. One embodiment of the method involves contacting a control cell that expresses the prototheramutein and is capable of exhibiting a responsive phenotypic characteristic (linked to the functioning of the prototheramutein in the cell) with the known modulator of the prototheramutein, contacting a test cell that expresses the theramutein and is also capable of exhibiting the responsive phenotypic characteristic (linked to the functioning of the theramutein in the cell) with the chemical agent, and comparing the response of the treated test cell with the response of the treated control cell; to determine that the chemical agent is at least as effective a modulator of the theramutein as the known substance is a modulator of the prototheramutein. In certain other embodiments, one type of control cell may not express the prototheramutein at all. In other embodiments, the control cell may express about the same amount of the prototheramutein as the test cell expresses of the theramutein. In still other embodiments, the control cell may be capable of exhibiting the responsive phenotypic characteristic to about the same extent as the test cell under certain conditions.
2. Theramuteins of the invention that are of particular interest are those involved in regulatory function, such as enzymes, protein kinases, tyrosine kinases, receptor tyrosine kinases, serine threonine protein kinases, dual specificity protein kinases, proteases, matrix metalloproteinases, phosphatases, cell cycle control proteins, docking proteins such as the IRS family members, cell-surface receptors, G-proteins, ion channels, DNA- and RNA-binding proteins, polymerases, and the like. No limitation is intended on the type of theramutein that may be used in the invention. At the present time, three theramuteins are known: BCR-ABL, c-Kit, and EGFR.
3. Any responsive phenotypic characteristic that can be linked to the presence of the theramutein (or prototheramutein) in the cell can be employed for use in the method, including, for example, growth or culture properties, the phosphorylation state (or other modification) of a substrate of the theramutein, and any type of transient characteristic of the cell, as will be defined and discussed in detail



DESCRIPTION OF THE FIGURES
FIG. 1 shows the effect on growth and viability of different concentrations of Compound 2 (C2) for non-transformed vector control Ba/F3 cells (which are IL-3 dependent) as well as Ba/F3 cells expressing the “wild type” p210Bcr-Abl (designated p210Bcr-Abl-wt), and Ba/F3 cells expressing the p210Bcr-Abl-T315I drug resistant mutant. Cell counts and viability were determined on an automated cell counter as discussed in detail in the specification. Cell counts are shown by the solid color bars; cell viability is shown by the hashed bars. Note that STI-571 potently inhibits growth of the P210 cell line (grey bar) whereas it is unable to inhibit the growth of the T315I cell line (white bar) even at 10 μM concentration. 500 nM C2 shows the largest specificity gap within this dose-response series. Compare STI-571 at 10 μM to C2 at 500 nM on the T315I cell line (white bars). Abbreviations: DMSO: dimethylsulfoxide (solvent used for drug dissolution).
FIG. 2 shows the effect on growth and viability of different concentrations of Compound 6 (C6) for non-transformed vector control Ba/F3 cells as well as Ba/F3 cells expressing the p210Bcr-Abl-T315I drug resistant mutant. All other details are as per FIG. 1.
FIG. 3 shows various determinations of the specificity gap by comparing the effects of various compounds identified in the screen in terms of their effects on the prototheramutein- and theramutein-expressing cell lines. Compound 3 (C3) shows the best example of the ability of the method to identify a compound that exerts an even greater effect on the theramutein than on its corresponding prototheramutein. (Panel E). Panel A: control DMSO treatments; B: negative heterologous specificity gap; C: slightly positive heterologous specificity gap; D: large positive homologous specificity gap; E: positive heterologous specificity gap. See text for explanations.
FIG. 4 shows an autoradiograph of recombinant P210 Bcr-Abl wild type and T3151 mutant kinase domains assayed for autophosphorylation activity. 200 ng of protein were preincubated with test substances for 10 minutes under standard autophosphoryation reaction conditions and then radiolabelled ATP was added and the reactions proceeded for 30 minutes at 30° C., after which the samples were separated by SDS-PAGE. The gels were silver-stained, dried down under vacuum and exposed to X-ray film. Note that whereas 10 μM STI 571 is effective against wild type P210 Bcr-Abl, it is virtually ineffective against the T3151 kinase domain, even at concentrations up to 100 μM. C2 and C6 are the best two compounds identified, followed by C5, C7 and C4. All of the compounds tested positively to some extent. “P210 cell line” refers to cells expressing p210BCR-ABL-wt. “T315I cell line” refers to cells expressing p210BCR-ABL-T315I.
FIG. 5 shows the chemical structures of representative compounds of the present invention.
FIG. 6 shows the chemical structures of representative compounds of the present invention.
FIG. 7 shows the chemical structures of representative compounds of the present invention.
FIG. 8 shows the chemical structures of representative compounds of the present invention.
FIG. 9 shows the chemical structures of representative compounds of the present invention.
FIG. 10 shows the chemical structures of representative compounds of the present invention.
FIG. 11 shows the chemical structures of representative compounds of the present invention.
FIG. 12 shows the chemical structures of representative compounds of the present invention.
FIG. 13 shows the chemical structures of representative compounds of the present invention.



DETAILED DESCRIPTION OF THE INVENTION
The term “halo” or “halogen” as used herein includes fluorine, chlorine, bromine and iodine.
The term “alkyl” as used herein contemplates substituted and unsubstituted, straight and branched chain alkyl radicals having from 1 to 6 carbon atoms. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like. Additionally, the alkyl group may be optionally substituted with one or more substituents selected from halo, CN, CO2R, C(O)R, C(O)NR2, NR2, cyclic-amino, NO2, and OR.
The term “cycloalkyl” as used herein contemplates substituted and unsubstituted cyclic alkyl radicals. Preferred cycloalkyl groups are those with a single ring containing 3 to 7 carbon atoms and includes cyclopropyl, cyclopentyl, cyclohexyl, and the like. Other cycloalkyl groups may be selected from C7 to C10 bicyclic systems or from C9 to C14 tricyclic systems. Additionally, the cycloalkyl group may be optionally substituted with one or more substituents selected from halo, CN, CO2R, C(O)R, C(O)NR2, NR2, cyclic-amino, NO2, and OR.
The term “alkenyl” as used herein contemplates substituted and unsubstituted, straight and branched chain alkene radicals. Preferred alkenyl groups are those containing two to six carbon atoms. Additionally, the alkenyl group may be optionally substituted with one or more substituents selected from halo, CN, CO2R, C(O)R, C(O)NR2, NR2, cyclic-amino, NO2, and OR.
The term “alkynyl” as used herein contemplates substituted and unsubstituted, straight and branched chain alkyne radicals. Preferred alkynyl groups are those containing two to six carbon atoms. Additionally, the alkynyl group may be optionally substituted with one or more substituents selected from halo, CN, CO2R, C(O)R, C(O)NR2, NR2, cyclic-amino, NO2, and OR.
The term “aralkyl” as used herein contemplates an alkyl group which has as a substituent an aromatic group, which aromatic group may be substituted and unsubstituted. The aralkyl group may be optionally substituted on the aryl with one or more substituents selected from halo, CN, CF3, NR2, cyclic-amino, NO2, OR, CF3, —(CH2)xC(O)(CH2)yR, —(CH2)xC(O)N(R)(R′′), —(CH2)x—C(O)O(CH2)yR, —(CH2)xN(R′)(R′′), —N(R)SO2R, —O(CH2)xC(O)N(R′)(R′′), —SO2N(R′)(R′′), —(CH2)xN(R)—(CH2)y—R, —(CH2)xN(R)—C(O)—(CH2)y—R, —(CH2)xN(R)—C(O)—O—(CH2)y—R, —(CH2)xC(O)—N(R)—(CH2)y—R, —(CH2)xC(O)N(R)—(CH2)y—R, —O—(CH2)x—C(O)—N(R)—(CH2)y—R, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl, and a substituted and unsubstituted heterocyclic ring, wherein the substituted alkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heterocyclic ring may be substituted with one of more halo, CN, CF3, CO2R, C(O)R, C(O)NR2, NR2, cyclic-amino, NO2, and OR.
The term “heterocyclic group” or “heterocyclic ring” as used herein contemplates aromatic and non-aromatic cyclic radicals having at least one heteroatom as a ring member. Preferred heterocyclic groups are those containing 5 or 6 ring atoms which includes at least one hetero atom, and includes cyclic amines such as morpholino, piperidino, pyrrolidino, and the like, and cyclic ethers, such as tetrahydropyran, tetrahydropyran, and the like. Aromatic heterocyclic groups, also termed “heteroaryl” groups contemplates single-ring hetero-aromatic groups that may include from one to three heteroatoms, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. The term heteroaryl also includes polycyclic hetero-aromatic systems having two or more rings in which two atoms are common to two adjoining rings (the rings are “fused”) wherein at least one of the rings is a heteroaryl, e.g., the other rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/or heteroaryls. Examples of polycyclic heteroaromatic systems include quinoline, isoquinoline, tetrahydroisoquinoline, quinoxaline, quinaxoline, benzimidazole, benzofuran, purine, imidazopyridine, benzotriazole, and the like. Additionally, the heterocyclic groups may be optionally substituted with halo, CN, CF3, NR2, cyclic-amino, NO2, OR, CF3, —(CH2)xC(O)(CH2)yR, —(CH2)xC(O)N(R′)(R′′), —(CH2)xC(O)O(CH2)yR, —(CH2)xN(R′)(R′′), —N(R)SO2R, —O(CH2)xC(O)N(R′)(R′′), —SO2N(R′)(R′′), —(CH2)xN(R)—(CH2)y—R, —(CH2)xN(R)—C(O)—(CH2)y—R, —(CH2)xN(R)—C(O)—O—(CH2)yR, —(CH2)x—C(O)—N(R)—(CH2)y—R, —(CH2)xC(O)N(R)—(CH2)y—R, —O—(CH2)x—C(O)—N(R)—(CH2)y—R, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl, and a substituted and unsubstituted heterocyclic ring, wherein the substituted alkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heterocyclic ring may be substituted with one of more halo, CN, CF3, CO2R, C(O)R, C(O)NR2, NR2, cyclic-amino, NO2, and OR.
The term “aryl” or “aromatic group” as used herein contemplates single-ring aromatic groups (for example, phenyl, pyridyl, pyrazole, etc.) and polycyclic ring systems (naphthyl, quinoline, etc.). The polycyclic rings may have two or more rings in which two atoms are common to two adjoining rings (the rings are “fused”) wherein at least one of the rings is aromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/or heteroaryls. Additionally, the aryl groups may be optionally substituted with one or more substituents selected from halo, CN, CF3, NR2, cyclic-amino, NO2, OR, CF3, —(CH2)xC(O)(CH2)yR, —(CH2)xC(O)N(R′)(R′′), —(CH2)xC(O)O(CH2)yR, —(CH2)xN(R′)(R′′), —N(R)SO2R, —O(CH2)xC(O)N(R′)(R′′), —SO2N(R′)(R′′), —(CH2)xN(R)—(CH2)y—R, —(CH2)xN(R)—C(O)—(CH2)y—R, —(CH2)xN(R)—C(O)—O—(CH2)y—R, —(CH2)x—C(O)—N(R)—(CH2)y—R, —(CH2)xC(O)N(R)—(CH2)y—R, —O—(CH2)x—C(O)—N(R)—(CH2)y—R, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted aryl, and a substituted and unsubstituted heterocyclic ring, wherein the substituted alkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl, substituted alkynyl, substituted aryl, and substituted heterocyclic ring may be substituted with one of more halo, CN, CF3, CO2R, C(O)R, C(O)NR2, NR2, cyclic-amino, NO2, and OR.
The term “heteroatom”, particularly as a ring heteroatom, refers to N, O, and S.
Each R is independently selected from H, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted aryl and a substituted and unsubstituted heterocyclic ring, wherein the substituted alkyl, substituted cycloalkyl, substituted aralkyl, substituted aryl and substituted heterocyclic ring may be substituted with one or more halo, CN, CF3, OH, CO2H, NO2, C1-6alkyl, —O—(C1-6alkyl), —NH2, —NH(C1-6alkyl) and —N(C1-6alkyl)2. Each R′ and R′′ are independently selected from H, or substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted aryl and a substituted and unsubstituted heterocyclic ring, wherein the substituted alkyl, substituted cycloalkyl, substituted aralkyl, substituted aryl and substituted heterocyclic ring may be substituted with one or more halo, CN, CF3, OH, CO2H, NO2, C1-6alkyl, —O—(C1-6alkyl), —NH2, —NH(C1-6alkyl) and —N(C1-6alkyl)2; or R′ and R′′ may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain up to three further heteroatoms. Each x and each y are independently selected from 0 to 4.
In a preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-315I theramutein having the formula I




