Compounds and methods of identifying, synthesizing, optimizing and profiling protein modulators

Abstract

This invention relates to methods of identifying, synthesizing, optimizing and profiling compounds that are inhibitors or activators of proteins, both naturally occurring endogenous proteins as well as certain variant forms of endogenous proteins, and novel methods of identifying such variants. The method accelerates the identification and development of compounds as potential therapeutically effective drugs by simplifying the pharmaceutical discovery and creation process through improvements in hit identification, lead optimization, biological profiling, and rapid elimination of toxic compounds. Implementation results in overall cost reductions in the drug discovery process resulting from the corresponding increases in efficiency.

Claims

1. A method of treating a condition in a human subject, comprising administering to the human subject a therapeutically effective amount of a compound, the compound capable of modulating a target protein in the human subject, wherein a cellular phenoresponse is linked to the presence and functional activity of the target protein, the target protein being associated with the condition, the condition being relapsing neoplastic disorders and proliferative disorders, including leukemia, the compound being selected for modulating the target protein using a method of identifying improved cellular specificity as compared to a known modulator of the target protein, comprising: a) measuring modulation of the phenoresponse of a test cell treated with the known modulator; b) measuring modulation of the phenoresponse of a control cell treated with the known modulator; c) measuring modulation of the phenoresponse of the test cell treated with the compound; d) measuring modulation of the phenoresponse of the control cell treated with the compound; e) determining the cellular specificity gap of the known modulator and the cellular specificity gap of the compound; and f) if the cellular specificity gap of the compound is greater than the cellular specificity gap of the second compound, identifying the compound as a modulator of the target protein.

2. The method as recited in claim 1, wherein the target protein is one of an enzyme, a cell cycle control protein, a docking protein, a surface receptor, and a component of a signal transduction cascade.

3. The method as recited in claim 1, wherein the selected target protein is a theramutein.

4. The method as recited in claim 1, wherein the phenoresponse of the test cell comprises at least one of a growth characteristic of the test cell, a transformation state of the test cell and a differentiation state of the test cell, and the phenoresponse of the control cell respectively comprises at least one of a growth characteristic of the control cell, a transformation state of the control cell, and a differentiation state of the control cell.

5. The method as recited in claim 1, wherein the phenoresponse of the test cell comprises at least one of a phosphorylation state of a second protein, that is not the target protein, in the test cell; a change in ion flux across a membrane of the test cell; a change in pH within the test cell; and a change in concentration of an intracellular chemical species within the test cell, and wherein the phenoresponse of the control cell respectively comprises at least one of a phosphorylation state of the second protein in the control cell; a change in the ion flux across a membrane of the control cell; a change in pH within the control cell; and a change in concentration of the intracellular chemical species within the control cell.

6. The method as recited in claim 5, wherein the intracellular chemical species comprises at least one of a phosphoinositide and a cyclic nucleotide.

7. The method as recited in claim 1, wherein the phenoresponse of the test cell comprises modulation of the genetic expression of the target protein within the test cell, and the phenoresponse of the control cell comprises modulation of the genetic expression of the target protein within the control cell.

Description

DESCRIPTION OF THE FIGURES

(1) 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” p210.sup.Bcr-Abl (designated p210.sup.Bcr-Abl-wt), and Ba/F3 cells expressing the p210.sup.Bcr-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).

(2) 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 p210.sup.Bcr-Abl-T315I drug resistant mutant. All other details are as per FIG. 1.

(3) 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.

(4) FIG. 4 shows an autoradiograph of recombinant P210 Bcr-Abl wild type and T315I 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 T315I kinase domain, even at concentrations up to 100 μM. “P210 cell line” refers to cells expressing p210.sup.BCR-ABL-wt. “T315I cell line” refers to cells expressing p210.sup.BCR-ABL-T315I.

(5) FIG. 5 shows the chemical structures of representative compounds of the present invention.

(6) FIG. 6 shows the chemical structures of representative compounds of the present invention.

(7) FIG. 7 shows the chemical structures of representative compounds of the present invention.

(8) FIG. 8 shows the chemical structures of representative compounds of the present invention.

(9) FIG. 9 shows the chemical structures of representative compounds of the present invention.

(10) FIG. 10 shows the chemical structures of representative compounds of the present invention.

(11) FIG. 11 shows the chemical structures of representative compounds of the present invention.

(12) FIG. 12 shows the chemical structures of representative compounds of the present invention.

(13) FIG. 13 shows the chemical structures of representative compounds of the present invention.

(14) FIG. 14 shows the inhibitory effect on growth rate of a hypothetical compound having a cellular specificity gap of 1 with respect to a test cell and a control cell.

(15) FIG. 15 shows the inhibitory effect on growth rate of a hypothetical compound having a cellular specificity gap of 40 with respect to a test cell and a control cell.

(16) FIG. 16 shows the growth inhibitory effect of imatinib mesylate at concentrations significantly below the apparent IC.sub.50 for cellular toxicity.

(17) FIG. 17 shows the effect on growth of different concentrations of C2 and various C2 analogues for Ba/F3 cells expressing the p210.sup.Bcr-Abl-T315I drug resistant mutant.

(18) FIG. 18 shows the results of a standard cell free protein kinase autophosphorylation assay for T315I mutant kinase domains in the presence of C2 and various C2 analogues at a concentration of 20 μM.

DETAILED DESCRIPTION OF THE INVENTION

(19) The term “halo” or “halogen” as used herein includes fluorine, chlorine, bromine and iodine.

(20) 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, CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, cyclic-amino, NO.sub.2, and OR.

(21) 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 include cyclopropyl, cyclopentyl, cyclohexyl, and the like. Other cycloalkyl groups may be selected from C.sub.7 to C.sub.10 bicyclic systems or from C.sub.9 to C.sub.14 tricyclic systems. Additionally, the cycloalkyl group may be optionally substituted with one or more substituents selected from halo, CN, CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, cyclic-amino, NO.sub.2, and OR.

(22) 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, CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, cyclic-amino, NO.sub.2, and OR.

(23) 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, CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, cyclic-amino, NO.sub.2, and OR.

(24) 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, CF.sub.3, NR.sub.2, cyclic-amino, NO.sub.2, OR, CF.sub.3, —(CH.sub.2).sub.xR, —(CH.sub.2).sub.xC(O)(CH.sub.2).sub.yR, —(CH.sub.2).sub.xC(O)N(R′)(R″), —(CH.sub.2).sub.xC(O)O(CH.sub.2).sub.yR, —(CH.sub.2).sub.xN(R′)(R″), —N(R)SO.sub.2R, —O(CH.sub.2).sub.xC(O)N(R′)(R″), —SO.sub.2N(R′)(R″), —(CH.sub.2).sub.xN(R)(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xN(R)—C(O)—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xN(R)—C(O)—O—(CH.sub.2).sub.y—R, —(CH.sub.2)—C(O)—N(R)—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xC(O)N(R)—(CH.sub.2).sub.y—R, —O—(CH.sub.2).sub.x—C(O)—N(R)—(CH.sub.2).sub.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, CF.sub.3a CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, cyclic-amino, NO.sub.2, and OR.

(25) 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 tetrahydrofuran, 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, CF.sub.3, NR.sub.2, cyclic-amino, NO.sub.2, OR, CF.sub.3, —(CH.sub.2).sub.xC(O)(CH.sub.2).sub.yR, —(CH.sub.2).sub.xC(O)N(R′)(R″), —(CH.sub.2).sub.xC(O)O(CH.sub.2).sub.yR, —(CH.sub.2).sub.xN(R′)(R″), —N(R)SO.sub.2R, —O(CH.sub.2).sub.xC(O)N(R′)(R″), —SO.sub.2N(R′)(R″), —(CH.sub.2).sub.xN(R)—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xN(R)—C(O)—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xN(R)—C(O)—O—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.x—C(O)—N(R)—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xC(O)N(R)—CH.sub.2).sub.y—R, —O—(CH.sub.2).sub.x—C(O)—N(R)—(CH.sub.2).sub.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, CF.sub.3, CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, cyclic-amino, NO.sub.2, and OR.

(26) The term “cyclic-amino” as used herein contemplates aromatic and non-aromatic cyclic radicals having at least one nitrogen as a ring member. Preferred cyclic amino groups are those containing 5 or 6 ring atoms, which includes at least one nitrogen, and includes morpholino, piperidino, pyrrolidino, piperazino, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine and the like. Additionally, the cyclic-amino may be optionally substituted with halo, CN, CF.sub.3, NR.sub.2, NO.sub.2, OR, CF.sub.3, 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 or more of halo, CN, CF.sub.3, CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, NO.sub.2, and OR.

(27) 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, CF.sub.3, NR.sub.2, cyclic-amino, NO.sub.2, OR, CF.sub.3, —(CH.sub.2).sub.xC(O)(CH.sub.2).sub.yR, —(CH.sub.2).sub.xC(O)N(R′)(R″), —(CH.sub.2).sub.xC(O)O(CH.sub.2).sub.yR, —(CH.sub.2).sub.xN(R′)(R″), —N(R)SO.sub.2R, —O(CH.sub.2).sub.xC(O)N(R′)(R″), —SO.sub.2N(R′)(R″), —(CH.sub.2).sub.xN(R)—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xN(R)—C(O)—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.xN(R)—C(O)—O—(CH.sub.2).sub.y—R, —(CH.sub.2).sub.x—C(O)—N(R)—(CH.sub.2).sub.y—R, —CH.sub.2).sub.xC(O)N(R)—(CH.sub.2).sub.y—R, —O—(CH.sub.2).sub.x—C(O)—N(R)—(CH.sub.2).sub.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, CF.sub.3, CO.sub.2R, C(O)R, C(O)NR.sub.2, NR.sub.2, cyclic-amino, NO.sub.2, and OR.

(28) The term “heteroatom”, particularly as a ring heteroatom, refers to N, O, and S.

(29) 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, CF.sub.3, OH, CO.sub.2H, NO.sub.2, C.sub.1-6alkyl, —O—(C.sub.1-6alkyl), —NH.sub.2, —NH(C.sub.1-6alkyl) and —N(C.sub.1-6alkyl).sub.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, CF.sub.3, OH, CO.sub.2H, NO.sub.2, C.sub.1-6alkyl, —O—(C.sub.1-6alkyl), —NH.sub.2, —NH(C.sub.1-6alkyl) and —N(C.sub.1-6alkyl).sub.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, which heteroatoms may be substituted by C.sub.1-6alkyl. Each x and each y are independently selected from 0 to 4.

(30) In a preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula I

(31) ##STR00002## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)(CH.sub.2).sub.qC(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; R.sup.2 is selected from —CR.sup.21.sub.a—, —NR.sup.22.sub.b—, and —(C═R.sup.23)—; each R.sup.21 is independently selected from H, halo, —NH.sub.2, —N(H)(C.sub.1-3 alkyl), —N(C.sub.1-3 alkyl).sub.2, —O—(C.sub.1-3 alkyl), OH and C.sub.1-3 alkyl; each R.sup.22 is independently selected from H and C.sub.1-3 alkyl; R.sup.23 is selected from O, S, N—R.sup.0, and N—OR.sup.0; R.sup.3 is selected from —CR.sup.31.sub.c—, —NR.sup.32.sub.d—, —SO.sub.2—, and —(C═R.sup.33)—; each R.sup.31 group is selected from H, halo, —NH.sub.2, —N(H)(R.sup.0), —N(R.sup.0).sub.2, —O—R.sup.0, OH and C.sub.1-3 alkyl; each R.sup.32 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO.sub.2R.sup.0, C(O)R.sup.0, aryl; and a heterocyclic ring; R.sup.33 is selected from O, S, N—R.sup.34, and N—OR.sup.0; R.sup.34 is selected from H, NO.sub.2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring; R.sup.4 is selected from —CR.sup.41.sub.e, —NR.sup.42.sub.f, —(C═R.sup.43)—, —SO.sub.2—, and —O—; each R.sup.41 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, C.sub.2R, C(O)R.sup.0, aralkyl, aryl, and a heterocyclic ring; each R.sup.42 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO.sub.2R.sup.0, C(O)R.sup.0, aryl, and a heterocyclic ring; each R.sup.43 is selected from O, S, N—R.sup.0, and N—OR.sup.0; with the provisos that when R.sup.2 is —NR.sup.22.sub.b— and R.sup.4 is —NR.sup.42.sub.f, then R.sup.3 is not —NR.sup.32.sub.d—; that both R.sup.3 and R.sup.4 are not simultaneously selected from —(C═R.sup.33)— and —(C═R.sup.43)—, respectively; and that R.sup.3 and R.sup.4 are not simultaneously selected from —SO.sub.2—; R.sup.5 is selected from —Y—R.sup.6 and —Z—R.sup.7; Y is selected from a chemical bond, O, NR.sup.0, R.sup.7 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, COR.sup.0, C(O)R.sup.0, C(O)N(R.sup.0).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.7 is H or is selected from aryl and a heterocyclic ring; each R.sup.0 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.

(32) An important component and conceptual teaching of the Invention described herein is that neither the R.sup.2 nor the R.sup.3 positions of the compounds of this invention are members of any aromatic or non-aromatic ring structure. We have discovered that compounds having the R.sup.2 and/or the R.sup.3 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.

(33) In preferred embodiments of the invention, ring A is an aromatic ring.

(34) In preferred embodiments of the invention, X.sup.1 or X.sup.2 is N. In another preferred embodiment, both X.sup.1 and X.sup.2 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:

(35) ##STR00003##

(36) In preferred embodiments of the invention, R.sup.5 is a group having the formula

(37) ##STR00004## wherein: X.sup.3 is N or CH; R.sup.61 is selected from aryl and a heterocyclic ring; Q is selected from a chemical bond or a group having the formula —O—, —(CH.sub.2).sub.i—, —(CH.sub.2).sub.iC(O)(CH.sub.2).sub.j—, —(CH.sub.2).sub.i—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iC(O)—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.i—C(O)O(CH.sub.2).sub.j—, —(CH.sub.2).sub.iN(R.sup.62)C(O)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iOC(O)N(R.sup.62)—(CH.sub.2).sub.j—, and —O—(CH.sub.2).sub.i—C(O)N(R.sup.62)—(CH.sub.2).sub.j—; R.sup.62 is selected from H, alkyl, aryl, and a heterocyclic ring; each R.sup.0 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.

(38) In further preferred embodiments of the invention, R.sup.5 is a group having the formula

(39) ##STR00005##
wherein:
X.sup.3 is N or CH;
Q.sup.1 is selected from a chemical bond or a group having the formula —O—, —CH.sub.2—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;
each R.sup.70 is selected from halo, alkyl, CN, N(R.sup.71).sub.2, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3,
each R.sup.71 is selected from H, alkyl, aryl, aralkyl and a heterocyclic ring; and
k is 0 to 4.

(40) In further preferred embodiments of the invention, R.sup.5 is a group having the formula

(41) ##STR00006##
wherein
X.sup.3 is N or CH;
Q.sup.1 is selected from a chemical bond or a group having the formula —O—, —CH.sub.2—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—;
R.sup.70 is selected from halo, alkyl, CN, N(R.sup.71).sub.2, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3; and
each R.sup.71 is selected from H, alkyl, aryl, aralkyl and a heterocyclic ring.
In particularly preferred embodiments one or more of the following selections is made: Q.sup.1 is —NH—; X.sup.3 is N; each R.sup.71 is independently selected from H, methyl, and ethyl, and preferably each R.sup.71 is methyl; and/or R.sup.71 is selected from OH, OCH.sub.3, halo, and CF.sub.3.

(42) In a preferred embodiment, if R.sup.2 or R.sup.4 is selected to be —NR.sup.22.sub.b— or —NR.sup.42—, respectively, then R.sup.31 is not selected from halo, —NH.sub.2, —N(H)(R.sup.0), —N(R.sup.0).sub.2, —O—R.sup.0, or OH.