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R2 is selected from —CR21a—, —NR22b—, and —(C═R23)—;
    
        each R21 is independently selected from H, halo, —NH2, —N(H)(C1-3—N(C1-3 alkyl)2, —O—(C1-3 alkyl), OH and C1-3 alkyl;
        each R22 is independently selected from H and C1-3 alkyl;
        R23 is selected from O, S, N—R0, and N—OR0;
    
    
    R3 is selected from —CR31, —NR32d—, and —(C═R33)—;
    
        each R31 group is selected from H, halo, —NH2, —N(H)(R0), —N(R0)2, —O—R0, OH and C1-3 alkyl;
        each R32 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, aryl, and a heterocyclic ring;
        R33 is selected from O, S, N—R34, and N—OR0;
        R34 is selected from H, NO2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring;
    
    
    R4 is selected from —CR41e—, —NR42f—, —(C═R43)—, and —O—;
    
        each R41 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO2R0, C(O)R0, aralkyl, aryl, and a heterocyclic ring;
        each R42 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, aryl, and a heterocyclic ring;
        each R43 is selected from O, S, N—R0, and N—OR0;

with the provisos that when R2 is —NR22b— and R4 is —NR42f—, then R3 is not —NR32d—; and that both R3 and R4 are not simultaneously selected from —(C═R33)— and —(C═R43)—, respectively;

    
    
    R5 is selected from —Y—R6 and —Z—R7;
    
        Y is selected from a chemical bond, O, NR0,
        R6 is selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
        R7 is H or is selected from aryl and a heterocyclic ring;
    
    
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring;
    a is 1 or 2;
    b is 0 or 1;
    c is 1 or 2;
    d is 0 or 1;
    e is 1 or 2; and
    f is 0 or 1.


An important component and conceptual teaching of the Invention described herein is that neither the R2 nor the R3 positions of the compounds of this invention are members of any aromatic or non-aromatic ring structure. We have discovered that compounds having the R2 and/or the R3 positions as members of any aromatic or non-aromatic ring structure do not effectively inhibit the T315I theramutein, whereas the compounds of the invention that lack such a ring component at these positions, in addition to having other preferred chemical groups, are potent inhibitors of the T315I theramutein.
In preferred embodiments of the invention, ring A is an aromatic ring.
In preferred embodiments of the invention, X1 or X2 is N. In another preferred embodiment, both X1 and X2 are N. In particularly preferred embodiments of the invention Ring A is a pyridine ring or a pyrimidine ring. In still further preferred embodiments, Ring A is selected from the structures provided below:




In a preferred embodiment, if R2 or R4 is selected to be —NR22b— or —NR42—, respectively, then R31 is not selected from halo, —NH2, —N(H)(R0), —N(R0)2, —O—R0, or OH.
In a further preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula Ia




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    each R22 is independently selected from H and C1-3 alkyl;
    R3 is selected from —CR31c—, —NR32d—, and —(C═R33)—;
    
        each R31 group is selected from H, halo, —NH2, —N(H)(R0), —N(R0)2, —O—R0, OH and C1-3 alkyl;
        each R32 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, aryl, and a heterocyclic ring;
        R33 is selected from O, S, N—R34, and N—OR0;
        R34 is selected from H, NO2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring;
    
    
    R4 is selected from —CR41e—, —NR42f—, —(C═R43)—, and —O—;
    
        each R41 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO2R0, C(O)R0, aralkyl, aryl, and a heterocyclic ring;
        each R42 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, aryl, and a heterocyclic ring;
        each R43 is selected from O, S, N—R0, and N—OR0; with the provisos that when R4 is —NR42f—, then R3 is not —NR32d—; and that both R3 and R4 are not simultaneously selected from —(C═R33)— and —(C═R43)—, respectively;
    
    
    R5 is selected from —Y—R6 and —Z—R7;
    
        Y is selected from a chemical bond, O, N—R0,
        R6 is selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
        R7 is H or is selected from aryl and a heterocyclic ring;
    
    
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring;
    a is 1 or 2;
    b is 0 or 1;
    c is 1 or 2;
    d is 0 or 1;
    e is 1 or 2; and
    f is 0 or 1.


In a further preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula Ib




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)pR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    each R22 is independently selected from H and C1-3 alkyl;
    each R32 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, aryl, and a heterocyclic ring;
    R4 is selected from —CR41e—, —(C═R43)—, and —O—;
    
        each R41 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO2R0, C(O)R0, aralkyl, aryl, and a heterocyclic ring;
        each R43 is selected from O, S, N—R0, and N—OR0;
    
    
    R5 is selected from —Y—R6 and —Z—R7;
    
        Y is selected from a chemical bond, O, N—R0,
        R6 is selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
        R7 is H or is selected from aryl and a heterocyclic ring;
    
    
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring;
    a is 1 or 2;
    b is 0 or 1;
    c is 1 or 2;
    d is 0 or 1;
    e is 1 or 2; and
    f is 0 or 1.


In preferred embodiments of the invention, R2, R3 and R4 of formula I are selected to give the following chemical groups:

    
    
        —N(R22)—N═C(R41)—
        —N(R22)—N(R32)—C(═O)—
        —N(R22)—N(R32)—C(R41)(R41)—
        —N(R22)—C(R31)(R31)—C(R41)(R41)—
        —N(R22)—C(R31)(R31)—C(═O)—
        —N═N—C(R41)(R41)—
        —C(R21)═C═C(R41)—
        —C(R21)═C(R31)—C(═O)—
        —C(R21)═C(R31)—C(R41)(R41)—
        —C(R21)R21)—C(R31)═C(R41)—
        —C(R21)(R21)—C(R31)(R31)—C(═O)—
        —C(R21)(R21)—C(R31)(R31)—C(R41)(R41)—
        —C(R21)(R21)—N(R32)—C(═O)—
        —C(R21)(R21)—N(R32)—C(R41)(R41)—
        —N(R22)—C(═O)—C(R41)(R41)—
        —N(R22)—C(═O)—N(R41)—
        —N(R22)—C(═O)—O—
        —C(R21)(R21)—C(═O)—C(R41)(R41)—
        —C(R21)(R21)—C(═O)—N(R42)—
        —N(R22)—C(═NR34)—N(R42)—
        —C(═O)—N(R32)—N(R42).

Particularly preferred chemical groups for R2, R3 and R4 include:

        —N(R22)—N═C(R41)—
        —N(R22)—N(R32)—C(═)—
        —N(R22)—C(R31)(R31)—C(R41)(R41)—
        —N(R22)—C(R31)(R31)—C(═O)—
        —C(R21)(R21)—C(═O)—C(R41)(R41)—
        —C(R21)(R21)—C(═O)—N(R42)—
        —N(R22)—C(═NR34)—N(R42)—
        —C(═O)—N(R32)—N(R42).
    
    


In further preferred embodiment, R6 or R7 is an aryl group, which may be optionally substituted. Particularly preferred aryl groups include substituted or unsubstituted phenyl and pyridyl. In additional or alternative embodiments, it is preferred that the substituents R21 and R22 are independently selected from groups which have small steric bulk and are preferably selected from H and CH3, and more preferably are H.
In a further preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula II




wherein

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R8 is selected from the group consisting of is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO2R0, C(O)R0, aralkyl, aryl, and a heterocyclic ring;
    R9 is selected from —Y—R6 and —Z—R7;
    
        Y is selected from a chemical bond, O, N—R0,
        R6 is selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
        R7 is H or is selected from aryl and a heterocyclic ring; and
    
    
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


In a further preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula IIa




wherein

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1,
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R8 is selected from the group consisting of is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO2R0, C(O)R0, aralkyl, aryl, and a heterocyclic ring;
    X3 is N, CH or C—R50;
    each R50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR51, —(CH2)rC(O)(CH2)sR51, —(CH2)rC(O)N(R52)(R53), —(CH2)rC(O)O(CH2)sR51, —(CH2)rN(R51)C(O)R51, —(CH2)rN(R52)(R53), —N(R51)SO2R51, —OC(O)N(R52)(R53), —SO2N(R52)(R53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    
        R51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        R52 and R53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R52 and R53 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        r is 0 to 4;
        s is 0 to 4;
    
    
    m is 0 to 4; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


In a further preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula IIb




wherein:

    R14 is selected from H and F;
    R8 is selected from the group consisting of is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO2R0, C(O)R0, aralkyl, aryl, and a heterocyclic ring;
    X3 is N, CH or C—R60;
    each R60 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR0, halo, aryl, and a heterocyclic ring;
    R61 is selected from aryl and a heterocyclic ring;
    Q is selected from a chemical bond or a group having the formula —O—, —(CH2)i—, —(CH2)iC(O)(CH2)j—, —(CH2)i—N(R62)—(CH2)j—, —(CH2)iC(O)—N(R62)—(CH2)j—, —(CH2)iC(O)O(CH2)j—, —(CH2)iN(R62)C(O)—(CH2)j—, —(CH2)iOC(O)N(R62)—(CH2)j—, and —O—(CH2)i—C(O)N(R62)—(CH2)j—;
    R62 is selected from aryl, and a heterocyclic ring;
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring;
    h is 0 to 4;
    i is 0 to 4; and
    j is 0 to 4.


In preferred embodiments of compounds of the formula IIb, R60 is selected from halo, CF3, and OH.
Exemplary compounds of the formula II, IIa or IIb includes the following structures:





































In a further preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula III




wherein

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R10 is selected from —Y′—R18;
    Y′ is selected from a chemical bond, O, NR0—, and a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
    R18 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF3, aryl, and a heterocyclic ring; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


In a further preferred embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula IIIa




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    X3 is N, CH or C—R50;
    each R50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR51, —(CH2)rC(O)(CH2)sR51, —(CH2)rC(O)N(R52)(R53), —(CH2)rC(O)O(CH2)sR51, —(CH2)rN(R51)C(O)R51, —(CH2)rN(R52)(R53), —N(R51)SO2R51, —OC(O)N(R52)(R53), —SO2N(R52)(R53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    
        R51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        R52 and R53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R52 and R53 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        r is 0 to 4;
        s is 0 to 4;
    
    
    m is 0 to 4; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


Exemplary compounds of the formula III or IIIa includes the following structures:







In a further embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula IV




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R22 is selected from H and C1-3 alkyl;
    R34 is selected from H, NO2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring;
    R44 is selected from H, alkyl, cycloalkyl, —(C═O)R0, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
    R45 is selected from —Y′′—R19;
    Y′′ is selected from a chemical bond, O, NR0—, and a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
    R19 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF3, aryl, and a heterocyclic ring; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


Exemplary compounds of the formula IV include the following structures:




In a further embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula V




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R22 is selected from H and C1-3 alkyl;
    R34 is selected from H, NO2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring;
    R55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
    R56 is selected from —Y′′—R19;
    Y′′ is selected from a chemical bond, O, NR0—, and a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R)2, NO2, and OR0;
    R19 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF3, aryl, and a heterocyclic ring; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


In a further embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula Va




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
    X3 is N or C—R50;
    each R50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR51, —(CH2)rC(O)(CH2)sR51, —(CH2)rC(O)NR52)(R53), —(CH2)rC(O)O(CH2)sR51, —(CH2)rN(R51)C(O)R51, —(CH2)rN(R52)(R53), —N(R51)SO2R51, —OC(O)N(R52)(R53), —SO2N(R52)(R53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    
        R51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        R52 and R53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R52 and R53 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        r is 0 to 4;
        s is 0 to 4;
    
    
    m is 0 to 4; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


Exemplary compounds of the formula V or Va include the following structures:




In a further embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula VI




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1;
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)qR11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, —OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
    R56 is selected from —Y′′—R19;
    Y′′ is selected from a chemical bond, O, NR0—, and a hydrocarbon chain having from 1 to 4 carbon atoms, and optionally substituted with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO2R0, C(O)R0, C(O)N(R0)2, CN, CF3, N(R0)2, NO2, and OR0;
    R19 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF3, aryl, and a heterocyclic ring; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


In a further embodiment, the present invention provides inhibitors of the P210BCR-ABL-T315I theramutein having the formula VIa




wherein:

    ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring;
    X1 is selected from N,N—R0 or C—R1,
    X2 is selected from N,N—R0 or C—R1;
    the dotted lines represent optional double bonds;
    each R1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR11, —(CH2)pC(O)(CH2)qR11, —(CH2)pC(O)N(R12)(R13), —(CH2)pC(O)O(CH2)9R11, —(CH2)pN(R11)C(O)R11, —(CH2)pN(R12)(R13), —N(R11)SO2R11, OC(O)N(R12)(R13), —SO2N(R12)(R13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R1 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    n is 0 to 6,
    
        each R11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        each R12 and R13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R12 and R13 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        p is 0 to 4;
        q is 0 to 4;
    
    
    R55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
    X3 is N or C—R50;
    each R50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF3, NO2, OR51, —(CH2)rC(O)(CH2)sR51, —(CH2)rC(O)N(R52)(R53), —(CH2)rC(O)O(CH2)sR51, —(CH2)rN(R51)C(O)R51, —(CH2)rN(R52)(R53), —N(R51)SO2R51, —OC(O)N(R52)(R53), —SO2N(R52)(R53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms;
    
        R51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;
        R52 and R53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R52 and R53 may be taken together with the nitrogen to which they are attached form a 5- to 7-membered ring which may optionally contain a further heteroatom;
        r is 0 to 4;
        s is 0 to 4;
    
    
    m is 0 to 4; and
    each R0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.


Exemplary compounds of the formula VI or VIa include the following structures:










As used herein, the definition of each expression, e.g. alkyl, m, n, R, R′ etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
For each of the above descriptions of compounds of the structures I, Ia, Ib, II, IIa, IIb, III, IIIa, IV, IVa, V, Va, VI, and VIa each recitation of the terms halo, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, heterocyclic group or heterocyclic ring, are independently selected from the definitions of these terms as provided in the beginning of this section.
It will be understood that chemical structures provided herein include the implicit proviso that substitution is in accordance with permitted valence of the substituted atom and the substituent(s), and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
When one or more chiral centers are present in the compounds of the present invention, the individual isomers and mixtures thereof (e.g., racemates, etc.) are intended to be encompassed by the formulae depicted herein.
When one or more double bonds are present in the compounds of the present invention, both the cis- and trans-isomers are intended to be encompassed by the formulae depicted herein. Although chemical structures (such as, for example, structures II, IIa, V, Va, VI, and VIa) are depicted herein in either cis of trans configuration, both configurations are meant to be encompassed by the each of the formulae.
In certain embodiments, compounds of the invention may exist in several tautomeric forms. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds.
The compounds of the invention may generally be prepared from commercially available starting materials and known chemical techniques. Embodiments of the invention may be synthesized as follows. One of skill in the art of medicinal or synthetic chemistry would be readily familiar with the procedures and techniques necessary to accomplish the synthetic approaches given below.
Embodiments wherein R2═NH, R3═N, R4═CH, and R5═-aryl may be prepared by reaction of an appropriate hydrazine compound, such as A, and an appropriate aldehyde, such as B, under conditions similar to those described on p. 562 of Gineinah, et al. (Arch. Pharm. Pharm. Med. Chem. 2002, 335, 556-562).




For example, heating A with 1.1 equivalents of B for 1 to 24 hours in a protic solvent such as a C1 to C6 alcohol, followed by cooling and collection of the precipitate, would afford C. Alternatively, product C may be isolated by evaporation of the solvent and purification by chromatography using silica gel, alumina, or C4 to C18 reverse phase medium. Similar methodology would be applicable in the cases where “Aryl” is replaced by other groups defined under R5.

Embodiments wherein R2═NH, R3═NR32, R4═C(O), and R5=a heterocyclic ring may be prepared by reaction of an appropriate hydrazine compound, such as D, and an activated carboxylic acid such as E, wherein LG is a leaving group such as halo, 1-oxybenztriazole, pentafluorophenoxy, p-nitrophenoxy, or the like, or Compound E may also be a symmetrical carboxylic acid anhydride, whereby conditions similar to those described on p. 408 of Nair and Mehta (Indian J. Chem. 1967 5, 403-408) may be used.




For example, treatment of D with an active ester such as Heterocycle-C(O)—OC6F5 in an inert solvent such as dichloromethane, 1,2-dichloroethane, or N,N-dimethylformamide, optionally in the presence of a base such as pyridine or another tertiary amine, and optionally in the presence of a catalyst such as 4-N,N-dimethylaminopyridine, at an appropriate temperature ranging from 0° C. to the boiling point of the solvent, would afford F, which may be isolated by evaporation of the solvent followed by chromatography using silica gel, alumina, or C4 to C18 reverse phase medium. The above active ester example of E would be readily prepared from the corresponding carboxylic acid and pentafluorophenol using a carbodiimide such as dicyclohexylcarbodiimide as a condensing agent. Similar methodology would be applicable in the cases where “Heterocycle” is replaced by other groups defined under R5.

Precursors such as A and D may be prepared by reaction of an appropriate nucleophile, for example, a hydrazine derivative, with a heteroaromatic compound bearing a halo substituent at a position adjacent to a nitrogen atom. For example, using methods analogous to those described by Wu, et al. (J. Heterocyclic Chem. 1990, 27, 1559-1563), Breshears, et al. (J. Am. Chem. Soc. 1959, 81, 3789-3792), or Gineinah, et al. (Arch. Pharm. Pharm. Med. Chem. 2002, 335, 556-562), examples of compounds A and D may be prepared starting from, for example, a 2,4-dihalopyrimidine derivative, many of which are commercially available or are otherwise readily prepared by one skilled in the art. Thus, treatment of an appropriate 2,4-dihalopyrimidine derivative G with an amine or other nucleophile (Z), optionally in the presence of an added base, selectively displaces the 4-halo substituent on the pyrimidine ring. Subsequent treatment of the product with a second nucleophilic reagent such as hydrazine or a hydrazine derivative, optionally in a solvent such as a C1 to C6 alcohol and optionally in the presence of an added base, displaces the 2-halo substituent on the pyrimidine ring, to afford compounds that are examples of structures A and D above.




Embodiments wherein R2 is —NR22 and R3 is —C(═R33) can be synthesized by methods such as the following, or straightforward modifications thereof. The synthesis may be conducted starting from an appropriate ring A derivative J that bears a leaving group (LG) adjacent to the requisite ring nitrogen. Structure G above and the product of reaction of structure G with nucleophile Z, as illustrated above, are examples of such appropriate Ring A derivatives J. Suitable LG′ groups are halo, alkylthio, alkylsulfonyl, alkylsulfonate or arylsulfonate. Treatment of J with an amine R12NH2 effects displacement of LG′ to afford intermediates K. An example of this chemical transformation wherein R12 is H and LG′ is CH3SO2— is reported by Capps, et al. J. Agric. Food Chem. 1993, 41, 2411-2415, and an example wherein R12 is H and LG′ is Cl is reported in Marshall, et al. J. Chem. Soc. 1951, 1004-1015.




Intermediates of structure K are transformed to compounds of the invention by simultaneous or sequential introduction of the elements, of R3, R4, and R5. For example, treatment of intermediates of structure % with individual isocyanates R6—N═C═O affords in a single step compounds of structure M, which are compounds of the invention wherein R2═—NR22—, R3═—C═O—, R4=—NH—, and R5═-chemical bond-R6. Alternative methods to convert compounds of structure K to compounds of structure M are well known to those skilled in the art, wherein R3 together with a leaving group (for example p-nitrophenoxy or chloro) is first introduced, followed by subsequent displacement of the leaving group by, for example, an amine R6—NH2, to introduce R5 and R6.