(43) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula I.sub.a

(44) ##STR00007## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)(CH.sub.2).sub.qC(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; e is 0 to 4; each R.sup.22 is independently selected from H and C.sub.1-3 alkyl; R.sup.3 is selected from —CR.sup.31.sub.c—, —NR.sup.32.sub.d, —SO.sub.2—, and —(C═R.sup.33)—; each R.sup.31 group is selected from H, halo, —NH.sub.2, —N(H)(R.sup.0), —N(R.sup.0).sub.2, —O—R.sup.0, OH and C.sub.1-3 alkyl; each R.sup.32 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO.sub.2R.sup.0, C(O)R.sup.0, aryl, and a heterocyclic ring; R.sup.33 is selected from O, S, N—R.sup.34, and N—OR.sup.0; R.sup.34 is selected from H, NO.sub.2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring; R.sup.4 is selected from —CR.sup.41.sub.e—, —NR.sup.42.sub.f, —(C═R.sup.43)—, —SO.sub.2—, and —O—; each R.sup.41 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO.sub.2R.sup.0, C(O)R.sup.0, aralkyl, aryl, and a heterocyclic ring; each R.sup.42 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO.sub.2R.sup.0, C(O)R.sup.0, aryl, and a heterocyclic ring; each R.sup.43 is selected from O, S, N—R.sup.0, and N—OR.sup.0; with the provisos that when R.sup.4 is —NR.sup.42.sub.f— then R.sup.3 is not —NR.sup.32.sub.d— and that both R.sup.3 and R.sup.4 are not simultaneously selected from —(C═R.sup.33)— and —(C═R.sup.43)—, respectively; and that R.sup.3 and R.sup.4 are not simultaneously selected from —SO.sub.2—; R.sup.5 is selected from —Y—R.sup.6 and —Z—R.sup.7; Y is selected from a chemical bond, O, N—R.sup.0, R.sup.6 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, CO.sub.2R.sup.0, C(O)R.sup.0, C(O)N(R.sup.0).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.7 is H or is selected from aryl and a heterocyclic ring; each R.sup.0 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.

(45) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula I.sub.b

(46) ##STR00008## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)(CH.sub.2).sub.qC(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; each R.sup.22 is independently selected from H and C.sub.1-3 alkyl; each R.sup.32 group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, C.sub.2R.sup.0, C(O)R.sup.0, aryl, and a heterocyclic ring; R.sup.4 is selected from —CR.sup.41.sub.e—, —(C═R.sup.43)—, —SO.sub.2—, and —O—; each R.sup.41 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO.sub.2R.sup.0, C(O)R.sup.0, aralkyl, aryl, and a heterocyclic ring; each R.sup.43 is selected from O, S, N—R.sup.0, and N—OR.sup.0; R.sup.5 is selected from —Y—R.sup.6 and —Z—R.sup.7; Y is selected from a chemical bond, 0, N—R.sup.0, R.sup.6 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, C.sub.2R.sup.0, C(O)R.sup.0, C(O)N(R.sup.0).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.7 is H or is selected from aryl and a heterocyclic ring; each R.sup.0 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.

(47) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula I.sub.c

(48) ##STR00009## wherein ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)(CH.sub.2).sub.qC(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; X.sup.3 is N, CH or C—R.sup.2; each R.sup.2 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.21, —(CH.sub.2).sub.rC(O)(CH.sub.2).sub.sR.sup.21, —(CH.sub.2).sub.rC(O)N(R.sup.22)(R.sup.2), —(CH.sub.2).sub.rC(O)O(CH.sub.2).sub.sR.sup.21, —(CH.sub.2).sub.rN(R.sup.21)C(O)R.sup.21, —(CH.sub.2), N(R.sup.22)(R.sup.3), —N(R.sup.21)S.sub.2R.sup.21, —OC(O)N(R.sup.22)(R.sup.23), —SO.sub.2N(R.sup.22)(R.sup.23), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.2 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms; R.sup.21 is selected from H, alkyl, cycloalkyl; alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.22 and R.sup.23 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.22 and RU 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; r is 0 to 4; s is 0 to 4; m is 0 to 4; R.sup.4 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO.sub.2R.sup.0, C(O)R.sup.0, aryl, and a heterocyclic ring; a is 0 or 1; X.sup.4 is selected from

(49) ##STR00010## each R.sup.3 is independently selected from the group consisting of H, —N(R.sup.0).sub.2, alkyl, cycloalkyl, alkenyl, alkynyl, CO.sub.2R.sup.0, C(O)R.sup.0, aralkyl, aryl, and a heterocyclic ring, R.sup.3′ is selected from H, —N(R.sup.0).sub.2, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring, and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(50) In preferred embodiments of the invention, R.sup.2, R.sup.3 and R.sup.4 of formula I are selected to give the following chemical groups:
—N(R.sup.22)—N—C(R.sup.41)—
—N(R.sup.22)—N(R.sup.32)—C(═O)—
—N(R.sup.22)—N(R.sup.32)—C(R.sup.41)(R.sup.41)—
—N(R.sup.22)—C(R.sup.31)(R.sup.31)—C(R.sup.41)(R.sup.41)—
—N(R.sup.22)—C(R.sup.31)(R.sup.31)—C(═O)—
—N═N—C(R.sup.41)(R.sup.41)—
—C(R.sup.21)═C═C(R.sup.41)—
—C(R.sup.21)═C(R.sup.31)—C(═O)—
—C(R.sup.21)═C(R.sup.31)—C(R.sup.41)—
—C(R.sup.21)(R.sup.21)—C(R.sup.31)═C(R.sup.41)—
—C(R.sup.21)(R.sup.21)—C(R.sup.31)(R.sup.31)—C(═O)—
—C(R.sup.21)(R.sup.21)—C(R.sup.31)(R.sup.31)—C(R.sup.41)(R.sup.41)—
—C(R.sup.21)R.sup.21)—N(R.sup.32)—C(═O)—
—C(R.sup.21)(R.sup.21)—N(R.sup.32)—C(R.sup.41)(R.sup.41)—
—N(R.sup.22)—C(═O)—C(R.sup.41)(R.sup.41)—
—N(R.sup.22)—C(═O)—N(R.sup.41)—
—N(R.sup.22)—C(═O)—O—
—C(R.sup.21)(R.sup.21)—C(═O)—C(R.sup.41)(R.sup.41)—
—C(R.sup.21)(R.sup.21)—C(═O)—N(R.sup.42)—
—N(R.sup.22)—C(═NR.sup.34)—N(R.sup.42)—
—C(═O)—N(R.sup.32)—N(R.sup.42).

(51) Particularly preferred chemical groups for R.sup.2, R.sup.3 and R.sup.4 include:
—N(R.sup.22)—N═C(R.sup.41)—
—N(R.sup.22)—N(R.sup.32)—C(═O)—
—N(R.sup.22)—C(R.sup.31)(R.sup.31)—C(R.sup.41)(R.sup.41)—
—N(R.sup.22)—C(R.sup.31)(R.sup.31)—C(═O)—
—C(R.sup.21)(R.sup.21)—C(═O)—C(R.sup.41)(R.sup.41)—
—C(R.sup.21)(R.sup.21)—C(═O)—N(R.sup.42)—
—N(R.sup.22)—C(═NR.sup.34)—N(R.sup.42)—
—C(═O)—N(R.sup.32)—N(R.sup.42).

(52) In further preferred embodiment, R.sup.6 or R.sup.7 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 R.sup.21 and R.sup.22 are independently selected from groups which have small steric bulk and are preferably selected from H and CH.sub.3, and more preferably are H.

(53) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II

(54) ##STR00011## wherein ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(CH.sub.2).sub.qC(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; R.sup.8 is selected from the group consisting of is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO.sub.2R.sup.0, C(O)R.sup.0, aralkyl, aryl, and a heterocyclic ring; R.sup.9 is selected from —Y—R.sup.6 and —Z—R.sup.7; Y is selected from a chemical bond, O, N—R.sup.0, R.sup.6 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, CO.sub.2R.sup.0, C(O)R.sup.0, C(O)N(R.sup.0).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.7 is H or is selected from aryl and a heterocyclic ring; and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(55) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II.sub.a

(56) ##STR00012## wherein ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; R.sup.8 is selected from the group consisting of is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO.sub.2R.sup.0, C(O)R.sup.0, aralkyl, aryl, and a heterocyclic ring; X.sup.3 is N, CH or C—R.sup.50; each R.sup.50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.51, —(CH.sub.2).sub.rC(O)(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rC(O)N(R.sup.52)(R.sup.53), —(CH.sub.2).sub.rC(O)O(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rN(R.sup.51)C(O)R.sup.51, —(CH.sub.2).sub.rN(R.sup.52)(R.sup.53), —N(R.sup.51)SO.sub.2R.sup.51, —OC(O)N(R.sup.52)(R.sup.53), —SO.sub.2N(R.sup.52)(R.sup.53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms; R.sup.51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.52 and R.sup.53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.52 and R.sup.53 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; r is 0 to 4; s is 0 to 4; m is 0 to 4; and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(57) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II.sub.b

(58) ##STR00013## wherein: R.sup.14 is selected from H and F; R.sup.8 is selected from the group consisting of is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO.sub.2R.sup.0, C(O)R.sup.0, aralkyl, aryl, and a heterocyclic ring; X.sup.3 is N, CH or C—R.sup.60; each R.sup.60 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, halo, aryl, and a heterocyclic ring; R.sup.61 is selected from aryl and a heterocyclic ring; Q is selected from a chemical bond or a group having the formula —O—, —(CH.sub.2).sub.i—, —(CH.sub.2).sub.iC(O)(CH.sub.2).sub.j—, —(CH.sub.2).sub.i—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iC(O)—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iC(O)O(CH.sub.2).sub.j—, —(CH.sub.2).sub.iN(R)C(O)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iOC(O)N(R.sup.62)(CH.sub.2).sub.j—, and —O—(CH.sub.2).sub.i—C(O)N(R.sup.62)—(CH.sub.2).sub.j—; R.sup.62 is selected from H, alkyl, aryl, and a heterocyclic ring; each R.sup.0 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.

(59) In preferred embodiments of compounds of the formula II.sub.b, R.sup.60 is selected from halo, CF.sub.3, and OH. In other preferred embodiments, R.sup.8 is selected from H and CH.sub.3.

(60) In preferred embodiments of compounds of the formula II.sub.b, X.sup.3 is N. In further preferred embodiments, Q is selected to be —(CH.sub.2).sub.i—N(R.sup.62)—(CH.sub.2).sub.j—, and particularly in preferred embodiments, Q is —N(R.sup.62)—.

(61) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II.sub.c

(62) ##STR00014## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.11, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; R.sup.8 is selected from H and methyl; X.sup.3 is N or CH; R.sup.61 is selected from aryl and a heterocyclic ring; Q is selected from a chemical bond or a group having the formula —O—, —(CH.sub.2).sub.i—, —(CH.sub.2).sub.iC(O)(CH.sub.2).sub.j—, —(CH.sub.2).sub.i—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iC(O)—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iC(O)O(CH.sub.2).sub.j—, —(CH.sub.2).sub.iN(R.sup.62)C(O)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iOC(O)N(R.sup.62)—(CH.sub.2).sub.j—, and —O—(CH.sub.2).sub.i—C(O)N(R.sup.62)—(CH.sub.2).sub.j—; R.sup.62 is selected from H, alkyl, aryl, and a heterocyclic ring; each R.sup.0 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.

(63) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II.sub.d

(64) ##STR00015## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3 NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring; R.sup.8 is selected from H and CH.sub.3; X.sup.3 is N or CH; Q.sup.1 is selected from a chemical bond or a group having the formula —O—, —CH.sub.2—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—; each R.sup.70 is selected from halo, alkyl, CN, N(R.sup.7%, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3, each R.sup.71 is selected from H, alkyl, aryl, aralkyl and a heterocyclic ring; and k is 0 to 4.

(65) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II.sub.e

(66) ##STR00016## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3 NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring; R.sup.8 is selected from H and CH.sub.3; X.sup.3 is N or CH; Q.sup.1 is selected from a chemical bond or a group having the formula —O—, —CH.sub.2—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—; each R.sup.70 is selected from halo, alkyl, CN, N(R.sup.71).sub.2, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3; and each R.sup.71 is selected from H and alkyl.

(67) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II.sub.f

(68) ##STR00017##
wherein
R.sup.14 is selected from H and F;
R.sup.8 is selected from H and CH.sub.3;
each R.sup.70 is selected from halo, alkyl, CN, N(R.sup.71).sub.2, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3,
each R.sup.71 is selected from H, alkyl, aryl, aralkyl and a heterocyclic ring; and
k is 0 to 4.

(69) Exemplary compounds of the formula II, II.sub.a, II.sub.b, II.sub.c, II.sub.d, II.sub.e or II.sub.f includes the following structures:

(70) ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115## ##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121## ##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135##

(71) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula III

(72) ##STR00136## wherein ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.12), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; R.sup.10 is selected from —Y—R.sup.18; Y′ is selected from a chemical bond, O, NR.sup.0—, 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, CO.sub.2R.sup.0, C(O)R.sup.0, C(O)N(R.sup.0).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.18 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF.sub.3, aryl, and a heterocyclic ring; and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(73) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula III.sub.a

(74) ##STR00137## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; X.sup.3 is N, CH or C—R.sup.50; each R.sup.50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF, NO.sub.2, OR.sup.51, —(CH.sub.2).sub.rC(O)(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rC(O)N(R.sup.52)(R.sup.53), —(CH.sub.2).sub.rC(O)O(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rN(R.sup.51)C(O)R.sup.51, —(CH.sub.2).sub.rN(R.sup.52)(R.sup.53), —N(R.sup.51)SO.sub.2R.sup.51, —OC(O)N(R.sup.52)(R.sup.53), —SO.sub.2N(R.sup.52)(R.sup.53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms; R.sup.51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.52 and R.sup.53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.52 and R.sup.53 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, C.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; r is 0 to 4; s is 0 to 4; m is 0 to 4; and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(75) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula III.sub.b

(76) ##STR00138## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; X.sup.3 is N or CH; R.sup.61 is selected from aryl and a heterocyclic ring; Q is selected from a chemical bond or a group having the formula —O—, —(CH.sub.2).sub.i—, —(CH.sub.2).sub.iC(O)(CH.sub.2).sub.j—, —(CH.sub.2).sub.i—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iC(O)—N(R.sup.62)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iC(O)O(CH.sub.2).sub.j—, —(CH.sub.2).sub.iN(R.sup.62)C(O)—(CH.sub.2).sub.j—, —(CH.sub.2).sub.iOC(O)N(R.sup.62)—(CH.sub.2).sub.j—, and —O—(CH.sub.2).sub.i—C(O)N(R.sup.62)—(CH.sub.2).sub.j—; R.sup.62 is selected from H, alkyl, aryl, and a heterocyclic ring; each R.sup.0 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.

(77) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula III.sub.c

(78) ##STR00139## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.11), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; X.sup.3 is N or CH; each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring; Q.sup.1 is selected from a chemical bond or a group having the formula —O—, —CH.sub.2—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—; each R.sup.70 is selected from halo, alkyl, CN, N(R.sup.71).sub.2, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3, each R.sup.71 is selected from H, alkyl, aryl, aralkyl and a heterocyclic ring; and k is 0 to 4.

(79) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula II.sub.d

(80) ##STR00140## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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, wherein the 5- to 7-membered ring may optionally be substituted with one to three substituents that are independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.0, CO.sub.2R.sup.0, C(O)R.sup.0, halo, aryl, and a heterocyclic ring; p is 0 to 4; q is 0 to 4; each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring; R.sup.8 is selected from H and CH.sub.3; X.sup.3 is N or CH; Q.sup.1 is selected from a chemical bond or a group having the formula —O—, —CH.sub.2—, —NH—, —C(O)—NH—, —C(O)O—, —NH—C(O)—, —OC(O)NH—, and —O—C(O)NH—; each R.sup.70 is selected from halo, alkyl, CN, N(R.sup.71).sub.2, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3; and each R.sup.71 is selected from H and alkyl.

(81) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula III.sub.e

(82) ##STR00141##
wherein
R.sup.14 is selected from H and F;
each R.sup.70 is selected from halo, alkyl, CN, N(R.sup.71).sub.2, cyclic-amino, NO.sub.2, OR.sup.71, and CF.sub.3,
each R.sup.71 is selected from H, alkyl, aryl, aralkyl and a heterocyclic ring; and
k is 0 to 4.

(83) Exemplary compounds of the formula III, III.sub.a, III.sub.b, III.sub.c, III.sub.d, or III.sub.e includes the following structures:

(84) ##STR00142## ##STR00143## ##STR00144##

(85) In a further embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula IV

(86) ##STR00145## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; e is 0 to 4; R.sup.22 is selected from H and C.sub.1-3 alkyl; R.sup.34 is selected from H, NO.sub.2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring; R.sup.44 is selected from H, alkyl, cycloalkyl, —(C═O)R.sup.0, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.45 is selected from —Y″—R.sup.19; Y″ is selected from a chemical bond, O, NR.sup.0—, 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, CO.sub.2R.sup.0, C(O)R.sup.0, C(O)N(R.sup.0).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.19 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF.sub.3, aryl, and a heterocyclic ring; and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(87) Exemplary compounds of the formula IV include the following structures:

(88) ##STR00146##

(89) In a further embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula V

(90) ##STR00147## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; R.sup.22 is selected from H and C.sub.1-3 alkyl; R.sup.34 is selected from H, NO.sub.2, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and a heterocyclic ring; R.sup.55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.56 is selected from —Y″—R.sup.19; Y″ is selected from a chemical bond; O, NR.sup.0—, 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, CO.sub.2R.sup.0, C(O)R.sup.0, C(O)N(R).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.19 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF.sub.3, aryl, and a heterocyclic ring; and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(91) In a further embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula V.