Alternatively, treatment of intermediates of structure % with a reagent such as cyanamide (NH2—CN), typically under conditions of heating and optionally in the presence of acid in a solvent such as ethyl acetate or dioxane, affords intermediates N. Alternatives to cyanamide are nitroguanidine or amidinosulfonic acid (NH2—C(═NH)—SO3H). An example of such a transformation using cyanamide is reported by Latham et al., J. Org. Chem. 1950, 15, 884. An example using nitroguanidine is reported by Davis, Proc. Natl. Acad. Sci. USA 1925, 11, 72. Use of amidinosulfonic acid was reported by Shearer, et al. Bioorg. Med. Chem. Lett. 1997, 7, 1763.




In analogy to the conversion of intermediates A or D to embodiments represented by C or F, intermediates K are converted, respectively, to compounds represented by P or Q, which are further embodiments of the invention.




Treatment of A or K with a ketone S, wherein R is as defined above, in place of an aldehyde B in the schemes above; affords compounds of structure T or U, respectively, which are further embodiments of the invention.




The non-guanidino carbon-nitrogen double bond of U can be selectively reduced by an appropriate reducing agent such as a metal (boron, aluminum, silicon, etc.) hydride reagents, preferably one with basic properties, to afford compounds V of the invention.




Embodiments of the invention wherein R2═CO, R3=—NR32−, R4 and R5═ZR7, wherein Z is a hydrocarbon chain and R7 is as defined above, may be prepared as follows. When R32═H, a Ring A-derived carboxylic acid W is activated by conversion to the corresponding acid chloride, or alternatively to an active ester, or to an analogous activated derivative, many of which are well known in the art. Treatment of the activated carboxylic acid with hydrazine affords the corresponding hydrazide Y. Treatment of Y with an aldehyde or ketone (under conditions of heating and/or mild acid catalysis if necessary) affords the desired final product Z.




If not commercially available, Ring A-derived carboxylic acids W may be prepared by treatment of starting material J above with cyanide ion, optionally with heating or transition metal catalysis, to replace the leaving group LG′ with a cyano residue. Basic or acidic hydrolysis of the cyano group affords the desired carboxylic acid intermediate W.
When R32 is not H, then a protected form of monosubstituted hydrazine may be used in the above scheme in place of hydrazine. Thus, treatment of the activated carboxylic acid from W with R32NHNH-PG, where PG is a nitrogen protecting group such as benzyloxycarbonyl or t-butyloxycarbonyl, followed by deprotection and treatment with an appropriate aldehyde or ketone as above affords Z′, a further embodiment of the invention.




It will be apparent to a practitioner skilled in the art of organic molecule synthesis that the reaction processes illustrated above are representative of a broader set of methods that are logical extensions of the illustrated processes. Thus, additional embodiments of the invention that incorporate additional variants in R2, R3, R4, and R5 claimed by this invention are prepared by obvious modifications of the above processes.
As would be recognized by a person of ordinary skill, it may be advantageous to employ a temporary protecting group in achieving the final product. The phrase “protecting group” as used herein means temporary modifications of a potentially reactive functional group which protect it from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).
A “mutein” is a protein having an amino acid sequence that is altered as a result of a mutation that has occurred in its corresponding gene (Weigel et al, 1989). Such mutations may result in changes in one or more of the characteristics of the encoded protein. For example, an enzyme variant that has modified catalytic activity resulting from a change in one or more amino acids is a mutein.
This invention is concerned with proteins harboring an alteration of at least one amino acid residue (the terms “amino acid sequence change” or “amino acid sequence alteration” include changes, deletions, or additions, of at least one amino acid residue, or any combination of deletions, additions, changes) such that the resulting mutein has become (as a result of the mutation) resistant to a known therapeutic agent relative to the sensitivity of the non-mutated version of said protein to the therapeutic agent. This specialized class of muteins is hereinafter referred to as a theramutein, and the corresponding protein lacking the mutation is referred to herein as a prototheramutein.
As used herein, “prototheramutein” refers to an endogenously occurring protein in a cell that is susceptible to mutation that confers relative insensitivity (i.e. resistance) to a therapeutic compound which otherwise inhibits or activates the protein. Accordingly, “theramutein” refers to an endogenously occurring protein or portion of a protein in a cell that contains at least one amino acid sequence alteration relative to an endogenous form of the protein, wherein the amino acid sequence change is or was identified or becomes identifiable, and is or has been shown to be clinically significant for the development or progression of a given disease, following exposure of at least one human being to a substance that is known to inhibit or activate the prototheramutein. Solely for the purposes of defining the preceding sentence, a substance need not be limited to a chemical agent for the purposes of first defining the existence of a theramutein. Thus, by definition, a theramutein is a protein which harbors a mutation in its corresponding endogenous gene, wherein said mutation is associated with the development of clinical resistance in a patient to a drug that is normally able to activate or inhibit the non-mutated protein. With respect to a given theramutein, the term “corresponding prototheramutein” refers to the prototheramutein which, through mutation, gives rise to said theramutein. Similarly, with respect to a given prototheramutein, the “corresponding theramutein” refers to the theramutein which has arisen by mutation from said prototheramutein.
Accordingly, it is apparent to a skilled artisan that, as the genes which encode theramuteins are limited to endogenously occurring genes, the definition of a theramutein excludes proteins encoded by disease-causing infectious agents such as viruses and bacteria. As used herein, the term “endogenous gene” refers to a gene that has been present in the chromosomes of the organism at least in its unmutated form, since inception. The term “cell” as used herein refers to a living eukaryotic cell whether in an organism or maintained under appropriate laboratory tissue or organ culture conditions outside of an organism.
In one aspect of the invention, a theramutein is a protein that is altered for the first time with respect to a commonly occurring “wild type” form of the protein (i.e. the prototheramutein). In another aspect of the invention, a theramutein is a variant of a protein (prototheramutein) that is, itself, already a mutein. In still another embodiment, a theramutein may be further mutated as compared to a previously existing theramutein. In such instances, the first theramutein (such as the T315I mutant of p210 BCR-ABL (see below), may be thought of as a “primary” theramutein, whereas subsequent mutations of the (already mutated) T315I variant may be termed a secondary theramutein, tertiary theramutein, etc. As exemplified below, a mutein of the invention is a variant of Bcr-Abl tyrosine kinase that escapes inhibition by an inhibitor of the “wild type” Bcr-Abl. Such a Bcr-Abl mutein is altered with respect to a more common or “wild type” form of Bcr-Abl (which is also a mutein as well) in such a way that a property of the protein is altered.
It will be understood that a mutein of primary interest is a theramutein that may have the same, increased, or decreased specific activity relative to its prototheramutein, and that it is not inhibited or is poorly inhibited by an agent that is capable of inhibiting the prototheramutein. Likewise, another theramutein of primary interest is one that has the same, increased or decreased specific activity (relative to its prototheramutein) and that is not activated or is poorly activated by an agent that is capable of activating the prototheramutein. Other variations are obvious to the skilled artisan. It will be further appreciated that theramuteins can include naturally occurring or commonly observed variants of a protein, for example, variants that are expressed from different alleles of a particular gene. In some cases such variants may be unremarkable with respect to their normal cellular function, with functional differences becoming apparent only in the presence of agents that differentially inhibit or activate the cellular function of the variants. For example, naturally occurring variants of a particular enzyme may have activity profiles that are not substantially different, but a therapeutic agent that modulates one may be ineffective in modulating the other.
It will be appreciated that, whereas one aspect of the invention is the identification of an agent that is active against a theramutein that arises or becomes dominant (by any mechanism) during the course of a treatment for a given disease, another aspect is the identification of an agent that is active against a mutein that is common within a population of unafflicted individuals, but wherein said mutein is less susceptible to modulation by an approved drug, and where the variation in the activity profile of the mutein becomes important (and is therefore first identified as being a theramutein) in a disease state such as where it is overexpressed or participates in a signaling process which has otherwise become abnormally regulated. For example, a neoplastic disease may be caused by abnormal regulation of a cellular component other than the theramutein or its prototheramutein, and still be treatable with an inhibitor of the prototheramutein, whereas the same treatment would be less effective or ineffective where the theramutein was present. This can be an issue where it is observed that the response of a particular tumor type to an anticancer agent varies among individuals that express different variants of an enzyme against which the anticancer agent is directed (Lynch et al., 2004). Here, the variants would not have arisen or become predominant during the course of treatment of the disease, but are preexisting in the healthy population and are detected only by their altered responsiveness to a particular course of established therapeutic treatment.
As used herein, the terms “agonist” and “activator” of a protein are used interchangeably. An activator (agonist) is limited to a substance that binds to and activates the functioning of a given protein. Unless explicitly stated otherwise, an “activator”, an “agonist”, and an “activator of a protein” are identical in meaning. The activation by an activator may be partial or complete. Likewise, as used herein, the terms “antagonist” and “inhibitor” of a protein are used interchangeably. An inhibitor (antagonist) is limited to a substance that binds to and inhibits the functioning of a given protein. To state that a substance “inhibit(s)” a protein means the substance binds to the protein and reduce(s) the protein's activity in the cell without materially reducing the amount of the protein in the cell. Similarly, to state that a substance “activate(s)” a protein, such as a prototheramutein or theramutein, is to state that the substance increased the defined function of the protein in the cell without substantially altering the level of the protein in the cell. Unless explicitly stated otherwise, an “inhibitor”, an “antagonist” and an “inhibitor of a protein” are also synonymous. The inhibition by an inhibitor may be partial or complete. A modulator is an activator or an inhibitor. By way of example, an “activator of PKCβ1” should be construed to mean a substance that binds to and activates PKCβ1. Similarly, an “inhibitor of p210Bcr-Abl” is a substance that binds to and inhibits the functioning of p210Bcr-Abl. To state that a substance “inhibits a protein” requires that the substance bind to the protein in order to exert its inhibitory effect. Similarly, to state that a substance “activates protein X” is to state that the substance binds to and activates protein X. The terms “bind(s),” “binding,” and “binds to” have their ordinary meanings in the field of biochemistry in terms of describing the interaction between two substances (e.g., enzyme-substrate, protein-DNA, receptor-ligand, etc.). As used herein, the term “binds to” is synonymous with “interacts with” in the context of discussing the relationship between a substance and its corresponding target protein. As used herein, to state that a substance “acts on” a protein, “affects” a protein, “exerts its effect on” a protein, etc., and all such related terms uniformly mean (as the skilled investigator is well aware) that said substance activates or inhibits said protein.
The concept of inhibition or activation of a mutated form of an endogenous protein to a greater extent than the corresponding non-mutated counterpart protein is defined for the first time and referred to herein as a positive “specificity gap.” In general terms, and using an inhibitor case as an example, the specificity gap refers to the difference between the ability of a given substance, under comparable conditions to inhibit the theramutein in a cell-based assay system as compared to either:
a) the ability of the same substance under comparable conditions to inhibit the prototheramutein, or
b) the ability of a second substance (usually a known inhibitor of the prototheramutein) to inhibit the theramutein under comparable conditions, or
c) the ability of the second substance to inhibit the prototheramutein under comparable conditions.
When the comparison is made between the effects of two distinct substances (tested individually) on the theramutein alone, the result is termed a homologous specificity gap determination.
Alternatively, when a comparison is made between the effects of two distinct substances (generally, but not always), one of which is tested on the theramutein and the other on the prototheramutein, respectively, the result is termed a heterologous specificity gap (SG) determination. Thus, (a) and (c) as given above are examples of heterologous specificity gap (SG) determinations (although (a) uses the same substance in both instances), whereas (b) is an example of a homologous specificity gap determination.
Reference to FIG. 3 is informative in understanding and elucidating these concepts.
Analogous issues apply when the case concerns an activator. It will be immediately obvious to the skilled artisan that the term “comparable conditions” includes testing two different compounds, for example, at the same concentration (such as comparing two closely related compounds to determine relative potency), or by comparing the effects of two different compounds tested at their respective IC50 values on the corresponding prototheramutein and theramutein. The skilled investigator will easily recognize other useful variations and comparable conditions.
Thus, in one embodiment of the application of this approach, substances that are more effective against a theramutein have a “positive specificity gap.” A “zero, null or no” specificity gap indicates that there is no significant measurable difference between the effect of a substance on the theramutein as compared to its effect on the prototheramutein (however such compounds may be quite useful in their ability to inhibit or activate both a theramutein and its corresponding prototheramutein), and a “negative specificity gap” indicates a substance that at a given concentration is less effective against the given theramutein than against a form of the corresponding prototheramutein or other comparative form of the theramutein (such as one that may harbor a different mutation). The latter category is generally of lesser interest than the former categories of compounds, except in the case where the compound is so potent that its relatively lesser effect on the theramutein is of no real concern from the perspective of therapeutic efficacy. The skilled investigator can easily recognize a variety of approaches to quantifying the specificity gap assessment in a manner tailored to his or her needs.
The invention also provides a means for identifying compounds that exhibit a desired specificity gap. Such compounds can be identified and their ability to inhibit or activate the theramutein determined using an in vitro cell-based assay system where the effect of a substance on the cellular functioning of the mutated endogenous form of the protein is compared to the effect of the same drug on the cellular functioning of a non-mutated endogenous form of the protein.
Thus, the system enables the discovery of compounds capable of binding to a theramutein and exerting a greater modulatory effect on the cellular functioning of said theramutein than on its corresponding prototheramutein. Further, the system enables the discovery of compounds capable of binding to a theramutein and exerting at least as great or greater modulatory effect on the cellular functioning of a theramutein than previously known compounds are able to exert on the corresponding prototheramutein. In a particular embodiment of the invention, a compound may be screened for and identified that 1) is at least as effective against the theramutein as the original drug is against the prototheramutein, and/or 2) is similarly effective against the prototheramutein as against the theramutein (i.e., displays a small or essentially zero specificity gap).
In an embodiment of the invention, cells that overexpress a theramutein of, interest are used to identify chemical agents that are inhibitors or activators of (i.e., that bind to and inhibit or that bind to and activate) at least the selected theramutein. The chemical agents may also be inhibitors or activators of the prototheramutein or even other theramuteins of the same prototheramutein. As used herein, the terms “chemical agent” and “compound” are used interchangeably, and both terms refer exclusively to substances that have a molecular weight up to, but not including, 2000 atomic mass units (Daltons). Such substances are sometimes referred to as “small molecules.” Unless otherwise stated herein, the term substance as used herein refers exclusively to chemical agents/compounds, and does not refer to biological agents. As used herein, “biological agents,” are molecules which include proteins, polypeptides, and nucleic acids, and have molecular weights equal to or greater than 2000 atomic mass units (Daltons).
According to the invention, a theramutein is selected and used in a cell-based assay system designed to identify agents that are inhibitors or activators of the theramutein. Where two or more distinct theramuteins originating from the same prototheramutein are known, it is preferable to select the most resistant theramutein available for use in the assay system. In general, the degree of resistance of a theramutein to a given chemical agent is determined relative to its non-mutated counterpart (prototheramutein) using the drug that was first administered and known to inhibit or activate the prototheramutein and against which the theramutein “arose.” The methods of determining the degree of such resistance, for example by analysis of IC50 or AC50 values, are well known and standard in the art and will not be reiterated herein. However, no causal relationship is necessary or should be inferred between the treatment of the patient with a given therapeutic agent per se and the subsequent appearance of a theramutein. Rather, what is required in order to practice the invention is that a true theramutein be properly selected according to the teachings herein.
Thus, for example, randomly generated site directed mutants of known proteins that are created in the laboratory but that have not been shown to be clinically relevant are not appropriate muteins for use within the scope of this invention. Such muteins would not, of course, be properly classified as theramuteins.
For example, in an effort to obtain potential inhibitors of mutants of p210Bcr-Abl, Huron et al. (2003) used a recombinant c-abl preparation and screened a series of compounds known to inhibit c-src tyrosine kinase activity. The authors performed c-abl kinase assays on their compounds and identified the most potent compound as an 8 nM inhibitor against c-abl. When this compound (PD166326) was tested against various p210Bcr-Abl theramuteins, however, it showed activity against some of the mutants such as p210Bcr-Abl-E255K, but the p210Bcr-Abl-T315I theramutein was found to remain 10 fold more resistant (Huron et al. 2003, Table 3). Furthermore, in each case the compound was still markedly less effective on the p210Bcr-Abl theramuteins than it was against the wild-type p210Bcr-Abl. When the compound was tested against p210Bcr-Abl-T315I mutant activity, it was unable to inhibit the activity to any appreciable extent (p. 1270, left hand column, second paragraph; see also FIG. 4.). Thus, the disclosed compound was able to inhibit a theramutein that is partially resistant to STI-571, but had no activity against the T315I mutant of Bcr-Abl, which was already known at that time to be the theramutein that exhibited the most resistance to STI-571. Hence purely and simply, the Huron methodology failed to identify an effective inhibitor of the p210Bcr-AblT315I theramutein.
Indeed, prior to the disclosure of this invention, including both the detailed methodology described for the first time herein as well as the compositions provided herein, no one anywhere in the world has been successful in identifying a chemical agent, let alone a methodology that is capable of identifying a chemical agent that effectively inhibits the p210Bcr-AblT315I theramutein to an equal or greater extent than STI-571 is able to do with respect to the wild type p210Bcr-Abl protein. (See Shah et al., Science, July, 2004; O'Hare et al., Blood, 2004; Tipping et al., Leukemia, 2004; Weisberg et al., Leukemia, 2004).
It cannot be overemphasized that such compounds would be immensely useful, because at the present time there is no alternative for patients who progress to p210Bcr-Abl-T315I theramutein-mediated imatinib mesylate-resistant status. Once patients develop such resistance, there is no other effective alternative treatment available, and death is certain. The method described herein provides the first reported approach to identify, pharmacologically characterize and chemically synthesize effective inhibitors of the p210Bcr-Abl-T315I theramutein. Moreover, the skilled investigator will immediately recognize the applicability and generalizability of this approach to any highly drug-resistant theramutein.
In the present invention, a test cell is used that displays a carefully selected phenotypic characteristic (as defined below) which is linked to the presence and functional activity of the particular theramutein-of-interest (TOI) in the cell under appropriate conditions. This should be qualitatively the same as the phenotypic characteristic displayed by a cell that expresses the prototheramutein. A phenotypic characteristic (i.e. a non-genotypic characteristic of the cell) is a property which is observed (measured), selected and/or defined for subsequent use in an assay method as described herein. Expression of the phenotypic characteristic is responsive to the total activity of the theramutein in the cell, and is a result of the absolute amount of the theramutein and its specific activity. Often, the phenotypic characteristic is observable as a result of elevated levels of theramutein activity and is not apparent in cells that express low amounts of the theramutein or low amounts of its corresponding prototheramutein. Further, it can often be demonstrated that the phenotypic characteristic is modulated by modulating the specific activity of the theramutein with an inhibitor or activator of the theramutein, although this is not always the case since an inhibitor or activator of the TOI may not always be available at the time the skilled investigator undertakes such a project. Thus, for the purpose of defining the phenotypic characteristic to be subsequently used with a given test cell for assay purposes, the skilled investigator may also use a substance capable of increasing or decreasing the expression of the theragene, which will in turn lead to increases or decreases of the level of the corresponding theramutein. This allows the skilled investigator to simulate the effects of certain types of activators or inhibitors of the theramutein (such as a suicide inhibitor of the theramutein, which is a class of chemical agent which binds irreversibly and covalently modifies the TOI, rendering it permanently inactive), without actually having access to such a compound, for the purposes of refining the appropriate phenotypic characteristic for subsequently establishing a useful cellular assay system. Examples known to one of ordinary skill that would be helpful for such purposes include the use of anti-sense DNA oligonucleotides, small interfering RNAs, other RNA interference-based methodologies, and vector constructs containing inducible promoter systems. In this manner, the selected phenotypic characteristic is linked to the activity of the theramutein in the test cell. Notably for theramuteins, the selected phenotypic characteristic is usually also displayed by a cell that overexpresses the prototheramutein and in which the phenotypic characteristic is modulated by known inhibitors or activators of the prototheramutein.
A phenotypic characteristic is simply a characteristic of a cell other than a genotypic characteristic of the cell. Except for the specific requirements of a properly defined phenotypic characteristic as disclosed herein for the purposes of creating useful cellular assay systems according to the teachings of certain of the embodiments of the invention, no other limitation of the term phenotypic characteristic of any kind or nature is intended or appropriate in order to properly and effectively practice the invention. Indeed, the skilled artisan must be able to select any characteristic of the cell that maximizes the utility of establishing the proper cell-based assay for his or her needs. The phenotypic characteristic can be quantitative or qualitative and be observable or measurable directly (e.g., observable with the naked eye or with a microscope), but most commonly the characteristic is measured indirectly using standard automated laboratory equipment and assay procedures which are known to those of skill in the art. The term “observable” means that a characteristic may be measured or is otherwise detectable under appropriate conditions by any means whatsoever, including the use of any type of laboratory instrumentation available. The term “detectable” is not the same as “detected”. A characteristic may be detectable to a skilled artisan without being detected at any given time, depending upon how the investigator chooses to design the assay system. For example, in searching for activators of a prototheramutein (or theramutein), it may be desirable to have the relevant phenotypic characteristic detected only after the addition of a known activator or test substance capable of activating the POI. This provides the ability to maximize the intensity of the signal that is generated by the test cell in the assay.
Phenotypic characteristics include but are not limited to growth characteristics, transformation state, differentiation state, substrate phosphorylation state, catalytic activity, ion flux across the cell membrane (calcium, sodium, chloride, potassium, hydrogen ions, etc.), pH changes, fluctuations of second messenger molecules or other intracellular chemical species such as cAMP, phosphoinositides, cyclic nucleotides, modulations of gene expression, and the like. The characteristic of the cell may be observable or measurable continuously (e.g., growth rate of a cell), or after a period of time (e.g., terminal density of a cell culture), or transiently (e.g., modulation of a mutein causes a transient change in phosphorylation of a substrate of the mutein, or a transient flux in ion flow across the membrane, or elevations or reductions in intracellular cAMP levels). In certain embodiments, a selected phenotypic characteristic may be detected only in the presence of a modulator of the prototheramutein or the theramutein. No limitations are intended with respect to a characteristic that may be selected for measurement. As used herein, the terms “characteristic of a cell” and “phenotypic characteristic”, and simply “characteristic”, when used to refer to the particular measurable property of the intact cell or a subcellular fraction of the cell following the treatment of a test cell with a substance, are identical. For example, a phenotypic characteristic can be focus formation that becomes observable when a cell that over expresses a selected protein is cultured in the presence of an activator of the protein, or it may be a transient increase or decrease in the level of an intracellular metabolite or ion, such as cAMP, calcium, sodium, chloride, potassium, lithium, phosphatidylinositol, cGMP, bicarbonate, etc. It is obvious to one of ordinary skill in the art that after a cell is exposed to a test substance, the characteristic so measured (assayed) may be determined on a sub-cellular fraction of the cell. However, the initial treatment of the cell with a substance, which thereby causes the substance to come into contact with the cell, must be performed on the intact cell, not a sub-cellular fraction.