(92) ##STR00148## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2)R.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; R.sup.55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; X.sup.3 is N or C—R; each R.sup.50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.51, —(CH.sub.2).sub.rC(O)(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rC(O)N(R.sup.52)(R.sup.53), —(CH.sub.2).sub.rC(O)O(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rN(R.sup.51)C(O)R.sup.51, —(CH.sub.2).sub.rN(R.sup.52)(R.sup.53), —N(R.sup.51)SO.sub.2R.sup.51, —OC(O)N(R.sup.52)(R.sup.53), —SO.sub.2N(R.sup.52)(R.sup.53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms; R.sup.51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.52 and R.sup.53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.52 and R.sup.53 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 R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(93) Exemplary compounds of the formula V or V.sub.a include the following structures:

(94) ##STR00149##

(95) In a further embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula VI

(96) ##STR00150## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 may betaken 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; e is 0 to 4; R.sup.55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.56 is selected from —Y″—R.sup.19; Y″ is selected from a chemical bond, O, NR.sup.0—, 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, CO.sub.2R.sup.0, C(O)R.sup.0, C(O)N(R.sup.11).sub.2, CN, CF.sub.3, N(R.sup.0).sub.2, NO.sub.2, and OR.sup.0; R.sup.19 is selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF.sub.3, aryl, and a heterocyclic ring; and each R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(97) In a further embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula VI,

(98) ##STR00151## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R.sup.1; X.sup.2 is selected from N, N—R.sup.0 or C—R.sup.1; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2).sub.pC(O)(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —S.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; R.sup.55 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; X.sup.3 is N or C—R.sup.50; each R.sup.50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.51, —(CH.sub.2).sub.rC(O)(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rC(O)N(R.sup.52)(R.sup.53), —(CH.sub.2).sub.rC(O)O(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rN(R.sup.51)C(O)R.sup.51, —(CH.sub.2).sub.rN(R.sup.52)(R.sup.53), —N(R.sup.51)SO.sub.2R.sup.51, —OC(O)N(R.sup.52)(R.sup.53), —SO.sub.2N(R.sup.52)(R.sup.53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.30 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms; R.sup.51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.52 and R.sup.53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.52 and R.sup.53 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 R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(99) Exemplary compounds of the formula VI or VI.sub.a include the following structures:

(100) ##STR00152## ##STR00153## ##STR00154## ##STR00155##

(101) In a further preferred embodiment, the present invention provides inhibitors of the P210.sup.BCR-ABL-T315I theramutein having the formula VII

(102) ##STR00156## wherein: ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused bicyclic ring; X.sup.1 is selected from N, N—R.sup.0 or C—R X.sup.2 is selected from N, N—R.sup.0 or C—R; the dotted lines represent optional double bonds; each R.sup.1 is independently selected from the group consisting of H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.11, —(CH.sub.2)C(O)(CH.sub.2).sub.pR.sup.11, —(CH.sub.2).sub.pC(O)N(R.sup.12)(R.sup.13), —(CH.sub.2).sub.pC(O)O(CH.sub.2).sub.qR.sup.11, —(CH.sub.2).sub.pN(R.sup.11)C(O)R.sup.11, —(CH.sub.2).sub.pN(R.sup.12)(R.sup.13), —N(R.sup.11)SO.sub.2R.sup.11, —OC(O)N(R.sup.12)(R.sup.13), —SO.sub.2N(R.sup.12)(R.sup.13), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.1 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 R.sup.11 is independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; each R.sup.12 and R.sup.13 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.12 and R.sup.13 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; e is 0 to 4; ring B is selected from a cycloalkyl group having 5 or 6 ring atoms, and a heterocyclic group containing 5 or 6 ring atoms which includes one to three hetero atoms; each R.sup.50 is independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF.sub.3, NO.sub.2, OR.sup.51, —(CH.sub.2).sub.rC(O)(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rC(O)N(R.sup.52)(R.sup.53), —(CH.sub.2).sub.rC(O)O(CH.sub.2).sub.sR.sup.51, —(CH.sub.2).sub.rN(R)C(O)R.sup.51, —(CH.sub.2).sub.rN(R.sup.52)(R.sup.53), —N(R.sup.51)SO.sub.2R.sup.51, —OC(O)N(R.sup.52)(R.sup.53), —SO.sub.2N(R.sup.52)(R.sup.53), halo, aryl, and a heterocyclic ring, and additionally or alternatively, two R.sup.50 groups on adjacent ring atoms form a 5- or 6-membered fused ring which contains from 0 to 3 heteroatoms; R.sup.51 is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; R.sup.52 and R.sup.53 are independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring; or R.sup.52 and R.sup.53 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 R.sup.0 is independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and a heterocyclic ring.

(103) Exemplary compounds of the formula VII include the following structures:

(104) ##STR00157## ##STR00158##

(105) 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.

(106) For each of the above descriptions of compounds of the structures I, I.sub.a, I.sub.b, II, II.sub.a, etc., 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.

(107) 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.

(108) 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.

(109) 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, II.sub.a, V, V.sub.a, VI, and VI.sub.a) are depicted herein in either cis of trans configuration, both configurations are meant to be encompassed by the each of the formulae.

(110) 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.

(111) 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.

(112) Compounds of the formula II 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. Med. Chem. 2002, 11, 556-562).

(113) ##STR00159##
For example, heating A with 1.1 equivalents of B for 1 to 24 hours in a protic solvent such as a C.sub.1 to C.sub.6 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 C.sub.4 to C.sub.18 reverse phase medium. Similar methodology would be applicable in the cases where “Aryl” is replaced by other groups defined under R.sup.5.

(114) Compounds of the formula III 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.

(115) ##STR00160##

(116) For example, treatment of D with an active ester such as Aryl-C(O)—OC.sub.6F.sub.5 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 C.sub.4 to C.sub.18 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.

(117) 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. Med. Chem. 2002, 11, 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 C.sub.6 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.

(118) ##STR00161##

(119) Embodiments wherein R.sup.2 is —NR.sup.22 and R.sup.3 is —C(═R.sup.33) 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 R.sup.12NH.sub.2 effects displacement of LG′ to afford intermediates K. An example of this chemical transformation wherein R.sup.12 is H and LG′ is CH.sub.3SO.sub.2— is reported by Capps, et al. J. Agric. Food Chem. 1993, 41, 2411-2415, and an example wherein R.sup.12 is H and LG′ is Cl is reported in Marshall, et al. J. Chem. Soc. 1951, 1004-1015.

(120) ##STR00162##

(121) Intermediates of structure K are transformed to compounds of the invention by simultaneous or sequential introduction of the elements, of R.sup.3, R.sup.4, and R.sup.5. For example, treatment of intermediates of structure K with individual isocyanates R.sup.6—N═C═O affords in a single step compounds of structure M, which are compounds of the invention wherein R.sup.2═—NR.sub.22—, R.sup.3═—C═O—, R.sup.4═—NH—, and R.sup.5=-chemical bond-R.sup.6. Alternative methods to convert compounds of structure K to compounds of structure M are well known to those skilled in the art, wherein R.sup.3 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 R.sup.6—NH.sub.2, to introduce R.sup.5 and R.sup.6.

(122) ##STR00163##

(123) Alternatively, treatment of intermediates of structure K with a reagent such as cyanamide (NH.sub.2—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 (NH.sub.2—C(—NH)—SO.sub.3H). 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.

(124) ##STR00164##

(125) 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.

(126) ##STR00165##

(127) 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 Tor U, respectively, which are further embodiments of the invention.

(128) ##STR00166##

(129) ##STR00167##

(130) 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.

(131) ##STR00168##

(132) Embodiments of the invention wherein R.sup.2═CO, R.sup.3═—NR.sup.32−, R.sup.4 ═N—, and R.sup.5═ZR.sup.7, wherein Z is a hydrocarbon chain and R.sup.7 is as defined above, may be prepared as follows. When R.sup.32═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 7.

(133) ##STR00169##

(134) 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.

(135) When R.sup.32 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 R.sup.32NHNH-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.

(136) ##STR00170##

(137) 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 R.sup.2, R.sup.3, R.sup.4, and R.sup.5 claimed by this invention are prepared by obvious modifications of the above processes.

(138) 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, 2.sup.nded.; Wiley: New York, 1991).

(139) One embodiment of this invention is directed to any endogenously occurring mammalian target protein selected by the skilled investigator to be of interest for the identification and/or optimization of a compound as an inhibitor or activator of said protein. In general such selected proteins will already be known to be involved in the etiology or pathogenesis of a human disease. In another embodiment, the invention is also directed toward mutant forms of such mammalian proteins. 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.

(140) 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.

(141) 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.

(142) 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.

(143) In one embodiment of the invention, the target protein (POI) may be any endogenously encoded mammalian protein. In another aspect of the invention, the POI is a theramutein, which is a protein that is altered for the first time with respect to a commonly occurring “wild type” form of the protein (i.e., a wild type protein is the prototheramutein from which the theramutein arises). In yet another aspect of the invention, a theramutein is a variant of a protein that is, itself, already a mutein (i.e., a mutein is the prototheramutein from which the theramutein arises). 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.

(144) It is understood that a protein of interest (POI) is an endogenously encoded mammalian protein. It will also 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 used to inhibit 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 used to activate 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.

(145) It will be appreciated that one aspect of the invention is the identification of an agent that is active against a selected POI whose cellular function contributes to a given disease state such that activators or inhibitors of said POI would be expected to be therapeutically effective during the course of treatment for the disease. No limitation of any kind or nature is intended on the type of disease that may be treated, nor on the type of protein that may be targeted for modulation according to the teachings herein, provided that all other limitations stated herein are met, including the fact that any such protein that is selected for targeting must be an endogenous protein. Obviously, the skilled investigator may use non-endogenously occurring nucleic acids such as cDNAs in order to practice the method taught herein provided that the amino acid sequence corresponds to an endogenously occurring POI.

(146) It will also be appreciated that, whereas one aspect of the invention is the identification of an agent that is active against a protein or theramutein that arises or becomes dominant (by any mechanism) prior to or 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.

(147) 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 increases 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 PKC.sub.β1” should be construed to mean a substance that binds to and activates PKC.sub.β1. Similarly, an “inhibitor of p210.sup.Bcr-Abl” is a substance that binds to and inhibits the functioning of p210.sup.Bcr-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.

(148) 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 of the invention as compared to either:

(149) a) the ability of the same substance under comparable conditions to inhibit the prototheramutein; or

(150) b) the ability of a second substance (usually a known inhibitor of the prototheramutein) to inhibit the theramutein under comparable conditions; or

(151) c) the ability of the second substance to inhibit the prototheramutein under comparable conditions.

(152) 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.

(153) 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.

(154) Reference to FIG. 3 is informative in understanding and elucidating these concepts.

(155) 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 IC.sub.50 values on the corresponding prototheramutein and theramutein. The skilled investigator will easily recognize other useful variations and comparable conditions.

(156) 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 give 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. Such an analysis may assist the skilled investigator in classifying various compounds into discrete categories that may be helpful in guiding further lead optimization or biological profiling studies on such compounds.

(157) 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.

(158) 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).

(159) 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).

(160) In one embodiment of the invention, a theramutein is selected and used in a phenoresponse-based cellular assay system of the present invention 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 IC.sub.50 or AC.sub.50 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 as it pertains to theramuteins is that a true theramutein be properly selected according to the teachings herein.

(161) Thus, for example, randomly generated site directed mutants of known proteins that are created in the laboratory but that have Mg 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 either.

(162) For example, in an effort to obtain potential inhibitors of mutants of p210.sup.Bcr-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 p210.sup.Bcr-Abl theramuteins, however, it showed activity against some of the mutants such as p210.sup.Bcr-Abl-E255K, but the p210.sup.Bcr-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 P210.sup.Bcr-Abl theramuteins than it was against the wild-type p210.sup.Bcr-Abl. When the compound was tested against p210.sup.Bcr-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 p210.sup.Bcr-AblT315I theramutein.

(163) 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 p210.sup.Bcr-AblT315I theramutein to an equal or greater extent than STI-571 is able to do with respect to the wild type p210.sup.Bcr-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).

(164) 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 p210.sup.Bcr-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 p210.sup.Bcr-AblT315I theramutein. Moreover, the skilled investigator will immediately recognize the applicability and generalizability of this approach to any highly drug-resistant theramutein. Finally, the skilled investigator will further recognize that linking a phenoresponse as defined herein to the increased presence and functional activity of a particular POI in the cell under appropriate conditions allows one to utilize the method with any given endogenous target protein for which a therapeutically effective compound is sought.

(165) 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 protein-of-interest (POI) or theramutein-of-interest (TOI) in the cell under appropriate conditions. With respect to a theramutein, 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 protein in the cell, and is a result of the absolute amount of the protein and its specific activity. Often, the phenotypic characteristic is observable as a result of elevated levels of protein activity and is not apparent in cells that express low amounts of the protein, or if the protein is also a theramutein, then the phenotypic characteristic will often not be apparent in cells expressing low amounts of either the theramutein or its corresponding prototheramutein. Further, it can often be demonstrated that the phenotypic characteristic is modulated by modulating the specific activity of the protein 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. (However, clearly a known inhibitor or activator of a given prototheramutein will always exist as a result of the intrinsic definition of the nature of a theramutein itself.) 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 gene encoding a given POI (such as a theragene in the case of a theramutein), 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.

(166) 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 POI such as 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.

(167) 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, V 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 protein causes a transient change in phosphorylation of a substrate of the protein, 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 protein. 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.

(168) The characteristic selected for measurement within the cell must not be an intrinsic physical or chemical property of the protein (or 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 protein (or theramutein or prototheramutein) 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 a 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.

(169) The term “responsive phenotypic characteristic” means a characteristic of the cell which is responsive to inhibitors or activators of a given protein (including, e.g., a 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.

(170) A useful phenotypic characteristic, as exemplified herein in association with p210.sup.Bcr-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 protein of interest (POI) 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, when also demonstrated to be responsive to a known modulator of the POI is referred to herein as a “phenoresponse.” In the special case of a theramutein which has no known modulator, a modulator of the prototheramutein must be utilized to establish a phenoresponse to be used with the theramutein. The conception and recognition of this highly useful property of a cell represents one of the substantial advances of this invention over the prior art, including Applicant's own prior original work in the general area of cell-based assays (U.S. Pat. Nos. 4,980,281; 5,266,464; 5,688,655; 5,877,007). The identification and selection of the phenoresponse provides the skilled investigator with a cellular assay system that is extremely sensitive in terms of its ability to identify inhibitors or activators of the POI, and therefore identifies such chemical agents with a much higher degree of assurance than any other related assay method disclosed in the prior art.

(171) Though not always necessary, it will often be advantageous to employ cells that express high levels of the POI, and to select a phenotypic characteristic that results from overexpression of the POI. This is because phenotypic characteristics linked to the functioning of the POI generally become more distinguishable (easier to measure) as a POI is overexpressed to a greater extent. Further, phenoresponses that are observed in response to modulators of the POI are often amplified as the functional level of the POI is increased. Expressed another way, the selected phenoresponse observed in cells that overexpress the protein (or theramutein) is particularly sensitive to modulators of the protein (or theramutein).

(172) Preferably, the protein is stably expressed in a test cell. Stable expression results in a level of the protein 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 proteins of the invention, such down-regulation is usually sufficiently overcome by artificially overexpressing the protein. 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 protein, rather than a change in its level, even if down-modulation of the protein subsequently occurs. For these reasons, although stable expression of the protein is preferred, transfection followed by transient expression of the protein 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 protein that 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).

(173) The term “cellular specificity” means the ability of a compound, at a given concentration, to modulate a selected phenoresponse of the Test cell without affecting the Control Cell to the same extent, if at all. The term “cellular specificity gap” (“CSG”) means a measurement of the ability of a selected compound to modulate the selected phenoresponse corresponding to a given target protein (not limited to a theramutein) in a test cell relative to the ability of said compound to modulate the same phenoresponse in a corresponding control cell. For the purposes of applying the CSG technique to non-theramutein endogenous target proteins, the selected phenoresponse must have been previously defined using a known inhibitor or activator of the target protein.

(174) Determination of the CSG provides the skilled investigator with a method of comparing the relative potential therapeutic value of different compounds within a group of compounds (two or more) by comparing their relative cross-reactivity with control cells irrespective of the potency against the target protein of any given compound within the group. Compounds that exhibit the greatest “specificity” in their activity against test cells relative to control cells are generally the most desirable compounds, since a “wide” CSG will assist in selecting a compound that may reasonably be assumed to have minimal potential side effects in patients as compared to other compounds within the aforementioned group that have “narrow” CSGs. The effects of the CSG measurement are seen most easily when comparing cell-based assay generated dose-response curves in their entirety, however the following hypothetical example is also instructive.

(175) Consider the following table of hypothetical compounds and their corresponding IC.sub.50 values using a cell-free assay system. This example uses a protein kinase as the target protein. This is the sort of situation that investigators skilled in the art are faced with on a daily basis when dealing with the problem of trying to perform lead optimization on a selected compound or group of compounds for the purpose of identifying a potential optimized lead candidate compound for subsequent pre-clinical (animal) and clinical studies.

(176) TABLE-US-00001 TABLE 1 Cell-Free Purified Protein Kinase Inhibition Assay. IC.sub.50 against Target IC.sub.50 against a Non-Target IC.sub.50 Compound Protein Kinase (nM) Protein Kinase (nM) Ratio A 0.2 10,000 50,000 B 3 10,000 3,333 C 250 10,000 40 D 500 10,000 20

(177) A standard approach in the art at the present time is to identify compounds that exhibit a high degree of potency with respect to inhibition of the target protein kinase's enzymatic activity in a cell-free assay system without showing significant inhibitory activity against a distinct but closely related protein kinase. As the results of the cell-free assay system shown above in Table 1 indicate, compound A is the most potent of the series of compounds (A,B,C,D) and also shows the largest difference between its IC.sub.50 against the target protein relative to its effect on the non-target protein. For example, if one were interested in identifying inhibitors of the Abl kinase, one might use another protein kinase, such as the EGF receptor, c-kit, or c-Src, as a “negative” control kinase in such an assay. As with c-Abl, all of these latter enzymes are tyrosine protein kinases. Indeed, it is commonplace in the field at the present time to use so-called “panels” of protein kinases, including serine/threonine kinases, tyrosine kinases, and dual-specificity kinases, in order to identify compounds that inhibit as few protein kinases as possible (other than the target protein kinase itself). The reasoning behind this approach is that the fewer the number of kinases that are inhibited in a cell-free system by a given compound, the less likely the compound is to have untoward side effects in the patient. However, there is very little clinical evidence that actually supports this view.

(178) Furthermore, in some cases it has been argued by others that compounds that target more than one kinase may have additional therapeutic effects as compared to those compounds that are highly specific for only a single target protein. There is some evidence for this being true in the case of imatinib, whose cross-reactivity with c-kit has resulted in beneficial effects for patients with certain histologic types of carcinoma of the small intestine, as discussed previously herein. Despite this cross-reactivity with c-kit, however, imatinib displays a high degree of cellular specificity in the assay systems of the present invention, which is consistent with its high degree of clinical efficacy and relatively modest side effect profile within the first three years of treatment. However, as the specificity of a given compound drops in the cellular systems of the present invention, the increased cross-reactivity with other targets such as (in this example) other protein kinase family members may result in untoward side effects in the patient. This is discussed in further detail below.

(179) TABLE-US-00002 TABLE 2 Results from Applying the Method(s) of the Invention by Utlilizing a Phenoresponse-Based Cellular Assay System and Measuring the Cellular Specificity Gap (CSG) IC.sub.50 against IC.sub.50 against Compound Test Cells (nM) Control Cells (nM) CSG A 1 1 1 B 10 100 10 C 500 20,000 40 D 10 200 20

(180) The conclusion that would be drawn by the investigator from the results of the cell-free assay shown in Table 1 above is that compound A is the most potent compound, showing a 50% inhibitory concentration (IC.sub.50) value of 0.2 nanomolar (0.2 nM). As shown in Table 2 and FIG. 14, however, this compound, shows no specificity for the Test cells relative to the Control Cells, since it's IC.sub.30 for the Control cells is also 1 nanomolar. Similarly, compounds B and D are still quite potent, with both having IC.sub.50's of 10 nM against the Test cells, whereas compound D is more specific than B since its IC.sub.50 for the Control cells is higher (200 nM). The CSG measurements of compounds B and D immediately reflect that compound D would be the preferred compound between these two, all other considerations being equal. Most importantly, however, is that compound C is shown in this example to be the best compound of the group in terms of its CSG, at 40 (Table 2, FIG. 15). This means that compound C shows the greatest specificity for the Test cells relative the Control cells, and would be expected to have the lowest incidence of inducing unwanted side effects in patients, since this finding is derived from a direct testing of the compound in a living cellular system, the ultimate point of pharmacological action for the overwhelming majority of medicines. The important point in this example is that the CSG measurement allows the skilled investigator to rank order the potential therapeutic value of a series of compounds independently of their potency in cell-free systems. Thus, although compound C is the least potent compound, it is the most specific in its ability to inhibit the Test cells while leaving the Control cells relatively unaffected over wide concentration range. This is reflected in its CSG of 40, and demonstrates that compound C, rather than compound A, is the compound that should be given the highest priority for further pre-clinical and clinical development efforts.

(181) Use of the method of present invention in this manner provides for an ability to rank order and prioritize the pharmaceutical discovery and development process in a manner which was not possible previously. Through iterative application of the approach given above, the skilled investigator working together with medicinal chemists may synthesize analogus of compounds that score positively in the assay systems described herein, test such compounds in the assay methods of the invention, rank order the compounds according to their CSG values, select the best ones for further development, and repeat the process as many times as necessary in order to fully develop and optimize compounds that exhibit a high degree of specificity against, a given Test cell relative to its corresponding Control cell. Once a given compound has been optimized using the system described herein, which generally means that the CSG between Control and Test cells (if measured using the cellular IC.sub.50 ratio method described above) is at least three to five fold, the skilled investigator can then proceed to complete the lead optimization process through additional chemical modifications of the compound selected in this way and tested for properties such as plasma half-life, oral bioavailability, and related parameters using appropriate animal models. Of course, the likelihood of such compounds having the desired effects on the target protein in the cellular environment is virtually assured as a result of the process of optimizing said compounds according to the methods described herein, rather than with older, cell-free methods.

(182) It will be immediately apparent to the skilled investigator that analogous approaches may also be utilized with activators of a given target protein. It will also be apparent to the skilled investigator that there are other ways of determining the CSG. For example, using the inhibitor example given above, another useful approach to determine the CSG is to measure the ratio of the highest concentration of a compound which results in 50% growth inhibition of the control cell line, divided by the lowest concentration of the compound at which at least 90% of the test cell line is inhibited. Other obvious modifications of this approach may be utilized as well, including computing the logarithm of the concentrations at which compounds show a given percentage of activity, normalization of either the control or test cell responses relative to one another, etc. No limitation is intended on the nature of the computed or observed comparison of the control cell responsiveness to the test cell responsiveness for the purposes of determining the CSG.

(183) If one uses the IC.sub.50-ratio of control cells/test cells method as described above, then compounds with CSG values less than or equal to 1 would not generally be considered to be good clinical candidate compounds, whereas compounds with CSG values of greater than approximately 10 would be quite promising and worthy of further consideration.

(184) This example also highlights the distinctions between the effects of a given compound in a cell-free system versus the more medically and physiologically relevant cell-based system of the present invention.

(185) In an embodiment of the invention, compound profiling is used to identify and/or minimize side effects associated with administration of a compound to a patient. The cell-based lead optimization method allows early identification of potential side effects as compared to the cell-free approach. For example, imatinib shows a wide cellular specificity gap according to the methods of the invention described herein. This is consistent with imatinib's significant advance in the area of anti-cancer drugs. However, it is not without side effects. Recent evidence demonstrates that imatinib is associated with cardiac toxicity in a small percentage of patients (Kerkelä et al., 2006). This group may increase over time as patients take imatinib for longer periods of time.

(186) Through the use of the methods of the invention, FIG. 16 shows that imatinib tested at various concentrations on the wild type Ba/F3 cell line shows a slight but significant growth inhibitory effect at concentrations that are substantially below the apparent IC.sub.50 for cellular toxicity (about 10 μM) on the control cell line that imatinib exhibits at markedly higher concentrations. Such results become even more evident when the comparison of the effects of other compounds on the Control cell line are compared to those of imatinib.

(187) In still another implementation of the method, compounds which may show promising activity in a cell-free system but have small CSG values (as discussed above) would be expected to have higher potential side effects in patients, especially over longer treatment periods. Compounds having low CSG values have been reported by others with respect to certain targets such at p210 Bcr-Abl.sup.T315I mutant (Carter et al., 2005), and still other groups have even entered such compounds into clinical trials. However, based upon the teachings of this invention it may be expected that such compounds will have an increased incidence of untoward side effects in patients.

(188) A preferred drug screening method of the present invention involves the following:

(189) 1) Identification of a protein of interest (POI), such as a theramutein for which a novel inhibitor or activator is desired. Often, the POI is implicated or suspected to be implicated in establishment or maintenance of a disease state, perhaps due to inappropriate expression or a mutation-induced change in specific activity. Identification of an appropriate theramutein, for example, 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.

(190) 2) Provision of a test cell that expresses the POI and displays an observable (measurable) phenotypic characteristic that is linked to expression of the POI. In the case of a theramutein, the phenotypic characteristic is usually one which has been previously shown to be responsive to inhibitors or activators of the theramutein or, more commonly, the corresponding prototheramutein. Such a phenotypic characteristic that is linked to expression of the POI and has been previously shown to be responsive to inhibitors or activators of the POI (or the prototheramutein-of-interest (pTOI)) is defined herein 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 TOI 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. (Gorre et al., 2001; Housey et al., 1988). In one embodiment, overexpression results in a level of the theramutein that is-Sat 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.

(191) 3) Provision of a control cell that expresses the POI to a lesser extent or not at all (e.g., an unmodified host cell or host cell harboring an expression vector that does not express the POI). In the case of a theramutein of interest, the control cell can also be a cell expressing the prototheramutein corresponding to the theramutein of interest.

(192) 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 and 2001).

(193) 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).

(194) 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).

(195) 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.

(196) 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.

(197) 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.

(198) 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.

(199) 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.

(200) In a particular embodiment of the invention, the method is used to identify A substances that are inhibitors of the p210.sup.Bcr-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.

(201) Another useful assay is the determination of the state of phosphorylation of a direct substrate of p210.sup.Bcr-Abl-T315I. One such substrate is Crkl (Gorre 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 p210.sup.Bcr-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.

(202) 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 p210.sup.Bcr-Abl-wt.

(203) According to the present invention, a therapeutically effective amount of one or more compounds that modulate the functional activity of a p210.sup.Bcr-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.

(204) 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 p210.sup.Bcr-Abl tyrosine kinase signaling in CML has been extensively demonstrated, as has the role of theramuteins of p210.sup.Bcr-Abl in drug resistant recurrence of CML. Further, different muteins of p210.sup.Bcr-Abl exhibit varying sensitivity to inhibitors of p210.sup.Bcr-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.

(205) 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 protein modulators.

(206) 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.

(207) 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.

(208) 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.

(209) 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.

(210) 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.

(211) 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.

(212) 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.

(213) 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.

(214) 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.

(215) 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 triethylamine, di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine, ethylenediamine or 2-phenylethylamine. (see, Berge et al. J. Pharm. Sci. 1977, 66, 1-19).

(216) 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.

(217) 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.

(218) 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, 2.sup.nd 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

(219) It is to be understood and expected that variations in the principles of the 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.

(220) Examples of the invention which follow are set forth to further illustrate the invention and should not be construed to limit the invention in any way.

Example 1: Identification of a Protein Modulator

(221) p210.sup.Bcr-Abl-T315I is a theramutein of the p210Bcr-Abl protein (p210.sup.Bcr-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.

(222) A phenoresponse was determined for a Ba/F3 cell line engineered to overexpress the p210.sup.Bcr-Abl-T315I theramutein. The phenoresponse was determined relative trp non-transformed Ba/F3 cells and Ba/F3 cells that express the p210.sup.Bcr-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.

(223) 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.

(224) 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.

(225) 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 T315I cells) overexpressing the p210.sup.Bcr-Abl-T315I theramutein by any means.

(226) 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 p210.sup.Bcr-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 p210.sup.Bcr-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.

(227) All five compounds inhibited p210.sup.Bcr-Abl-T315I 120 Kd activity as measured by inhibition of autophosphorylation activity. Thus, of the 6 highest scoring compounds out of more than 113,000 compounds screened, at least 5 of the six directly inhibited the p210.sup.Bcr-AblT315I mutant directly. One compound appeared 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.

(228) 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 or other protein with only obvious, minor modifications.

(229) 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 T315I theramutein at concentrations tinder which the growth and viability of the wild type Ba/F3 non-transformed cells (not expressing either, p210.sup.Bcr-Abl-wt or p210.sup.Bcr-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.

(230) 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 or other disease associated protein of clinical significance.

(231) 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 protein 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 protein, including theramuteins and protothermuteins, expressed in any mammalian cell type for which a corresponding phenoresponse is detectable.

Example 2: Phenoresponse-Based Optimization of a Protein Modulator

(232) In this example, a compound previously identified according to the teachings of this invention is optimized for activity against its selected protein target. However, unlike methods typically used in the prior art, the optimization process herein is also performed entirely through the use of the phenoresponse-based cellular assay system. For completeness sake and to demonstrate the power of the methodology to refine, a cell-free assay system using recombinantly produced target enzyme is also used to independently demonstrate that the compounds that score positively in the phenoresponse-based cellular assay indeed also score positively in a cell-free assay system format that is standard in the art and uses recombinantly produced enzyme.

(233) Compound C2 which was originally identified as an inhibitor of the T315I theramutein was subjected to a novel lead optimization program as follows. Various chemical modifications were introduced into the basic scaffold structure of compound C2 using standard medicinal chemistry synthetic methods. Once synthesized, the various analogues (chemical variants) were tested using the phenoresponse-based cellular assay system described above in Example 1.

(234) Based upon the original structure of compound C2, the contribution(s) to pharmacological activity arising from the phenyl ring that contained the bromo, chloro, and hydroxyl substituents were analyzed. An initial series of analogues were synthesized that consisted of either the unsubstituted phenyl ring (C2-01), or various substituents on the phenyl ring, such as bromo, chloro, and hydroxyl, etc. located various positions around the phenyl ring. Detailed chemical structures are shown in Table 3.

(235) TABLE-US-00003 TABLE 3 Optimization of C2 Compounds C2 C2-27 C2-21 C2-01 C2-02 0 C2-05 C2-86 C2-87 C2-109 C2-112 0 C2-121 C2-122 C2-128

(236) These compounds were then tested in the phenoresponse-based cellular assay system, as shown in FIG. 17. Each of the compounds was also tested in a standard cell free protein kinase autophosphorylation assay at a concentration of 20 μM as shown in FIG. 18. As a comparison of FIG. 17 and FIG. 18 will indicate, there was a striking, essentially complete qualitative correlation between the activity of the compounds in the phenoresponse-based (cellular) assay system and their corresponding activity in the cell free purified protein kinase autophosphorylation assay.

(237) Additional chemical modifications were made to the same phenyl ring discussed above for a variety of medicinal chemistry purposes and for reasons that are beyond the scope of this invention, but were related to enhancing potency against the p210.sup.Bcr-Abl-T315I target while simultaneously limiting cross-reactivity with the control wild type Ba/F3 non-transformed cells (not expressing either p210.sup.Bcr-Abl-wt or p210.sup.Bcr-Abl-T315I), as well as to improve selectivity, minimize potential side effects in the patient, etc. As shown in FIGS. 17 and 18 taken together with Table 3, additional compounds synthesized and tested included C2-109, C2-112, C2-122, and C2-128. A detailed comparison of FIGS. 17 and 18 reveals once again that all of the compounds that score positively in the cellular assay system also exhibited protein kinase inhibitory activity in the cell free system, whereas those that were essentially inactive in the phenoresponse-based cellular assay were also essentially inactive in the cell-free system. These results conclusively demonstrate that the compounds showing inhibitory activity in the phenoresponse-based assay system were entirely consistent qualitatively with the results obtained in the cell free protein kinase autophosphorylation assay.