The characteristic selected for measurement within the cell must not be an intrinsic physical or chemical property of the theramutein or prototheramutein itself (such as the mere amount (mass) of the protein inside the cell), but rather must be a characteristic that results from the activity of the theramutein inside the cell, thus affecting a characteristic of the cell which is distinct from the theramutein itself, as discussed in detail above. For example, where the theramutein is a protein kinase that is capable of undergoing autophosphorylation, a process whereby the enzyme is capable of catalyzing the phosphorylation of itself by transferring a terminal phosphate group from ATP onto itself, it would NOT be appropriate to select the phosphorylation state of the TOI as an appropriate phenotypic characteristic of the cell for measurement. This is because such a characteristic does not reflect the activity of the TOI on other cellular components. As the skilled investigator knows, autophosphorylation is not necessarily reflective of the activity of a protein kinase in a cell, since mutants of protein kinases are known that retain enzymatic activity sufficient to undergo autophosphorylation, yet have lost the capability to engage in signal transduction events within the cell. The classic paper by White et al. (1988) is both educational and noteworthy in this respect.
The term “responsive phenotypic characteristic” means a characteristic of the cell which is responsive to inhibitors or activators of a given protein (prototheramutein or theramutein). The term “known therapeutic agent” is defined as any agent that has been administered to a human being for the treatment of a disease in a country of the world.
A useful phenotypic characteristic, as exemplified herein in association with p210Bcr-Abl and theramuteins thereof, is disregulation of cell growth and proliferation. It is noted that the same or similar assay may be appropriate for use with many different proteins of interest. For example, disregulations of growth, proliferation, and/or differentiation are common phenotypic characteristics that may result from overexpression of a variety of different cellular proteins. It is an important teaching of this invention that by overexpressing a selected protein in order to cause the appearance of such a phenotypic characteristic, the characteristic becomes linked to the presence, amount, and specific activity of that selected protein under suitable conditions, and this linkage allows the skilled investigator to identify inhibitors or activators of a theramutein of interest (TOI) as desired. Accordingly, the phenotypic characteristic is responsive to changes in the level and/or specific activity of the selected protein. Such a responsive phenotypic characteristic is referred to herein as a “phenoresponse.”
Though not always necessary, it will often be advantageous to employ cells that express high levels of the theramutein, and to select a phenotypic characteristic that results from overexpression of the theramutein. This is because phenotypic characteristics linked to the functioning of the theramutein generally become more distinguishable (easier to measure) as a theramutein is overexpressed to a greater extent. Further, phenoresponses that are observed in response to modulators of the theramutein are often amplified as the functional level of the theramutein is increased. Expressed another way, the selected phenoresponse observed in cells that overexpress the theramutein is particularly sensitive to modulators of the theramutein.
Preferably, the theramutein is stably expressed in a test cell. Stable expression results in a level of the theramutein in the cell that remains relatively unchanged during the course of an assay. For example, stimulation or activation of a component of a signaling pathway may be followed by a refractory period during which signaling is inhibited due to down-regulation of the component. For theramuteins of the invention, such down-regulation is usually sufficiently overcome by artificially overexpressing the theramutein. Expressed another way, the expression is sufficiently maintained that changes in a phenotypic characteristic that are observed during the course of an assay are due primarily to inhibition or activation of the theramutein, rather than a change in its level, even if down-modulation of the theramutein subsequently occurs. For these reasons, although stable expression of the theramutein is preferred, transfection followed by transient expression of the theramutein may be employed provided that the selected phenotypic characteristic is measurable and the duration of the assay system is short relative to the progressive decline in the levels of the transiently expressed theramutein which is to be expected in such systems over time. For these reasons, stably expressing cell lines are preferred (U.S. Pat. No. 4,980,281).
A preferred drug screening method of the present invention involves the following:
1) Identification of a theramutein for which a novel inhibitor or activator is desired. Identification of an appropriate theramutein may be performed using standard techniques (See, Gorre et al., Science, 2001; see also PCT/US02/18729). Briefly, patients that have been given a course of a therapeutically effective treatment using an activator or inhibitor of a known or suspected prototheramutein and have subsequently shown clinical signs and symptoms consistent with disease relapse are identified, and cells or tissue samples derived from such patients are obtained. Using standard laboratory techniques such as RT-PCR, the sequence of the prototheramutein is determined and compared to the previously determined nucleic acid sequence of the known prototheramutein gene or cDNA sequence. Mutations, if present, are identified and are correlated with functional resistance of the prototheramutein's function either in cell-based or, more commonly, cell-free assay systems, again using standard methodology. Once resistance-inducing mutations are confirmed, then said one or more confirmed mutants comprise a defined theramutein which may be used in the subsequent methods as described herein.
2) Provision of a test cell that expresses a theramutein of interest and displays an observable (measurable) phenotypic characteristic which has been previously shown to be responsive to inhibitors or activators of the theramutein or, more commonly, the corresponding prototheramutein. Said specific phenotypic characteristic that has been previously shown to be responsive to inhibitors or activators of the theramutein-of-interest (TOI), and/or the prototheramutein-of-interest (pTOI) is defined herein for the first time as a “phenoresponse.” One embodiment of this invention is the definitive use of the phenoresponse for the purpose of identifying compounds that are likely to be inhibitors or activators of the TOI. This may be accomplished through the use of a high-throughput screen using a cell line overproducing a given TOT and for which an appropriate phenoresponse has been identified and characterized. Alternatively, one may utilize a high-throughput primary screen using a more generic phenotypic characteristic of a cell line (that does not qualify as a phenoresponse according to the teachings herein) and then utilize a secondary screen according to the teachings herein to distinguish between compounds that are true positive “hits”, i.e. inhibitors or activators of the theramutein of interest, from false positive compounds that are not inhibitors or activators of the theramutein of interest. In one embodiment, a cell is selected that naturally expresses the theramutein such that a responsive phenotypic characteristic is present under suitable culture conditions which are obvious to one of ordinary skill in the art. In other embodiments, the theramutein is overexpressed, in some instances in a host cell that does not otherwise express the theramutein at all. This usually involves construction of an expression vector from which the theramutein can be introduced into a suitable host cell and overexpressed using standard vector systems and methodology. (Gone et al., 2001; Housey et al., 1988). In one embodiment, overexpression results in a level of the theramutein that is at least about 3 times the amount of the protein usually present in a cell. Alternatively, the amount is at least about 10 times the amount usually present in a cell. In another embodiment, the amount is at least about 20 times or more preferably at least about 50 times the amount usually present in a cell.
3) Provision of a control cell that expresses the prototheramutein corresponding to the theramutein of interest. As some of the muteins that are described herein are also enzymes, they usually retain catalytic activity, and therefore the control cell usually displays substantially the same phenotypic characteristic as the test cell. The phenotypic characteristic need not be quantitatively alike in both cells, however. For example, a mutation that leads to reactivation of the prototheramutein may also increase, decrease, or otherwise affect its specific activity with respect to one or more of its substrates in the cell. As a result, it may exhibit the selected phenotypic characteristic to a greater or lesser extent. Accordingly, it may be desirable in some cases to adjust expression of either or both of the prototheramutein and the theramutein such that test and control cells exhibit the phenotypic characteristic to approximately the same degree. This may be done, for example, by expressing the proteins from promoters whose activity can be adjusted by adjusting the amount of inducer present, all using standard methodology (see, for example, Sambrook et al. 1989 & 2001).
It will be obvious to one of ordinary skill in the art that a properly defined phenoresponse may be quantitatively different between the prototheramutein- and the theramutein-expressing cell lines as a result of differences in the specific activity (if any) between the theramutein and its corresponding prototheramutein. Theramutein-inducing mutations may increase or decrease the specific activity of said theramutein relative to the corresponding prototheramutein. When comparing a theramutein expressing cell line with a prototheramutein expressing cell line, it is preferable that the selected phenoresponse is qualitatively the same in both cell types. Thus, the skilled investigator may choose to normalize the activity of the theramutein-expressing cell line to that of the prototheramutein-expressing cell line, or vice versa. Such normalization methods are standard in the art. See, for example, Bolstad et al. (2003).
Alternatively, the skilled investigator may also wish to use unmodified host cells or host cells harboring the expression vector only as control cells for certain experimental procedures. (The host cells are the cells into which an expression vector encoding the theramutein was introduced in order to generate the test cells.) This may be the case where the investigator is only interested in identifying a specific inhibitor or activator of the theramutein of interest, irrespective of whether or not said compound is also effective against the prototheramutein of interest (pTOI).
4) The test and control cells are then maintained or propagated (although not necessarily at the same time) in growth media (or even in intact animals) under suitable conditions such that the phenoresponse may be expressed and assayed. Control cells that are expressing the prototheramutein may be treated with a known modulator of the prototheramutein, or with a test substance, and test cells are treated with test compounds to determine whether they are active against the theramutein, as measured by the ability of said substances to modulate the phenoresponse in the expected manner. Alternatively, control cells not expressing the prototheramutein may also be substituted, depending upon the particular phenoresponse that the skilled investigator has chosen for study. Substances may then be assayed on the test cells and, optionally, on the control cells at the same time, or at another time, and the results compared.
In one embodiment of the invention, substances that are active with regard to the test cells can be rapidly identified by their ability to modulate the phenoresponse of the test cells in the same manner as, for example, the known modulator of the prototheramutein alters the phenoresponse of prototheramutein-expressing control cells. In another embodiment, active substances may be identified by their ability to modulate the activity of the theramutein in the test cells while having little or no effect on the unmodified (prototheramutein and/or theramutein non-expressing) control cells. The skilled investigator will readily appreciate the many variations of this approach that may be utilized to identify, for example, modulators that are more effective against the theramutein, or that are equally effective against both the prototheramutein and one or more corresponding specific theramuteins.
Other phenoresponses can be observed and/or measured and include, for example, detection of substrates of the prototheramutein, and detection of gene expression changes that are regulated by the activity of the theramutein. In the simplest terms, any characteristic of the cell that the skilled investigator has previously correlated with the functional activity of the theramutein may be suitable for use with such methods. However, in selecting a given characteristic, the skilled investigator must first verify that said characteristic fulfills the criteria of being a phenoresponse according the teachings as given in detail herein. The skilled investigator may also wish to normalize the phenoresponse with the theramutein expressing cells to that of the prototheramutein expressing cells.
Characteristics suitable for detection may be measured by a variety of methods very well known to those of skill in the art. Such methods include, but are not limited to, detection of fluorescence of suitably labeled proteins (FACS), immunohistochemistry (IHC) for detection of protein expression, competitive radioligand binding assays, solid matrix blotting techniques, such as Northern, Southern, and Western blots of cell extracts, reverse transcriptase polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assays (ELISA), phosphorylation assays, gel retardation assays, membrane potential perturbations, and the like. The relevant phenotypic characteristic may be detected either on the intact cell after treatment with a test substance or, alternatively, on a subcellular fraction of the cell after treatment of the intact cell with a test substance.
Once compounds are identified that have the desired effect on the theramutein expressing test cells, it may be desirable (but not necessary) to independently verify that the compounds identified are exerting their effects on the theramutein through a direct binding mechanism, i.e. that the compounds fulfill the criteria of being inhibitors or activators (as desired) of the theramutein according to the teachings of the invention (the reader is referred to the definitions of the terms “activator” and “inhibitor” as given above). This may be accomplished with numerous standard binding assays that are known to one of ordinary skill in the art, involving either purified protein samples or intact cellular binding assays using cells transfected with the appropriate prototheramutein or theramutein together with appropriate controls as dictated by sound scientific methods. Since such methods are well established in the art they will not be reiterated here. Numerous reference texts comprehensively discuss such techniques (see, for example, Foreman and Johansen, 2002; Enna S. J. et al. (1991) Current Protocols in Pharmacology, Wiley & Sons, Incorporated; Bonifacino, J. S. et al. (1999) Current Protocols in Cell Biology, Wiley & Sons, Incorporated). See also Housey, G. M. 1988, Chapter 4, and references therein; see also Horowitz et al., 1981.
In a particular embodiment of the invention, the method is used to identify substances that are inhibitors of the p210Bcr-Abl-T315I theramutein. The prototheramutein and theramutein are each expressed in Ba/F3 (murine) cells using standard methodology and the phenoresponses that are observed are growth characteristics (terminal cell density for a carefully defined cell culture, and growth in the absence of Interleukin-3 (IL-3). Unmodified host cells, or host cells containing the expression vector only or both, may optionally also be used. In still another embodiment, the test cells alone may be used with or without reference to a known inhibitor or activator.
Another useful assay is the determination of the state of phosphorylation of a direct substrate of p210Bcr-Abl-T315I. One such substrate is Crkl (Gone et al., Science 293:876-80 (2001)), an adapter protein which mediates the connection between Bcr-Abl and Ras. The phosphorylation state of CRKL is representative of the signaling activity of p210Bcr-Abl in a cell. Another downstream substrate is p62DOK. Any such substrate would suffice for these purposes, provided of course that phosphorylation of said substrate has been shown to occur inside the cell, and is not simply an autophosphorylation event of the TOI or PTOI as discussed above. Other signal transduction cascade components may also be monitored, including src family kinases, STATS, PI3 Kinase, raf kinase, RAS, MEK, ERK1 and ERK2, JNK1, 2 and 3, MLK1, 2 and 3, MKK4, MKK7, AKT, mTOR, HSP90, and others.
As exemplified herein, inhibitors of the T315I theramutein have been identified. Furthermore, these inhibitors are also active to differing extents against the wild type prototheramutein p210Bcr-Abl-wt.
According to the present invention, a therapeutically effective amount of one or more compounds that modulate the functional activity of a p210Bcr-Abl theramutein is administered to a mammal in need thereof. The term “administering” as used herein means delivering the compounds of the present invention to a mammal by any method that may achieve the result sought. They may be administered, for example, orally, parenterally (intravenously or intramuscularly), topically, transdermally or by inhalation. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets and farm animals. “Therapeutically effective amount” means an amount of a compound that, when administered to a mammal, is effective in producing the desired therapeutic effect, such as inhibiting kinase activity, inhibiting cancer cell growth and division, etc.
The invention provides a method of treating disease in a mammal by administering to the mammal an effective amount of a modulator of a theramutein. Suitable diseases to be treated according to the present invention include, but are not limited to, relapsing neoplastic or other proliferative disorders that have become resistant to previously administered drugs. The method is also useful for overcoming variation among individuals with respect to susceptibility to drug treatment that results from allelic differences among therapy targets. For example, the role of p210Bcr-Abl tyrosine kinase signaling in CML has been extensively demonstrated, as has the role of theramuteins of p210Bcr-Abl in drug resistant recurrence of CML. Further, different muteins of p210Bcr-Abl exhibit varying sensitivity to inhibitors of p210Bcr-Abl. Although some theramuteins arise during drug therapy, others may preexist in the population. These latter examples will not be recognized as theramuteins until such time as the disease state ensues and is followed by treatment with a known class of therapeutic agents. Only after said treatment will such preexisting theramuteins reveal themselves as being clinically significant in terms of relative non-responsiveness leading to the progression of the disease in the patient harboring the theramutein.
In an embodiment of the invention, theramutein modulators are administered in combination with one or more other anti-neoplastic agents. Any suitable anti-neoplastic agent can be used, such as a chemotherapeutic agent, radiation or combinations thereof. The anti-neoplastic agent can be an alkylating agent or an anti-metabolite. Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples of anti-metabolites include, but not limited to, doxorubicin, daunorubicin, and paclitaxel, gemcitabine, and topoisomerase inhibitors irinotecan (CPT-11), aminocamptothecin, camptothecin, DX-8951f, topotecan (topoisomerase I inhibitor), and etoposide (VP-16; topoisomerase II inhibitor) and teniposide (VM-26; topoisomerase II inhibitor). When the anti-neoplastic agent is radiation, the source of the radiation can be either external (external beam radiation therapy—EBRT) or internal (brachytherapy—BT) to the patient being treated. The dose of anti-neoplastic agent administered depends on numerous factors, including, for example, the type of agent, the type and severity of the tumor being treated and the route of administration of the agent. It should be emphasized, however, that the present invention is not limited to any particular dose, route of administration, or combination of chemotherapeutic agents or other therapeutic regimens that are combined with the administration of theramutein modulators.
Anti-neoplastic agents which are presently known in the art or being evaluated can be grouped into a variety of classes including, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti survival agents, biological response modifiers, anti-hormones, and anti-angiogenesis agents, all of which can be administered with inhibitors or activators of theramuteins.
A modulator of a theramutein can be administered with antibodies that neutralize other receptors involved in tumor growth. Further, a modulator of a theramutein can be administered with a compound that otherwise modulates a component of a signal transduction pathway, preferably a component of the signal transduction pathway in which the theramutein is active and which is common to one or more other signal transduction pathways. In an embodiment of the invention, a theramutein modulator is used in combination with a receptor antagonist that binds specifically to the Epidermal Growth Factor Receptor (EGFR). Particularly preferred are antigen-binding proteins that bind to the extracellular domain of EGFR and block binding of one or more of its ligands and/or neutralize ligand-induced activation of EGFR. An EGFR antagonist can be an antibody that binds to EGFR or a ligand of EGFR and inhibits binding of EGFR to its ligand. Ligands for EGFR include, for example, EGF, TGF-α, amphiregulin, heparin-binding EGF (HB-EGF) and betacellulin. EGF and TGF-α are thought to be the main endogenous ligands that result in EGFR-mediated stimulation, although TGF-α has been shown to be more potent in promoting angiogenesis. It should be appreciated that the EGFR antagonist can bind externally to the extracellular portion of EGFR, which can or can not inhibit binding of the ligand, or internally to the tyrosine kinase domain in the case of chemical agents. Examples of EGFR antagonists that bind EGFR include, without limitation, biological agents such as antibodies (and functional equivalents thereof) specific for EGFR, and chemical agents (small molecules), such as synthetic kinase inhibitors that act directly on the cytoplasmic domain of EGFR.
Other examples of growth factor receptors involved in tumorigenesis are the receptors for vascular endothelial growth factor (VEGFR-1 and VEGFR-2), platelet-derived growth factor (PDGFR), nerve growth factor (NGFR), fibroblast growth factor (FGFR), and others.
In a combination therapy, the theramutein inhibitor is administered before, during, or after commencing therapy with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after commencing the anti-neoplastic agent therapy. For example, the theramutein inhibitor can be administered between 1 and 30 days, preferably 3 and 20 days, more preferably between 5 and 12 days before commencing radiation therapy. In a preferred embodiment of the invention, chemotherapy is administered prior to, concurrently with or, more preferably, subsequent to antibody therapy.
In the present invention, any suitable method or route can be used to administer theramutein inhibitors of the invention, and optionally, to co-administer anti-neoplastic agents and/or antagonists of other receptors. The anti-neoplastic agent regimens utilized according to the invention, include any regimen believed to be optimally suitable for the treatment of the patient's neoplastic condition. Different malignancies can require use of specific anti-tumor antibodies and specific anti-neoplastic agents, which will be determined on a patient to patient basis. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The dose of antagonist administered depends on numerous factors, including, for example, the type of antagonists, the type and severity of the tumor being treated and the route of administration of the antagonists. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration.
Suitable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Carriers can further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the theramutein modulator as the active ingredient. The compositions can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.
The compositions of this invention can be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.
Such compositions of the present invention are prepared in a manner well known in the pharmaceutical art. In making the composition the active ingredient will usually be mixed with a carrier, or diluted by a carrier and/or enclosed within a carrier which can, for example, be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, excipient or medium for the active ingredient. Thus, the composition can be in the form of tablets, lozenges, sachets, cachets, elixirs, suspensions, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, injection solutions, suspensions, sterile packaged powders and as a topical patch.
It should be appreciated that the methods and compositions of the present invention can be administered to any suitable mammal, such as a rabbit, rat, or mouse. More preferably, the mammal is a human.
The compounds according to the invention may also be present as salts. In the context of the invention, preference is given to pharmaceutically acceptable salts. Pharmaceutically acceptable salts refers to an acid addition salt or a basic addition salt of a compound of the invention in which the resulting counter ion is understood in the art to be generally acceptable for pharmaceutical uses. Pharmaceutically acceptable salts can be salts of the compounds according to the invention with inorganic or organic acids. Preference is given to salts with inorganic acids, such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or to salts with organic carboxylic or sulfonic acids, such as, for example, acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, phenylsulfonic acid, toluenesulfonic acid or naphthalenedisulfonic acid. Pharmaceutically acceptable salts can also be metal or ammonium salts of the compounds according to the invention. Particular preference is given to, for example, sodium, potassium, magnesium or calcium salts, and also to ammonium salts which are derived from ammonia or organic amines, such as, for example, ethylamine, di- or triethylamide, di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine, ethylenediamine or 2-phenylethylamine. (see, Berge et al. J. Pharm. Sci. 1977, 66, 1-19).