(238) Furthermore, where there are modest differences in relative potency between the cell free assay results and the phenoresponse-based assay results (see for example, compound C2-122 which appears to be more potent in the cell-free assay than in the phenoresponse-based cellular system, such distinctions point out the enhanced ability of the cellular assay system to predict in vivo efficacy of a given compound as compared to classical cell-free assay systems. Using a classical cell-free screen, one might have considered C2-122 to be an important compound, yet the phenoresponse-based assay immediately rules it out as being less potent than several of the other compounds tested. This type of lead optimization strategy employing a cellular system without dependence and reliance upon a cell-free radioligand or other binding assay has not been reported before in the prior art.

(239) In summary, the present invention provides the skilled investigator with a powerful and rapid method for lead optimization which supplants the necessity for repeated cell free in vitro verifications of the ability of a compound to hit its corresponding target. A Thus the optimization process may be performed essentially entirely with reliance upon the phenoresponse-based assay systems results, obviating the need for repetitive confirmatory cell free assay determinations. While such confirmatory experiments may be performed if the skilled investigator chooses to do so, they are generally unnecessary with this method, as the results given above unequivocally demonstrate.

(240) The skilled investigator is well aware that no assay system of any kind or nature, whether it be a radioligand binding assay, ELISA, ligand binding assay, or cellular assay, is free of false-positive results. This assay system, while surprisingly robust, will not be free of the possibility of false positive results either, and the skilled investigator knows that independent verification of unusual results is simply good science and should be taken into consideration where appropriate.

Example 3: Phenoresponse-Based Profiling of a Protein Modulator

(241) The phenoresponse-based assay system of the invention can be used to profile the biological activity of a given compound with respect to its ability to inhibit or activate multiple distinct protein targets to differing extents. For example, in certain instances the skilled artisan may be interested in identifying or optimizing modulators of a given target protein where additional proteins are known that are distinct but highly related to the target POI. Such protein families may consist of two or more members that share a high degree of homology at both the DNA and amino acid sequence levels, yet the family members may have distinct functions within the cell. Through iterative application of the phenoresponse-based system described herein, one could create individual Test Cells expressing each of the distinct family members and then utilize three or four or more distinct Test Cell lines with corresponding defined phenoresponses to identify or optimize compounds that are selective for one particular family member.

(242) In yet another embodiment of the present invention, the skilled artisan may also choose to express two or three or even four distinct protein targets in a single Test Cell (or Test Cell line) and create a phenoresponse-based assay system useful for identifying compounds that are NOT selective among individual isozymes of a given protein family. In certain therapeutic situations, lack of selectivity among individual family members may be preferable. Ibuprofen, for example, is an established, low-cost safe and effective non-steroidal anti-inflammatory drug that does not significantly discriminate between the cyclooxygenase type 1 (COX-1) and COX-2 family members. Such lack of discrimination may in some instances be beneficial and may reduce the likelihood of certain unwanted side effects that may occur with an overly selective chemical agent.

(243) Profiling the biological effects of a given compound with respect to its ability to inhibit or activate certain related protein targets, whether or not such targets are members of the same protein family, also has substantial value from the perspective of understanding the molecular and cellular mechanism(s) of action of a given chemical agent. For example, in the case of imatinib, not only does the compound inhibit the wild type version of the P210 Bcr/Abl protein (p210.sup.Bcr-Abl-wt), it also cross reacts with and is capable of inhibiting the c-kit oncoprotein as well. As discussed above in the background of the invention, this cross-reactive inhibition of the kit oncoprotein is serendipitous, because gastrointestinal stromal tumors (GIST), a type of tumor arising in the small intestine, are driven by kit activity and are thus responsive to imatinib treatment as well (NEJM paper). Thus, such cross reactivity with other related proteins need not always be associated with toxicity of a drug. In some instances such cross-reactivities can be therapeutically effective.

(244) Representative Compounds of the Invention corresponding to the various chemical formulae given above were tested in the cellular assay system described elsewhere herein (see Example 1) and assigned activity categories as shown in Table 4. The assigned activity categories are represented by the following designations, wherein the IC.sub.50 for a given cell line is the concentration at which a given compound inhibits the growth of that cell line by 50% in the cellular assay system. Compounds tested on a given cell line that exhibited an IC.sub.50 value that was <300 nM (less than 300 nanomolar) were designated as Category “A” compounds. Compounds tested on a given cell line that exhibited an IC.sub.50 value that was <1 μM (less than 1 micromolar) were designated as Category “B” compounds. Compounds tested on a given cell line that exhibited an IC.sub.50 value that was <10 μM (less than 10 micromolar) were designated as Category “C” compounds. Compounds tested on a given cell line that exhibited an IC.sub.50 value that was ≥10 μM (greater than or equal to 10 micromolar) were designated as Category “D” compounds.

(245) TABLE-US-00004 TABLE 4 Structure: wt BaF3 T315I B A C C 00 B B 01 B B 02 D D 03 D D 04 D D 05 B A 06 B B 07 D D 08 C B 09 B B 0 D D A A D D A A A A B B B A A A B B D D 0 B A B B C B B B D D C B A A A A A A A A 0 A A B B C C B B

Example 4

(246) ##STR00234##

(247) Reaction Scheme:

(248) ##STR00235##

(249) Experimental Details: The mixture of compound 1 (25 g) and N, N-dimethylaniline (24.2 g) in POCl.sub.3 (110 mL) was refluxed for 5 h. POCl.sub.3 was removed by evaporation at reduced pressure and the residue was poured into ice-water (500 g) cautiously and stirred for 1 h. The mixture was then filtered and the solid was washed with water to give compound 2 as a yellow solid.

(250) 2. Reaction Scheme:

(251) ##STR00236##

(252) Experimental Details: To a solution of compound 2 (1.04 g) in 15 ml of ethanol, 1.08 g (2 eq) of morphine was added dropwise at −10° C. in 15 min. The mixture was stirred for 0.5 h and heated at 50° C. for 15 min. After cooling and dilution with water (50 ml), compound 3 was obtained as a yellow solid powder after filtration.

(253) 3. Reaction Scheme:

(254) ##STR00237##

(255) Experimental Details: To 1.1 g of compound 3 was added 8 ml of NH.sub.2NH.sub.2.H.sub.2O. The mixture was refluxed for 2 h. After cooling, the crude product was obtained after filtration. Purification by column chromatography gave pure compound 4 as a light yellow solid.

(256) 4. Reaction Scheme:

(257) ##STR00238##

(258) Experimental Details: To the solution of compound 5 (1.0 g, 1.0 eq) and DMF (0.05 g, cat. amount) in 20 mL of dichloromethane was added (COCl).sub.2 (0.81 g, 1.1 eq) dropwise. The reaction mixture was stirred at r.t. for 2 h and then concentrated to give 1.2 g of crude product of compound 6, which was used for the next step without further purification.

(259) 5. Reaction Scheme:

(260) ##STR00239##

(261) Experimental Details: To the crude product of compound 6 (1.2 g, 1.0 eq) in 20 mL of dichloromethane was added 3-trifluoromethyl-phenylamine (0.94 g, 1.0 eq) and triethylamine (0.71 g, 1.2 eq). The reaction mixture was stirred at r.t. overnight, washed with 1 N NaOH solution, 1 N HCl solution and brine. The organic layer was collected, dried over Na.sub.2SO.sub.4, and concentrated to give crude product of compound 7. After purification by column chromatography, 1.1 g of compound 7 was obtained.

(262) 6. Reaction Scheme:

(263) ##STR00240##

(264) Experimental Details: To the mixture of compound 7 (0.3 g, 1.0 eq) and 3 mL of trifluoroacetic acid was added hexamethylenetetramine (0.53 g, 4.0 eq). The reaction mixture was immediately sealed and heated to 90° C. for 20 h. After cooling, the reaction mixture was adjusted to pH 8 with 1 N NaOH solution, extracted with dichloromethane and the organic phase was dried, concentrated to give a brown solid. Purification by preparative TLC gave compound 8 as a yellow solid.

(265) 7. Reaction Scheme:

(266) ##STR00241##

(267) Experimental Details: The mixture of compound 4 (30 mg, 1.0 eq) and compound 8 (19 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight. The precipitates were collected and washed with dichloromethane thoroughly, dried under vacuum to give the desired compound.

Example 5

(268) ##STR00242##

(269) 1. Reaction Scheme:

(270) ##STR00243##

(271) Experimental Details: To the mixture of compound 1 (1.0 g, 1.0 eq), compound 2 (0.92 g, 1.0 eq), Na.sub.2CO.sub.3 (0.77 g, 1.5 eq) in 15 mL of dioxane was added Pd(PPh.sub.3).sub.4 (0.56 g, 0.1 eq), and the reaction mixture was refluxed under N.sub.2 for 16 h. After cooling, the mixture was filtered and the filtrate was evaporated to dryness and purified by column chromatography to give compound 3.

(272) 2. Reaction Scheme:

(273) ##STR00244##

(274) Experimental Details: To the mixture of compound 3 (0.3 g, 1.0 eq) and 3 mL of trifluoroacetic acid was added hexamethylenetetramine (0.62 g, 4.0 eq). The reaction mixture was immediately sealed and heated to 90° C. for 20 h. After cooling, the reaction mixture was adjusted to pH 8 with 1 N NaOH solution, extracted with dichloromethane and the organic phase was dried, concentrated to give a brown solid. Purification by preparative TLC gave compound 4 as a yellow solid.

(275) 3. Reaction Scheme:

(276) ##STR00245##

(277) Experimental Details: The mixture of compound 4 (30 mg, 1.0 eq) and compound 5 (19 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight. The precipitates were collected and washed with dichloromethane, dried under vacuum to give the desired compound.

Example 6

(278) ##STR00246##

(279) 1. Reaction Scheme:

(280) ##STR00247##

(281) Experimental Details: To the solution of compound 1 (0.6 g, 1.0 eq) in 10 mL of methanol was added 4 mL of 1 N NaOH solution, and the mixture was stirred at r.t. overnight. Solvent was evaporated and the residue was acidified to pH 6 with 5% citric acid, extracted with dichloromethane. The organic layer was dried, concentrated to give compound 2.

(282) 2. Reaction Scheme:

(283) ##STR00248##

(284) Experimental Details: The mixture of compound 2 (0.4 g, 1.0 eq), trifluoromethyl phenylamine (0.39 g, 1.0 eq), EDC (0.71 g, 1.5 eq), HOBt (33 mg, 0.1 eq) in 10 mL of dichloromethane was stirred at r.t. overnight. The mixture was washed with 1 N NaOH solution, water, extracted with dichloromethane. The organic layer was dried over Na.sub.2SO.sub.4, concentrated to dryness, and purified by column chromatography to give compound 3.

(285) 3. Reaction Scheme:

(286) ##STR00249##

(287) Experimental Details: The solution of compound 3 (0.2 g, 1.0 eq) in 10 mL of dioxane was treated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2 h. After cooled, pH was adjusted to 8 by addition of NaHCO.sub.3. The mixture was extracted with dichloromethane, washed the organic layer with water, dried with Na.sub.2SO.sub.4 and evaporated to dryness. The crude product was purified by column chromatography to give compound 4.

(288) 4. Reaction Scheme:

(289) ##STR00250##

(290) Experimental Details: The mixture of compound 4 (40 mg, 1.0 eq) and compound 5 (30 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight. The mixture was concentrated to dryness and purified by preparative HPLC to give the desired compound.

Example 7

(291) ##STR00251##

(292) 1. Reaction Scheme:

(293) ##STR00252##

(294) Experimental Details: The mixture of compound 2 (0.3 g, 1.0 eq), 2-chloro-6-methyl-phenylamine (0.26 g, 1.0 eq), EDC (0.53 g, 1.5 eq), HOBt (25 mg, 0.1 eq) in 10 mL of dichloromethane was stirred at r.t. overnight. The mixture was washed with 1 N NaOH solution, water, and extracted with dichloromethane. The organic layer was dried over Na.sub.2SO.sub.4, concentrated to dryness, purified by column chromatography to give compound 3.

(295) 2. Reaction Scheme:

(296) ##STR00253##

(297) Experimental Details: The solution of compound 3 (0.2 g, eq) in 10 mL of dioxane was treated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2 h. After cooling, the pH was adjusted to 8 by addition of NaHCO.sub.3. The mixture was extracted with dichloromethane, washed the organic layer with water, dried with Na.sub.2SO.sub.4 and evaporated to dryness. The crude product was purified by column chromatography to give compound 4.

(298) 3. Reaction Scheme:

(299) ##STR00254##

(300) Experimental Details: The mixture of compound 4 (40 mg, 1.0 eq) and compound 5 (30 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight. The mixture was concentrated to dryness and purified by preparative HPLC to give the desired compound.

Example 8

(301) ##STR00255##

(302) 1. Reaction Scheme:

(303) ##STR00256##

(304) Experimental Details: To a solution of g (5.5 mmol) of 5-bromo-2-cyanopyridine, 0.97 g of (6.05 mmol 1.1 eq) 3-(trifluoromethyl)aniline in 100 ml of toluene was added 3 eq of t-BuONa, 0.2 eq of BINAP and 0.1 eq of Pd.sub.2(dba).sub.3. Then the solution was heated to reflux overnight. The reaction was monitored by LC/MS. The volatiles were removed under reduced pressure. The crude product was purified by flash chromatography to afford compound 2.

(305) 2. Reaction Scheme:

(306) ##STR00257##

(307) Experimental Details: 250 mg (0.95 mmol) of compound 2 was added to 20 mL of concentrated HCl, then the solution was heated to reflux until the starting material disappeared. The mixture was concentrated under reduced pressure to obtain compound 3 as a yellow solid without purification.

(308) 3. Reaction Scheme:

(309) ##STR00258##

(310) Experimental Details: A solution of 50 mg (0.18 mmol) of compound 3 in 3 mL of dichloromethane was added to 0.5 mL of thionyl dichloride. The mixture was heated and stirred for 3 h. Finally the solution was evaporated under reduced pressure. Compound. 4 was obtained and used in next step without purification.

(311) 4. Reaction Scheme:

(312) ##STR00259##

(313) Experimental Details: A solution of 50 mg of compound 4 and 43 mg (1.2 eq) of hydrazine in 5 mL of DCM was stirred at 25° C. for 3 hours. The reaction mixture was concentrated and the residue was purified by preparative HPLC to give the desired compound.

Example 9

(314) ##STR00260##

(315) 1. Reaction Scheme:

(316) ##STR00261##

(317) Experimental Details: A suspension of compound 1 (25 g, 0.145 mol) and SeO.sub.2 (27.5 g, 0.247 mol) in acetic acid (1200 mL) was heated to reflux for 12 h. The reaction mixture was concentrated under reduced pressure to dryness. The residue was dissolved into water and brought to pH=9 by adding K.sub.2CO.sub.3. The resulted mixture was extracted with EA (100 mL×3). The combined EA was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated under reduced pressure to give the crude product 2, which was used in next step without purification.

(318) 2. Reaction Scheme:

(319) ##STR00262##

(320) Experimental Details: A solution of 2 above prepared in ethanol triethyl orthoformate (10 mL) was refluxed 4 h. After removing off the solvent, the residue was separated by column to give the product 3 as oil.

(321) 3. Reaction Scheme:

(322) ##STR00263##

(323) Experimental Details: To a stirred and degassed mixture of compound 3 (73 mg, 0.28 mmol) and compound 4 (50 mg, 0.28 mmol), and .sub.tBuONa (27 mg, 0.56 mmol) and BINAP (70.4 mg, 1.12 mol) in toluene (15 mL) was added Pd.sub.2(dba).sub.3 (26 mg, 0.028 mmol) under N.sub.2 atmosphere and stirred at 80° C. for 48 h. After filtering off the solid, the filtrate was concentrated to give the crude product 5 which was used in the next step without purification.

(324) 4. Reaction Scheme:

(325) ##STR00264##

(326) Experimental Details: A solution of compound 5 (335 mg, 0.1 mmol) in dichloromethane (10 mL) was treated with BBr.sub.3 (146 mg, 0.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to give the crude product 6 which was done next step without purification.

(327) 5. Reaction Scheme:

(328) ##STR00265##

(329) Experimental Details: A solution of compound 6 (27.74 mg, 0.1 mmol) and compound 7 (21 mg, 0.1 mmol) in anhydrous CH.sub.2C12 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

Example 10

(330) ##STR00266##

(331) 1. Reaction Scheme:

(332) ##STR00267##

(333) Experimental Details: To a stirred and degassed mixture of compound 1 (5 g, 25 mmol) and compound 2 (3.4 g, 27 mmol) in DMF (50 mL) and aqueous Na.sub.2CO.sub.3 (20 mL, 2 M) was added Pd.sub.2(dbbf).sub.3 (26 mg, 0.028 mmol) under N.sub.2 atmosphere and stirred at 100° C. for 18 h. After cooling to room temperature and filtrating off the solid, the filtrate was extracted with EA (200 mL). The organic layer was concentrated to dryness. The residue was purified by column to give the crude product 3.

(334) 2. Reaction Scheme:

(335) ##STR00268##

(336) Experimental Details: A mixture of compound 3 (2 g, 0.1 mol) and Na.sub.2S.sub.2O.sub.4 (5.2 g, 0.3 mol) in methanol (80 mL) and H.sub.2O (20 mL) was heated to reflux for 3 h. The reaction was concentrated to dryness under reduced pressure. The residue was dissolved into water and then was extracted with EA (150 mL). The organic layer was washed with brine twice and dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to give the product 4.

(337) 3. Reaction Scheme:

(338) ##STR00269##

(339) Experimental Details: To a stirred and degassed mixture of compound 5 (228 mg, 88 mmol) and compound 4 (150 mg, 88 mmol), and .sub.tBuONa (170 mg, 176 mmol) and BINAP (210 mg, 176 mmol) in toluene (25 mL) was added Pd.sub.2(dba).sub.3 (79 mg, 0.88 mmol) under N.sub.2 atmosphere and stirred at 80° C. for 48 h. After filtering off the solid, the filtrate was concentrated to give the crude product 6.

(340) 4. Reaction Scheme:

(341) ##STR00270##

(342) Experimental Details: A solution of compound 6 (140 mg, 4 mmol) in dichloromethane (20 mL) was treated with BBr.sub.3 (600 mg, 10.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought pH=9 by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to dryness. The residue was purified by column to give the product 7.

(343) 5. Reaction Scheme:

(344) ##STR00271##

(345) Experimental Details: A solution of compound 7 (98 mg, 0.36 mmol) and compound 8 (64 mg, 0.3 mmol) in anhydrous CH.sub.2Cl.sub.2 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

Example 11

(346) ##STR00272##

(347) 1. Reaction Scheme:

(348) ##STR00273##

(349) Experimental Details: To a stirred and degassed mixture of compound 1 (5.9 g, 25 mmol) and compound 2 (3.3 g, 27 mmol) in DMF (50 mL) and aqueous Na.sub.2CO.sub.3 (20 mL, 2 M) was added Pd.sub.2(dbbf).sub.3 (26 mg, 0.028 mmol) under N.sub.2 atmosphere and stirred at 100° C. for 18 h. After cooling to room temperature and filtrating off the solid, the filtrate was extracted with EA (200 mL). The organic layer was concentrated to give the crude product 3 which is done next step without purification.

(350) 2. Reaction Scheme:

(351) ##STR00274##

(352) Experimental Details: A mixture of 3 (6.5 g, 27.9 mmol) and Pd(OH).sub.2 (10%, 0.5 g) in ethanol (200 mL) was stirred under hydrogen atmosphere (20 psi) at room temperature for 2 hour. The catalyst was filtrated off, and the filtrate was removed under vacuum to afford the product 4 as a colorless oil.

(353) 3. Reaction Scheme:

(354) ##STR00275##

(355) Experimental Details: To a stirred and degassed mixture of compound 4 (406 mg, 20 mmol) and compound 5 (520 mg, 20 mmol), and .sub.tBuONa (170 mg, 176 mmol) and BINAP (210 mg, 176 mmol) in toluene (25 mL) was added Pd.sub.2(dba).sub.3 (79 mg, 0.88 mmol) under N.sub.2 atmosphere and stirred at 80° C. for 48 h. After filtrating off the solid, the filtrate was concentrated to give the crude product 6 which is used in next step without purification.

(356) 4 Reaction Scheme:

(357) ##STR00276##

(358) Experimental Details: A solution of compound 6 (383 mg, 10 mmol) in dichloromethane (20 mL) was treated with BBr.sub.3 (600 mg, 10.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought to pH 9 by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to dryness. The residue was purified by column to give the product 7.

(359) 5. Reaction Scheme:

(360) ##STR00277##

(361) Experimental Details: A mixture of 7 (60 mg, 0.22 mmol) nicotinaldehyde (33 mg, 0.15 m mol) in dichloromethane (10 mL) was heated to reflux for 3 hr. After removing off solvent, the residue was purified by chromatography to give the desired compound.

Example 12

(362) ##STR00278##

(363) 1. Reaction Scheme

(364) ##STR00279##

(365) Experimental Details: A solution of DMAP (9.3 g, 0.077 mol) and compound 1 (10 g, 0.051 mol) and Boc.sub.2O (12 g, 0.051 mol) in .sub.tBuOH (200 mL) was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by flash chromatography on silica gel. (ethyl acetate/petroleum ether=10:1) to give 2 as a colorless oil.

(366) 2. Reaction Scheme:

(367) ##STR00280##

(368) Experimental Details: A mixture of 2 (6.17 g, 27.9 mmol) and Pd(OH).sub.2 (10%, 1 g) in ethanol (200 mL) was stirred under hydrogen atmosphere (50 psi) at room temperature for 4 hour. The catalyst was filtrated off, and the filtrate was removed under vacuum to afford the product 3 as a colorless oil.

(369) 3. Reaction Scheme:

(370) ##STR00281##

(371) Experimental Details: A mixture of compound 3 (2.65 g, 12 mmol) and compound 4 (2.6 g, 10 mmol) and BuONa (1.34 g, 14 mmol,) and Pd.sub.2(dba).sub.3 (46.5 mg, 50 mmol,) and DCHPB (70 mg, 0.2 mmol) in dry toluene (50 mL) was heated to 80-90° C. under N.sub.2 for 24 hours. The precipitation was filtrated and the filtrate was removed in vacuo and the residue was purified by chromatography on silica gel (ethyl acetate/petroleum ether=10:1) to give afford 5 as a yellow oil.

(372) 4. Reaction Scheme:

(373) ##STR00282##

(374) Experimental Details: To a solution of 5 (0.9 g, 2.24 mmol) in CHCl.sub.3 (50 mL), CF.sub.3COOH (40 mL) was added at 0° C. After addition complete, the resulting mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure to dryness. The residue was recrystallized from ether to yield an off-white solid. The solid was dissolve in ammonia (10 mL). The mixture was brought the pH=7.0 by adding 1M HCl and precipitated. The precipitate was collected and washed with cold water (5 mL), and dried under reduced pressure to afford 6 as a dark solid.

(375) 5. Reaction Scheme:

(376) ##STR00283##

(377) Experimental Details: A mixture of 6 (60 mg, 0.22 mmol), nicotinaldehyde (33 mg, 0.15 m mol) in dichloromethane (10 mL) was heated to reflux for 3 hr. After removing off solvent, the residue was purified by chromatography to give the desired compound as a yellow solid.

Example 13

(378) ##STR00284##

(379) 1. Reaction Scheme:

(380) ##STR00285##

(381) Experimental Details: To a stirred and degassed mixture of compound 1 (6.7 g, 25 mmol) and compound 2 (4.1 g, 27 mmol) in DMF (50 mL) and aqueous Na.sub.2CO.sub.3 (20 mL, 2 M) was added Pd.sub.2(dbbf).sub.3 (26 mg, 0.028 mmol) under N.sub.2 atmosphere and stirred at 100° C. for 18 h. After cooling to room temperature and filtrating off the solid, the filtrate was extracted with EA (200 mL). The organic layer was concentrated to give the crude product 3.

(382) 2. Reaction Scheme:

(383) ##STR00286##

(384) Experimental Details: A mixture of 3 (3.2 g, 10 mmol) and Pd(OH).sub.2 (10%, 0.5 g) in ethanol (200 mL) was stirred under hydrogen atmosphere (20 psi) at room temperature for 2 hour. The catalyst was filtrated off, and the filtrate was removed under vacuum to afford the product 4 as colorless oil.

(385) 3. Reaction Scheme:

(386) ##STR00287##

(387) Experimental Details: To a stirred and degassed mixture of compound 4 (297 mg, 10 mmol) and compound 5 (259 mg, 10 mmol), and .sub.tBuONa (170 mg, 17.6 mmol) and BINAP (210 mg, 17.6 mmol) in toluene (25 mL) was added Pd.sub.2(dba).sub.3 (79 mg, 0.88 mmol) under N.sub.2 atmosphere and stirred at 80° C. for 48 h. After filtrating off the solid, the filtrate was concentrated to give the crude product 6 which is done next step without purification.

(388) 4. Reaction Scheme:

(389) ##STR00288##

(390) Experimental Details: A solution of compound 6 (446 mg, 10 mmol) in dichloromethane (20 mL) was treated with BBr.sub.3 (600 mg, 10.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought pH=9 by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to dryness. The residue was purified by column to give the product 7.

(391) 5. Reaction Scheme:

(392) ##STR00289##

(393) Experimental Details: A mixture of 7 (67 mg, 0.15 mmol), nicotinaldehyde (33 mg, 0.15 m mol) in dichloromethane (10 mL) was heated to reflux for 3 hr. After removing off solvent, the residue was purified by chromatography to give the desired compound as a yellow solid.

Example 14

(394) ##STR00290##

(395) 1. Reaction Scheme:

(396) ##STR00291##

(397) Experimental Details: To a stirred and degassed mixture of compound 1 (3 g, 0.02 mol) and compound 2 (3.4 g, 0.02 mol), and KOH (5.28 g, 0.1 mol) and TBBA (6.44 g, 0.02 mol) in anhydrous THF (100 mL) was added Pd (PPh.sub.3).sub.4 (2.31 g, 2 mmol) under N.sub.2 atmosphere and stirred under reflux for 12 h. After filtrating off the solid, the filtrate was concentrated to dryness. The residue was purified by column to give the product 3, which is used in next step without purification.

(398) 2. Reaction Scheme:

(399) ##STR00292##

(400) Experimental Details: To a stirred and degassed mixture of compound 3 (334 mg, 1.7 mmol) and compound 4 (434 mg, 1.7 mmol), and t-BuONa (322 mg, 3.4 mmol) and BINAP (420 mg, 0.67 mol) in toluene (60 mL) was added Pd.sub.2(dba).sub.3 (156 mg, 0.017 mmol) under N.sub.2 atmosphere and stirred at 80° C. for 48 h. After filtrating off the solid, the filtrate was concentrated to dryness. The residue was purified by column to give the product 5.

(401) 3. Reaction Scheme:

(402) ##STR00293##

(403) Experimental Details: A solution of compound 5 (100 mg, 0.3 mmol) in dichloromethane (10 mL) was treated with BBr.sub.3 (393 mg, 0.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to give the crude product 6.

(404) 4. Reaction Scheme:

(405) ##STR00294##

(406) Experimental Details: A mixture of 6 (39 mg, 0.13 mmol), nicotinaldehyde (29 mg, 0.13 m mol) in dichloromethane (10 mL) was heated to reflux for 3 hr. After removing off solvent, the residue was purified by chromatography to give the desired compound as a yellow solid.

Example 15

(407) ##STR00295##

(408) 1. Reaction Scheme:

(409) ##STR00296##

(410) Experimental Details: Paraformaldehyde (1.8 g, 59.3 mmol) was added to a solution of compound 1 (1.0 g, 5.9 mmol) in acetic acid (40 mL), followed by NaCNBH.sub.3 (1.8 g, 28.8 mmol) at 10° C. After stirring of 16 h at room temperature, the solution was poured into ice/water (100 mL), and the PH adjusted to 10 with concentrated NaOH. The solution was extracted with DCM (3×100 mL). The combined organic layers were dried (MgSO.sub.4), filtrated and concentrated in vacuo to give compound 2.

(411) 2. Reaction Scheme:

(412) ##STR00297##

(413) Experimental Details: 0.84 g (4.3 mmol) of compound 2 was hydrogenated under 1 atm hydrogen with Pd/C for 16 hour. The reaction mixture was filtered and the filtrate was concentrated to afford compound 3 without further purification.

(414) 3. Reaction Scheme:

(415) ##STR00298##

(416) Experimental Details: A solution of 250 mg (1.51 mmol) of compound 3, 0.2 eq of BINAP, 0.1 eq of Pd.sub.2(dba).sub.3, 3 eq of Cs.sub.2CO.sub.3 and 5-Bromo-2-diethoxymethyl-pyridine (0.783 g, 3.01 mmol) in 10 mL of 1,4-dioxane was reacted at 150° C. under microwave for 2 hours. The reaction was monitored by LC-Ms. The mixture was concentrated and the residue was purified by preparative TLC to afford compound 4.

(417) 4. Reaction Scheme:

(418) ##STR00299##

(419) Experimental Details: A solution of 300 mg (0.55 mmol) of compound 4 in 5 mL of DCM was added 4 mL of TFA. The reaction mixture was stirred for 30 min at r.t. The mixture was added ice/water and basified by NaHCO.sub.3 to PH=10 and extracted with DCM (15 mL*3). The combined organic layer was washed with water and brine, dried over MgSO.sub.4, filtered and concentrated to afford compound 5.

(420) 5. Reaction Scheme:

(421) ##STR00300##

(422) Experimental Details: A solution of 80 mg (0.295 mmol) of compound 5 and (5-Fluoro-4-morpholin-4-yl-pyrimidin-2-yl)-hydrazin in 10 mL of DCM was stirred at 25° C. for 15 hours. The reaction mixture was concentrated and the residue was purified by preparative HPLC to afford compound 6.

(423) 6. Reaction Scheme:

(424) ##STR00301##

(425) Experimental Details: To a solution of compound 6 (40 mg, 0.086 mmol) in dry DCM (5 mL) was added drop wise BBr (22 mg, 0.258 mmol) at 0° C. The reaction mixture was stirred for 3 h at r.t. The reaction was quenched with methanol, and the mixture was concentrated. The residue was purified by preparative HPLC to give the desired compound.

Example 16

(426) ##STR00302##

(427) 1. Reaction Scheme:

(428) ##STR00303##

(429) Experimental details: To a stirred solution of 3-Bromoaniline (0.86 g) in 30 ml of toluene were added 0.1 eq of Pd(PPh.sub.3).sub.4, 5 ml of sat. aq. of Na.sub.2CO.sub.3 and a solution of 3-methoxyphenyl boronic acid (0.75 g) in 10 ml of EtOH under N.sub.2 atmosphere. The mixture was vigorously stirred under reflux for 15 h and cooled, 10 ml of H.sub.2O was added, and the mixture was extracted with CH.sub.2Cl.sub.2 (20 ml×3). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography (PE/EA-10:1) to get pure compound 2.

(430) 2. Reaction Scheme:

(431) ##STR00304##

(432) Reaction details: A mixture of compound 2 (59.5 mg), 5-bromo-2-diethoxymethyl-pyridine (116.7 mg), t-BuONa (86.4 mg), BINAP (36.7 mg) and Pd.sub.2(dba).sub.3 (27.4 mg) in dioxane (2 ml) was microwaved for 2 hs at 150° C., the solution was filtered and concentrated. The residue was purified by preparative TLC to afford compound 3.

(433) 3. Reaction Scheme:

(434) ##STR00305##

(435) Reaction details: To a solution of compound 3 (120 mg) in 5 ml of DCM was added 1 ml of BBr.sub.3, the reaction was stirred overnight at r.t. Then added 5 ml of H.sub.2O to the mixture, extracted with EtOAc and concentrated. The crude product was purified by pre. TLC to afford compound 4.

(436) 4. Reaction Scheme:

(437) ##STR00306##

(438) Experimental details: A mixture of 43.5 mg of compound 4 and 32 mg of hydrazine in 5 ml DCM was stirred overnight at r.t. and concentrated. The crude product was purified by prereparative TLC to afford the desired compound.

Example 17

(439) ##STR00307##

(440) 1. Reaction Scheme:

(441) ##STR00308##

(442) Experimental Details: A solution of 2.0 g (16.2 mmol, 1.0 eq) of compound 1, 0.05 eq of BINAP, 0.05 eq of Pd.sub.2(dba).sub.3, 1.2 eq of t-BuONa and 1-Bromo-3-nitro-benzene (3.28 g, 16.2 mmol, 1.0 eq) in 20 mL of anhydrous toluene was reacted at 100° C. for 24 hours. The reaction was monitored by LC-MS. The mixture was concentrated and the residue was purified by column chromatography to afford compound 2.

(443) 2. Reaction Scheme:

(444) ##STR00309##

(445) Experimental Details: 2.5 g of compound 2 was hydrogenated under 1 atm of hydrogen with Pd/C (0.25 g) for 16 hour. The reaction mixture was filtered and the filtrate was concentrated to afford compound 3 without further purification.