    1. Throughout this application, various publications, reference texts, textbooks, technical manuals, patents, and patent applications have been referred to. The teachings and disclosures of these publications, patents, patent applications and other documents in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which the present invention pertains.
    2. It is to be understood and expected that variations in the principles of invention herein disclosed may be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.
    3. The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, the introduction of plasmids into host cells, and the expression and determination thereof of genes and gene products can be obtained from numerous publications, including Sambrook, J et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Coligan, J. et al. (1994) Current Protocols in Immunology, Wiley & Sons, Incorporated; Enna, S. J. et al. (1991) Current Protocols in Pharmacology, Wiley & Sons, Bonifacino, J. S. et al. (1999) Current Protocols in Cell Biology, Wiley & Sons, and U.S. Pat. No. 4,980,281. All references mentioned herein are incorporated in their entirety.


EXAMPLES
p210Bcr-Abl-T315I is a theramutein of the p210Bcr-Abl protein (p210Bcr-Abl) that is resistant to inhibition by imatinib mesylate (Gleevec, STI-571). The mutation at position 315 converts a threonine to an isoleucine residue and is one of several mutations that are observed among resistant or relapsed patients. This particular mutant, however, is the most resistant such theramutein yet identified.
A phenoresponse was determined for a Ba/F3 cell line engineered to overexpress the p210Bcr-Abl-T315I theramutein. The phenoresponse was determined relative to non-transformed Ba/F3 cells and Ba/F3 cells that express the p210Bcr-Abl-wt prototheramutein. The phenoresponse was the ability of the T315I mutants to grow to a higher cell saturation density under analogous culture conditions as compared to the control non-transformed Ba/F3 cell line, and to grow in the absence of interleukin 3 (IL-3), which is required for maintenance of the control non-transformed Ba/F3 cell line. The phenoresponse was defined and characterized according to the teachings given above.
The detection system utilized was a high speed cell imaging and counting system in which 3 μl sample volumes of cells were sequentially injected through a 5 μl optical microcell, digitally imaged and electronically stored, scanned, and then counted, all under a microcomputer-based control system. The system has the capacity to perform direct cell counts on samples from cultures as small as 500 μl and provides statistically significant total cell counts from culture samples containing as few as 12,500 cells. All of the figures displaying cell count and viability assays utilized this system for data acquisition and analysis. Simultaneously with the cell count performed, the system is also capable of determining overall cell viability by distinguishing counted, imaged cells that have excluded trypan blue (counted as “viable” cells) from cells which have taken up the trypan blue dye (counted as “non-viable” cells). Injection of trypan blue into the cell sample occurs immediately prior to the sample being sequentially injected into the microcell for simultaneous cell counting and imaging.
The system may be integrated into the workflow of high-throughput screening devices to provide a sensitive and precise cell counting and cell viability assay system that is more reliable and less prone to confounding effects of metabolic viability-based cellular assays such as XTT or Alamar blue.
Initially, approximately 113,000 compounds were screened at concentrations generally ranging from 10 to 20 μM to identify a subset that was capable of affecting growth of Ba/F3 cells (Ba/F3 T3151 cells) overexpressing the p210Bcr-Abl-T315I theramutein by any means.
A total of approximately 11,760 compounds showed greater than 50% growth inhibition, which were thought to correspond to approximately 4500 distinct chemical classes. Retesting of these compounds with the same cell line yielded a database of compound responsiveness which was then sorted and rank ordered according to those compounds exhibiting the highest overall growth inhibition. From this rank ordered database, the highest scoring 130 compounds (based upon the greatest degree of growth inhibition observed at the lowest concentrations that compounds were tested) were then rescreened in a defined cell-based assay system using Ba/F3 T315I as test cells and wild type Ba/F3 as control cells according to the methods of the present invention. Compounds of interest were those that differentially inhibited growth of Ba/F3 cells expressing the p210Bcr-Abl-T315I theramutein relative to non-transformed wild type Ba/F3 cells. Six compounds were identified that fulfilled the desired criteria, and some of these compounds were analyzed in further detail using the Ba/F3 p210Bcr-Abl-wt cells line (Ba/F3 P210 cells) as well. One compound was unavailable for further testing due to lack of availability of additional material from the chemical supplier. The remaining five compounds were independently evaluated in additional cell-based assays using the aforementioned cell lines as well as in a cell-free purified protein kinase assay using human recombinantly produced 120 Kd kinase domain fragments isolated from both wild type P210 Bcr-Abl as well as P210 T315I mutant kinase domain.
All five compounds inhibited p210Bcr-Abl-T315I 120 Kd activity as measured by inhibition of autophosphorylation activity, as shown in FIG. 4. Thus, of the 6 highest scoring compounds out of more than 113,000 compounds screened, at least 5 of the six directly inhibited the p210Bcr-Abl-T315I mutant. It is noteworthy that Compound 5 appears to spread the recombinant protein band out on the SDS page gel. This was also evident on the silver-stained gel (data not shown). It is possible that this compound may actually be a “suicide” inhibitor that is able to covalently cross-link the POI in order to permanently inhibit its activity, but this will require further study.
Taken together, the teachings and the results described herein provide conclusive proof that the system is capable of identifying inhibitors or activators of the selected theramutein, and the skilled investigator will immediately recognize that such a system may be easily applied to any other theramutein with only obvious, minor modifications.
Representative examples of the cell-based assay results demonstrating selective inhibition of growth of the Ba/F3 T315I cell line relative to the wild type non-transformed Ba/F3 cells are shown in FIGS. 1 and 2. The compounds inhibited growth and reduced the viability of cells expressing the T3151 theramutein at concentrations under which the growth and viability of the wild type Ba/F3 non-transformed cells (not expressing either p210Bcr-Abl-wt or p210Bcr-Abl-T315I) were relatively unaffected, whereas cells expressing both the prototheramutein as well as the theramutein were substantially inhibited. In some instances, the T315I expressing cells were inhibited to an even greater extent than the P210 prototheramutein expressing cells. (See, for example, FIG. 3, right hand side, Compound 3 results against P210 and T315I cells.
In summary, the methods presented herein provide a fundamental advance in the form of a generalizable approach for creating or identifying modulators of any given theramutein. The results demonstrate conclusively the power of the method to identify critically needed compounds to overcome a specific type of acquired drug resistance that is uniformly fatal in certain patient populations and is presently untreatable. Furthermore, it is evident to one of skill in this art that the techniques and methods described herein may, using obvious modifications, be straighforwardly generalized to any potential theramutein of clinical significance.
It is remarkable that out of a primary screen of more than 100,000 compounds where approximately 10,000 compounds exhibited some degree of growth inhibition, when the most potent growth inhibitory substances were rescreened using the Method described in detail herein, 6 distinct compounds were identified and all of the compounds that were subsequently tested exhibited inhibitory activity in a cell-free purified protein kinase assay using the T315I mutant (one compound was unavailable for further testing). Based upon such remarkable results, it becomes immediately clear to the skilled artisan that the method may be effectively applied toward the identification of inhibitors or activators of any theramutein based upon the proper selection and definition of the phenoresponse according to the teachings in the sections given above and the documents incorporated by reference herein. For example, with knowledge of the foregoing, one of ordinary skill in the art could easily design an assay system to identify inhibitors of theramuteins derived from other prototheramuteins known to exhibit mutations that confer drug resistance such as the c-kit gene product or the Epidermal Growth Factor (EGF) Receptor (EGFR), or the Platelet Derived Growth Factor (PDGF) Receptor α and β. No limitation should be inferred upon the utility of the method with respect to its ability to be utilized with any given theramutein expressed in any mammalian cell type for which a corresponding phenoresponse is detectable.
All references to any publication, patent, or other citation are hereby incorporated by reference.
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## Claims