(446) 3. Reaction Scheme:

(447) ##STR00310##

(448) Experimental Details: A solution of 500 mg (2.33 mmol) of compound 3, 5-Bromo-2-diethoxymethylpyridine (607 mg, 2.33 mmol), 0.05 eq of xantphos, 0.05 eq of Pd.sub.2(dba).sub.3 and 1.5 eq of t-BuONa in 10 mL of toluene was refluxed at 100° C. for 24 hours. The reaction was monitored by LC-MS. The mixture was concentrated and the residue was purified by column chromatography to afford compound 4.

(449) 4. Reaction Scheme:

(450) ##STR00311##

(451) Experimental Details: 100 mg of compound 4 was treated with 1 mL of HCl (1N aqueous solution) and 10 mL of dioxane. The mixture was stirred at rt for 4 h, adjusted to pH 8-9 with 0.5 N NaOH solution. After extracted with DCM, dried organic layer with Na.sub.2SO.sub.4 and concentrated to give compound 5.

(452) 5. Reaction Scheme:

(453) ##STR00312##

(454) Experimental Details: A solution of 50 mg (0.16 mmol) of compound 5 and (5-Fluoro-4-morpholin-4-ylpyrimidin-2-yl)-hydrazine (50 mg, 0.23 mmol) in 5 mL of DCM was stirred at 25° C. for 15 hours. The reaction mixture was concentrated and the residue was purified by preparative HPLC to afford compound 6.

(455) 6. Reaction Scheme:

(456) ##STR00313##

(457) Experimental Details: To a solution of compound 6 (50 mg, 0.09 mmol) in dry DCM (5 mL) was added dropwise BBr.sub.3 (20 mg, 0.25 mmol) at 0° C. The reaction mixture was stirred for 3 hr at r.t. The reaction was quenched with methanol, and the mixture was concentrated. The residue was purified by preparative HPLC to give the desired compound.

Example 18

(458) ##STR00314##

(459) 1. Reaction Scheme:

(460) ##STR00315##

(461) Experimental Details: A solution of 10 g (70.92 mmol) of 3-fluoro-nitrobenzene, 1 eq imidazole and 2 eq K.sub.2CO.sub.3 in 100 ml of DMSO was heated at 130° C. for 5 hours. The reaction was monitored by LC-MS. Then 500 mL of water was added and the precipitate was filtered and the solid was washed with water and dried to afford compound 2 without further purification.

(462) 2. Reaction Scheme:

(463) ##STR00316##

(464) Experimental Details: 5 g (31.4 mmol) of compound 2 was hydrogenated under 1 atm hydrogen with Pd/C for 0.5 hour. The reaction mixture was filtered and the filtrate was concentrated to afford compound 3 without further purification.

(465) 3. Reaction Scheme:

(466) ##STR00317##

(467) Experimental Details: A solution of 500 mg (3.14 mmol) of compound 3, 0.1 eq of xantphose, 0.1 eq of Pd.sub.2(dba).sub.3 and 1.5 eq of t-BuONa in 10 mL of toluene was refluxed at 130° C. for 15 hours. The reaction was monitored by LC-Ms and washed with water and extracted with EtOAc. The combined organic layer was washed with brine and dried over MgSO.sub.4. Filtered and concentrated, residue was purified by preparative TLC to afford compound 4.

(468) 4. Reaction Scheme:

(469) ##STR00318##

(470) Experimental Details: A solution of 200 mg of compound 4 in 5 mL of 1,4-dioxane was added 8 mL of 4N HCl and heated at 80° C. for 2 hours. The reaction mixture was basified by 2N NaOH to ph-10 and extracted with DCM (15 mL*3). The combined organic layer was washed with water and brine, dried over MgSO.sub.4, filtered and concentrated to afford 250 mg of crude product. The crude product was purified by preparative TLC to afford compound 5.

(471) 5. Reaction Scheme:

(472) ##STR00319##

(473) Experimental Details: A solution of 80 mg of compound 5 and 1 eq of compound 6 in 5 mL of DCM was stirred at 25° C. for 15 hours. The reaction mixture was concentrated and the residue was purified by preparative HPLC to afford the desired compound.

Example 19

(474) ##STR00320##

(475) 1. Reaction Scheme:

(476) ##STR00321##

(477) Experimental Details: A solution of 1.50 g of 1-Bromo-3-methyl-5-nitro-benzene and 1.60 g (1.2 eq) of Piperazine-1-carboxylic acid tert-butyl ester, 0.1 eq of xantphos, 0.1 eq of Pd.sub.2(dba).sub.3 and 1.5 eq of t-BuONa in 20 mL of toluene was refluxed at 130° C. for 4 hours. The reaction was monitored by LC-Ms and washed with water and extracted with EtOAc. The combined organic layer was washed with brine and dried over Na.sub.2SO.sub.4. Filtered and concentrated, residue was purified with column chromatography on silica gel using 10:1 PA:EA as an eluant. The appropriate fractions were combined and concentrated under reduced pressure, to give intermediate 1.

(478) 2. Reaction Scheme:

(479) ##STR00322##

(480) Experimental Details: 1.6 g of intermediate of 1 was hydrogenated under 1 atm hydrogen with Raney/Ni for 2 hours. The reaction mixture was filtered and the filtrate was concentrated to afford intermediate 2 without further purification.

(481) 3. Reaction Scheme:

(482) ##STR00323##

(483) Experimental Details: A solution of 1.20 g of Intermediate of 2, and 1.30 g (1.20 eq) of 5-Bromo-2-diethoxymethyl-pyridine, 0.1 eq of xantphose, 0.1 eq of Pd.sub.2(dba).sub.3 and 1.5 eq of t-BuONa in 20 mL of toluene was refluxed at 130° C. for 4 hours. The reaction was monitored by LC-MS and washed with water and extracted with EtOAc. The combined organic layer was washed with brine and dried over Na.sub.2SO.sub.4. Filtered and concentrated, residue was purified with column chromatography on silica gel using 4:1 PA:EA as an eluant. The appropriate fractions were combined and concentrated under reduced pressure to give intermediate 3.

(484) 4. Reaction Scheme:

(485) ##STR00324##

(486) Experimental Details: 200 mg of intermediate 3 was dissolved in 10 ml of DCM. 130 mg of TFA was added to the reaction mixture solution dropwise and stirred for 3 h at r.t. The reaction mixture was based with NaHCO.sub.3 solution to neutral and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to afford 220 mg of crude product. The crude product was purified by preparative TLC to afford intermediate 4.

(487) 5. Reaction Scheme:

(488) ##STR00325##

(489) Experimental Details: A solution of 50 mg of intermediate 4 and 1.0 eq of (5-Fluoro-4-morpholin-4-ylpyrimidin-2-yl)-hydrazine in 5 mL of DCM was stirred at r.t. for 3 hours. The reaction mixture was washed with water and brine, concentrated it to give the residue and purified by preparative HPLC to afford the desired compound.

Example 20

(490) ##STR00326##

(491) 1. Reaction Scheme:

(492) ##STR00327##

(493) Experimental Details: 15 ml of 1M BH.sub.3/THF was added dropwise into a solution of 3 g (13.95 mmol) of 4-bromo-2-methyl-benzoic acid in 20 ml of THF at 0° C. The reaction solution was allowed to reach room temperature for 1 hour and quenched by the dropwise addition of 50 ml of 50% aqueous THF. The mixture was treated with Na.sub.2CO.sub.3 and concentrated. The residue was extracted with Et.sub.2O. The organic layer was dried to give compound 2.

(494) 2. Reaction Scheme:

(495) ##STR00328##

(496) Experimental Details: A solution of 2.4 g (11.9 mmol) of compound 2 in 20 ml of DCM was added a slurry of 5.1 g (23.8 mmol) of PCC in 60 ml of DCM. The reaction solution was stirred for 1 hour at r.t. diluted with 300 ml of Et.sub.2O and filtered. The filtrate was concentrated to give compound 3.

(497) 3. Reaction Scheme:

(498) ##STR00329##

(499) Experimental Details: A solution of 2.1 g (14 mmol) was added into a solution of ethanol which containing 1.9 g (9.55 mmol) of compound 3. The reaction solution was heated to reflux for 3 hours, and then concentrated. The solid was washed with NaHCO.sub.3 and extracted with acetic ether. The organic layer was dried to give compound 4.

(500) 4 Reaction Scheme:

(501) ##STR00330##

(502) Experimental Details: 0.7 g of compound 4, 0.41 g of 3-trifluromethyl-phenylamine 0.32 g of Pd.sub.2(dba).sub.3, 0.21 g of binap and 0.02 g of t-BuONa were added into 35 ml of toluene. The reaction solution was heated to reflux overnight, and concentrated. The crude product was purified by column chromatography (ethyl acetate/hexane-1:1) to give compound 5.

(503) 5. Reaction Scheme:

(504) ##STR00331##

(505) Experimental Details: The solution of compound 5 (0.2 g, 1.0 eq) in 10 mL of dioxane was treated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2 h. After cooling, the pH was adjusted to 8 by addition of NaHCO.sub.3. The mixture was extracted with dichloromethane, washed the organic layer with water, dried with Na.sub.2SO.sub.4 and evaporated to dryness. The crude product was purified by column chromatography to give compound 6.

(506) 6. Reaction Scheme:

(507) ##STR00332##

(508) Experimental Details: A solution of 80 mg of compound 6 and 1 eq of compound 7 in 5 mL of DCM was stirred at 25° C. for 15 hours. The reaction mixture was concentrated and the residue was purified by preparative HPLC to afford the desired compound.

Example 21

(509) ##STR00333##

(510) 1. Reaction Scheme:

(511) ##STR00334##

(512) Experimental Details: To the mixture of compound 1 (0.5 g, 1.0 eq), 3-trifluoromethyl phenyl boronic acid (0.63 g, 1.0 eq), Na.sub.2CO.sub.3 (0.46 g, 1.5 eq) in 15 mL of dioxane was added Pd(PPh.sub.3).sub.4 (0.33 g, 0.1 eq), and the reaction mixture was refluxed under N.sub.2 for 16 h. After cooling, the mixture was filtered and the filtrate was evaporated to dryness and purified by column chromatography to give compound 2.

(513) 2. Reaction Scheme:

(514) ##STR00335##

(515) Experimental Details: A mixture of compound 2 (0.2 g, 1.0 eq), SeO.sub.2 (0.19 g, 2.0 eq) in 10 mL of acetic acid was refluxed under N.sub.2 for 48 h. Solvent was removed by evaporation and the residue was dissolved in water and adjusted to pH 6 with saturated NaHCO.sub.3 solution, extracted with dichloromethane. The organic layers were collected, dried and evaporated to dryness. The crude product was purified by column chromatography to$ give compound 3.

(516) 3. Reaction Scheme:

(517) ##STR00336##

(518) Experimental Details: The mixture of compound 3 (30 mg, 1.0 eq) and compound 4 (19 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight. The mixture was concentrated to dryness and purified by preparative HPLC to give the desired compound.

Example 22

(519) ##STR00337##

(520) 1. Reaction Scheme:

(521) ##STR00338##

(522) Experimental Details: The mixture of compound 1 (0.5 g, 1.0 eq), trifluoromethyl phenylamine (0.58 g, 1.0 eq), EDC (1.05 g, 1.5 eq), HOBt (50 mg, 0.1 eq) in 15 mL of dichloromethane was stirred at r.t. overnight. The mixture was washed with 1 N NaOH solution, water, extracted with dichloromethane. The organic layer was dried over Na.sub.2SO.sub.4, concentrated to dryness, purified by column chromatography to give compound 2.

(523) 2. Reaction Scheme:

(524) ##STR00339##

(525) Experimental Details: A mixture of compound 2 (0.2 g, 1.0 eq), SeO.sub.2 (0.16 g, 2.0 eq) in 10 mL of acetic acid was refluxed under N.sub.2 for 48 h. Solvent was removed by evaporation and the residue was dissolved in water and adjusted to pH 6 with saturated NaHCO.sub.3 solution, extracted with dichloromethane. The organic layers were collected, dried and evaporated to dryness. The crude product was purified by column chromatography to; give compound 3.

(526) 3. Reaction Scheme:

(527) ##STR00340##

(528) Experimental Details: The mixture of compound 3 (40 mg, 1.0 eq) and compound 4 (30 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight. The precipitates were collected and washed with dichloromethane, dried under vacuum to give the desired compound.

Example 23

(529) ##STR00341##

(530) 1. Reaction Scheme:

(531) ##STR00342##

(532) Experimental Details: The solution of compound 1 (3.0 g, 1.0 eq) and 2 mL 98% H.sub.2SO.sub.4 in 10 mL of EtOH was refluxed for 4 h, cooled to r.t., evaporated to dryness, diluted with water, adjusted to pH 8 with NaHCO.sub.3, extracted with dichloromethane. The organic layer was dried and concentrated to give compound 2.

(533) 2. Reaction Scheme:

(534) ##STR00343##

(535) Experimental Details: A mixture of compound 2 (1.7 g, 1.0 eq), SeO.sub.2 (2.29 g, 2.0 eq) in 80 mL of acetic acid was refluxed under N.sub.2 for 48 h. Solvent was removed by evaporation and the residue was dissolved in water and adjusted to pH 6 with saturated NaHCO.sub.3 solution, extracted with dichloromethane. The organic layers were collected, dried and evaporated to dryness. The crude product was purified by column chromatography to give compound 3.

(536) 3. Reaction Scheme:

(537) ##STR00344##

(538) Experimental Details: The solution of compound 3 (1.1 g, 1.0 eq), diethoxymethoxy-ethane (2.3 g, 2.5 eq) and TsOH.H.sub.2 (0.12 g, 0.1 eq) in 20 mL of ethanol was refluxed for 5 h. The solvent was evaporated and the solid was dissolved in EtOAc, washed with water. The organic layer was dried over Na.sub.2SO.sub.4 and evaporated to give compound 4.

(539) 4. Reaction Scheme:

(540) ##STR00345##

(541) Experimental Details: To the solution of compound 4 (0.6 g, 1.0 eq) in 10 mL of methanol was added 4 mL of 1 N NaOH solution, and the mixture was stirred at r.t. overnight. Solvent was evaporated and the residue was acidified to pH 6 with 5% citric acid, extracted with dichloromethane. The organic layer was dried, concentrated to give compound 5.

(542) 5. Reaction Scheme:

(543) ##STR00346##

(544) Experimental Details: The mixture of compound 5 (0.4 g, 1.0 eq), trifluoromethyl phenylamine (0.29 g, 1.0 eq), EDC (0.51 g, 1.5 eq), HOBt (25 mg, 0.1 eq) in 10 mL of dichloromethane was stirred at r.t. overnight. The mixture was washed with 1 N NaOH solution, water, extracted with dichloromethane. The organic layer was dried over Na.sub.2SO.sub.4, concentrated to dryness, purified by column chromatography to give compound 6.

(545) 6. Reaction Scheme:

(546) ##STR00347##

(547) Experimental Details: The solution of compound 6 (0.2 g, 1.0 eq) in 10 mL of dioxane was treated with 4 mL of 1 N HCl, and the mixture Was heated to 60° C. for 2 h. After cooling, pH was adjusted to 8 by addition of NaHCO.sub.3. The mixture was extracted with dichloromethane, washed the organic layer with water, dried with Na.sub.2SO.sub.4 and evaporated to dryness. The crude product was purified by column chromatography to give compound 7.

(548) 7. Reaction Scheme:

(549) ##STR00348##

(550) Experimental Details: The mixture of compound 7 (30 mg, 1.0 eq) an compound 8 (19 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight. The precipitates were collected and washed with dichloromethane, dried under vacuum to give the desired compound.

Example 24

(551) ##STR00349##

(552) 1. Reaction Scheme:

(553) ##STR00350##

(554) Experimental Details: A mixture of compound 1 (2.0 g, 1.0 eq), SeO.sub.2 (2.6 g, 2.0 eq) in 80 mL of acetic acid was refluxed under N.sub.2 for 36 h. Solvent was removed by evaporation and the residue was dissolved in water and adjusted to pH 6 with saturated NaHCO.sub.3 solution, extracted with dichloromethane. The organic layers were collected, dried and evaporated to dryness. The crude product was purified by column chromatography to give compound 2.

(555) 2. Reaction Scheme:

(556) ##STR00351##

(557) Experimental Details: The solution of compound 2 (0.5 g, 1.0 eq), diethoxymethoxy-ethane (1.0 g, 2.5 eq) and TsOH.H.sub.2O (0.05 g, 0.1 eq) in 8 mL of ethanol was refluxed for 3 h. The solvent was evaporated and the solid was dissolved in EtOAc, washed with water. The organic layer was dried over Na.sub.2SO.sub.4 and evaporated to give compound 3.