1. A method for determining whether a substance is an inhibitor or an activator of a theramutein which is capable of eliciting a detectable phenoresponse, which comprises:
a) incubating a first cell which expresses the theramutein at a substantially constant level with the substance;
b) incubating a second cell which expresses a corresponding prototheramutein at a substantially constant level with a known inhibitor or activator of the prototheramutein;
c) comparing a phenoresponse of the second cell to the known inhibitor or activator of the prototheramutein to the phenoresponse of the first cell to the substance; and
d) determining that the phenoresponse of the first cell is inhibited or activated to at least the same degree as the phenoresponse of the second cell is inhibited or activated by the known inhibitor or activator of the prototheramutein, thereby identifying the substance as an inhibitor or an activator of the theramutein.

2. The method of claim 1, wherein the phenoresponse of the cell expressing the theramutein to the substance is greater than the phenoresponse of the cell expressing the prototheramutein to the known inhibitor or activator of the theramutein.

3. A method for determining whether a substance is a specific inhibitor or specific activator of a theramutein, which comprises:
a) providing a test cell which expresses the theramutein and which gives rise to a detectable phenoresponse;
b) treating the test cell with the substance;
c) examining the treated cell to determine whether the phenoresponse is modulated by treatment with the substance.

4. The method of claim 1 or 3, wherein the theramutein or prototheramutein is a component of a signal transduction cascade.

5. The method of claim 1 or 3, wherein the theramutein or prototheramutein is an enzyme.

6. The method of claim 1 or 3, wherein the theramutein or prototheramutein is a protein kinase.

7. The method of claim 1 or 3, wherein the theramutein or prototheramutein is a tyrosine kinase.

8. The method of claim 1 or 3, wherein the theramutein or prototheramutein is a receptor tyrosine kinase.

9. The method of claim 1 or 3, wherein the prototheramutein is p210Bcr-Abl.

10. The method of claim 1 or 3, wherein the theramutein is the T315I mutant of p210Bcr-Abl.

11. The method of claim 1 or 3, wherein the phenoresponse is a change in a cultural, morphological, or transient characteristic of the cell.

12. The method of claim 1 or 3, wherein the phenoresponse includes phosphorylation of an intracellular substrate of the theramutein.

13. The method of claim 1 or 3, wherein the phenoresponse is detected on a subcellular fraction of the cell.