(558) 3. Reaction Scheme:

(559) ##STR00352##

(560) Experimental Details: To the mixture of compound 3 (0.6 g, 1.0 eq), 3-trifluoromethyl-phenylamine (0.37 g, 1.0 eq), t-BuONa (0.26 g, 1.2 eq) in 15 mL of toluene was added under N.sub.2Pd.sub.2(dba).sub.3 (42 mg, 0.02 eq) and xantphos (28 mg, 0.02 eq). The mixture was refluxed under N.sub.2 for 16 h, cooled, filtered. The filtrate was concentrated and purified by column chromatography to give compound 4.

(561) 4. Reaction Scheme:

(562) ##STR00353##

(563) Experimental Details: The solution of compound 4 (0.25 g, 1.0 eq) in 10 mL of dioxane was treated with 4 mL of 1 N HCl, and the mixture was heated to 60° C. for 2 h. After cooling, the pH was adjusted to 8 by addition of NaHCO.sub.3. The mixture was extracted with dichloromethane, washed the organic layer with water, dried with Na.sub.2SO.sub.4 and evaporated to dryness. The crude product was purified by column chromatography to give compound 5.

(564) 5. Reaction Scheme:

(565) ##STR00354##

(566) Experimental Details: The mixture of compound 5 (30 ng, 1.0 eq) and compound 6 (19 mg, 1.0 eq) in 5 mL of dichloromethane was stirred at r.t. overnight: The precipitates were collected and washed with dichloromethane, dried under vacuum to give the desired compound.

Example 25

(567) ##STR00355##

(568) 1. Reaction Scheme:

(569) ##STR00356##

(570) Experimental Details: To a solution of compound 1 (14 g, 0.1 mol) in aqueous HBr (30 mL) was added a solution of NaNO.sub.2 (8.3 g 0.15 mol) in H.sub.2O (10 mL) at 0° C. over a period of 30 min. After stirring for 60 min, the reaction mixture was added to a solution of CuBr (14 g, 0.1 mol) in aqueous HBr (16 mL) at 80° C. After complete addition, the reaction mixture was stirred at the same temperature for 2 h. After cooling to room temperature, the reaction mixture was extracted with EA (100 mL×3). The combined organic layer were washed with brine and dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to dryness. The residue was purified by column to give the product 2.

(571) 2. Reaction Scheme:

(572) ##STR00357##

(573) Experimental Details: To a stirred and degassed mixture of compound 2 (4.11 g, 0.02 mol) and compound 3 (4.74 g, 0.02 mol), and KOH (5.28 g, 0.1 mol) and TBBA (6.44 g, 0.02 mol) in anhydrous THF (100 mL) was added Pd (PPh.sub.3).sub.4 (2.31 g, 2 mmol) under N.sub.2 atmosphere and stirred under reflux for 12 h. After filtrating off the solid, the filtrate was concentrated to dryness. The residue was purified by column to give the product 4.

(574) 3. Reaction Scheme:

(575) ##STR00358##

(576) Experimental Details: A solution of 4 (1 g, 3 mmol) in dichloromethane (10 mL) was treated with TFA (1 mL) and then stirred for 6 h at room temperature. The solvent was removed under reduced pressure to give the product 5 which is done next step without purification.

(577) 4. Reaction Scheme:

(578) ##STR00359##

(579) Experimental Details: To a stirred and degassed mixture of compound 5 (619 mg, 2.4 mmol) and compound 6 (520 mg, 2.4 mmol), and .sub.tBuONa (460 mg, 4.8 mmol) and BINAP (599 mg, 6.9 mol) in toluene (60 mL) was added Pd.sub.2(dba).sub.3 (221 mg, 0.024 mmol) under N.sub.2 atmosphere and stirred at 80° C. for 48 h. After filtrating off the solid, the filtrate was concentrated to dryness. The residue was purified by column to give the product 7.

(580) 5. Reaction Scheme:

(581) ##STR00360##

(582) Experimental Details: A solution of compound 7 (396 mg. 0.1 mmol) in dichloromethane (10 mL) was treated with BBr.sub.3 (146 mg, 0.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to give the crude product 8, which was done next step without purification.

(583) 6. Reaction Scheme:

(584) ##STR00361##

(585) Experimental Details: A solution of compound 8 (32.2 mg, 0.1 mmol) and compound 9 (21 mg, 0.1 mmol) in anhydrous CH.sub.2C2 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

Example 26

(586) ##STR00362##

(587) 1. Reaction Scheme:

(588) ##STR00363##

(589) Experimental Details: A solution of 5-fluoro-1H-pyrimidine-2,4-dione (113 g, 0.5 mol) in N,N-dimethylaniline (70 mL) was treated with POCl.sub.3 (500 mL), then was reflux for 2 h. After cooling to room temperature, the reaction mixture was poured onto ice-water. The resulting mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with saturated aqueous of NaHCO.sub.3, then brine. The solvent was removed under reduced pressure to give compound 2.

(590) 2. Reaction Scheme:

(591) ##STR00364##

(592) Experimental Details: To a solution of compound 2 (20.8 g, 0.194 mol) in ethanol (300 mL) was added morpholine (21.6 g, 0.25 mol) drop wise at −10° C. over a period of 15 min. This mixture was stirred at room temperature for 0.5 h, heated then to 50° C. for 15 min. After cooling to room temperature and dilution with water, solid was precipitated. The solid was collected by filtrate and washed with water to give compound 3.

(593) 3. Reaction Scheme:

(594) ##STR00365##

(595) Experimental Details: A solution of 3 (4.6 g, 17.5 mmol) and hydrazine (8.75 g, 87.5 mmol) in ethanol (40 mL) was heated to reflux for 6 h. After cooling and precipitating, the precipitate was collected by filtrate and washed with ethanol to give compound 4

(596) 4. Reaction Scheme:

(597) ##STR00366##

(598) Experimental Details: A solution of compound 5 (14 g, 50 mmol) in anhydrous THF (100 mL) was treated with BuMgCl (37.5 mL, 60 mmol) at −15° C. under N.sub.2 atmosphere. After complete addition, this mixture was stirred at this temperature for 1 h. Anhydrous DMF (0.54 g, 75 mmol) was added to the reaction mixture at 0° C. over a period of 30 min, warmed then to room temperature for 1 h. The reaction mixture was quenched by adding 2 M HCl (80 mL). The result mixture was extracted with ethyl acetate (50 mL×3). The combined organic layers were dried over Na.sub.2SO.sub.4. The solvent was concentrated to dryness. The residue was separated by column to give compound 6.

(599) 5. Reaction Scheme:

(600) ##STR00367##

(601) Experimental Details: A solution of compound 6 (4.5 g, 22.5 mmol) in triethyl orthoformate (15 mL) was heated in the presence of a trace of TsOH over night. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with an aqueous of 5% Na.sub.2CO.sub.3. The organic layer was separated and dried over Na.sub.2SO.sub.4. The solvent was concentrated to give compound 7.

(602) 6. Reaction Scheme:

(603) ##STR00368##

(604) Experimental Details: To a stirred and degassed mixture of compound 7 (1.3 g, 5 mmol) and compound 8 (0.97 g, 6 mmol), and t BuONa (0.7 g, 7 mmol) and P(t-Bu).sub.3 (15 mg) in toluene (60 mL) was added Pd.sub.2(dba).sub.3 (23 mgl) under N.sub.2 atmosphere and stirred under reflux for 12 h. After filtrating off the solid, the filtrate was concentrated to dryness. The residue was purified by column to give product 9.

(605) 7. Reaction Scheme:

(606) ##STR00369##

(607) Experimental Details: A solution of compound 9 (200 mg, 0.58 mmol) in dichloromethane (10 mL) was treated with BBr.sub.3 (146 mg, 0.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to give the crude product 10.

(608) 8. Reaction Scheme:

(609) ##STR00370##

(610) Experimental Details: A solution of compound 10 (48.7 mg, 0.2 mmol) and compound 4 (63 mg, 0.2 mmol) in anhydrous CH.sub.2C12 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

Example 27

(611) ##STR00371##

(612) 1. Reaction Scheme:

(613) ##STR00372##

(614) Experimental Details: A solution of 3 (2.4 g, 11 mmol) and methylhydrazine (2 g, 45 mmol) in ethanol (40 mL) was heated to reflux for 6 h. After cooling and precipitating, the precipitate was collected by filtrate and washed with ethanol to give compound 2.

(615) 2. Reaction Scheme:

(616) ##STR00373##

(617) Experimental Details: A solution of compound 2 (28 mg, 0.1 mmol) and compound 3 (37 mg, 0.1 mmol) in anhydrous CH.sub.2Cl.sub.2 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

Example 28

(618) ##STR00374##

(619) 1. Reaction Scheme:

(620) ##STR00375##

(621) Experimental Details: A solution of 1 (2 g, 0.011 mol) in anhydrous THF (100 mL) was treated with MeMgCl (15 mL. 0.038 mol), at −20° C. and stirred for 2 h at this temperature. This reaction mixture was quenched by adding the saturated aqueous of NH.sub.4Cl. The resulting mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine. The solvent was removed under reduced pressure to give compound 2.

(622) 2. Reaction Scheme:

(623) ##STR00376##

(624) Experimental Details: A solution of compound 2 (2 g, 0.01 mol) and ethylene glycol (3 g, 0.048 mol) in aniline (100 mL) was heated in the presence of a trace of TsOH for 3 h. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with an aqueous of 5% Na.sub.2CO.sub.3. The organic layer was separated and dried over Na.sub.2SO.sub.4. The solvent was concentrated to give compound 3.

(625) 3. Reaction Scheme:

(626) ##STR00377##

(627) Experimental Details: To a stirred and degassed mixture of compound 3 (0.4 g, 1.6 mmol) and compound 4 (0.3 g, 1.9 mmol), and .sub.tBuONa (0.22 g, 2 mmol) and P(t:Bu).sub.3 (59 mg) in toluene (30 mL) was added Pd.sub.2(dba).sub.3 (29 mgl) under N.sub.2 atmosphere and stirred under reflux for 12 h. After filtrating off the solid, the filtrate was concentrated to dryness. The residue was purified by column to give product 5.

(628) 4. Reaction Scheme:

(629) ##STR00378##

(630) Experimental Details: A solution of compound 5 (100 mg, 0.3 mmol) in dichloromethane (10 mL) was treated with BBr.sub.3 (146 mg, 0.6 mmol) at −30° C. under N.sub.2 atmosphere, then was stirred at room temperature for 4 h. The reaction was poured unto ice-water and then was brought by adding Na.sub.2CO.sub.3. The resulting mixture was extracted with dichloromethane (25 mL×3), the combined organic layer was dried over Na.sub.2SO.sub.4. After filtrating off the Na.sub.2SO.sub.4, the filtrate was concentrated to give the crude product 6.

(631) 5. Reaction Scheme:

(632) ##STR00379##

(633) Experimental Details: A solution of compound 6 (64 mg, 0.2 mmol) and compound 7 (45 mg, 0.2 mmol) in anhydrous CH.sub.2Cl.sub.2 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

Example 29

(634) ##STR00380##

(635) 1. Reaction Scheme:

(636) ##STR00381##

(637) Experimental Details: A mixture of 1 (5.2 g, 22 mmol) and 2 (2.44 g, 20 mmol) in an aqueous of 2 M Na.sub.2CO.sub.3 (25 mL) and toluene (40 mL) was stirred with Pd(PPh.sub.3).sub.4 (0.57 g, 0.05 mmol) under reflux over night. The reaction mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine. The solvent was removed under reduced pressure to dryness. The residue was purified by column to give 3.

(638) 2. Reaction Scheme:

(639) ##STR00382##

(640) Experimental Details: A solution of compound 3 (0.78 g, 3.3 mmol) and triisopropyl borate (1 mL, 4 mmol) in anhydrous toluene (50 mL) was treated with n-BuLi (1.5 mL, 3.75 mmol) at −60° C. under N.sub.2 atmosphere. After complete addition, theis mixture was stirred at −10° C. for 1 h. The reaction mixture was quenched by adding aqueous of 2 M HCL, and washed with toluene. The aqueous layer was brought pH=8 by adding Na.sub.2CO.sub.3, extracted then with ethyl acetate (50 mL×3). The combined organic layers were washed with brine. The solvent was removed under reduced pressure to dryness. The residue was separated by column to give 4.

(641) 3. Reaction Scheme:

(642) ##STR00383##

(643) Experimental Details: To a stirred and degassed mixture of compound 4 (2.8 g, 14 mmol) and compound 5 (7 g, 42 mmol) in an aqueous of 2 M Na.sub.2CO (250 mL) and toluene (40 mL) was stirred with Pd(PPh.sub.3).sub.4 (0.57 g, 0.05 mmol) under reflux over night. The reaction mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine. The solvent was removed under reduced pressure to dryness. The residue was separated by column to give 6.

(644) 4 Reaction Scheme:

(645) ##STR00384##

(646) Experimental Details: A solution of 6 (0.53 g, 1.9 mmol) and hydrazine (0.52 g, 8.8 mmol) in ethanol (50 mL) was stirred under reflux for 6 h. After cooling and precipitating, the precipitate was collected by filtrate and washed with ethanol to give compound 7.

(647) 5. Reaction Scheme:

(648) ##STR00385##

(649) Experimental Details: A solution of compound 7 (53 mg, 0.13 mmol) and compound 8 (79 mg, 0.13 mmol) in anhydrous CH.sub.2Cl.sub.2 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was purified by prep-TLC to give the desired compound.

Example 30

(650) ##STR00386##

(651) 1. Reaction Scheme:

(652) ##STR00387##

(653) Experimental Details: A mixture of 1 (3.1 g, 13 mmol) and 2 (1 g, 12 mmpl) in an aqueous of 2 M Na.sub.2CO.sub.3 (15 mL) and toluene (30 mL) was stirred with Pd(PPh.sub.3).sub.4 (0.4 g, 0.029 mmol) under reflux over night. The reaction mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine. The solvent was removed under reduced pressure to dryness. The residue was separated by column to give 3.

(654) 2. Reaction Scheme:

(655) ##STR00388##

(656) Experimental Details: A solution of compound 3 (2.0 g, 10 mmol) and triisopropyl borate (7 mL, 30 mmol) in anhydrous toluene (50 mL) was treated with n-BuLi (12 mL, 30 mmol) at −60° C. under N.sub.2 atmosphere. After complete addition, this mixture was stirred at −10° C. for 1 h. The reaction mixture was quenched by adding an aqueous of 2 n HCl, and washed with toluene. The aqueous layer was brought pH=8 by adding Na.sub.2CO.sub.3, extracted then with ethyl acetate (50 mL×3). The combined organic layers were washed with brine. The solvent was removed under reduced pressure to dryness. The residue was separated by column to give 4.

(657) 3. Reaction Scheme:

(658) ##STR00389##

(659) Experimental Details: To a stirred and degassed mixture of compound 4 (0.5 g, 3 mmol) and compound 5 (1.5 g, 9 mmol) in an aqueous of 2 M Na.sub.2CO.sub.3 (3.5 mL) and toluene (40 mL) was stirred with Pd(PPh.sub.3).sub.4 (94 mg, 0.003 mmol) under reflux over night. The reaction mixture was extracted with ethyl acetate (100 mL×3). The combined organic layers were washed with brine. The solvent was removed under reduced pressure to dryness. The residue was purified by column to give 6.

(660) 4. Reaction Scheme:

(661) ##STR00390##

(662) Experimental Details: A solution of 6 (0.24 g, 1 mmol) and hydrazine (0.3 g, 4.7 mmol) in ethanol (50 mL) was stirred under reflux for 6 h. After cooling and precipitating, the precipitate was collected by filtrate and washed with ethanol to give compound 7.

(663) 5. Reaction Scheme:

(664) ##STR00391##

(665) Experimental Details: A solution of compound 7 (70 mg, 0.29 mmol) and compound 8 (83 mg, 0.3 mmol) in anhydrous CH.sub.2C12 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was purified by prep-TLC to give the desired compound.

Example 31

(666) ##STR00392##

(667) 1. Reaction Scheme:

(668) ##STR00393##

(669) Experimental Details: To a solution of compound 2 (0.83 g, 5 mmol) in ethanol (100 mL) was added benzylamine (0.54 g, 5 mmol) drop wise. After stirring for 2 h, the reaction mixture was diluted with water. The resulting mixture was extracted with ethyl acetate (50 mL×3). The combined organic layers were dried over Na.sub.2SO.sub.4. The solvent was concentrated to give compound 3.

(670) 2. Reaction Scheme:

(671) ##STR00394##

(672) Experimental Details: A solution of 3 (1.18 g, 5 mmol) and hydrazine (5 ml) in ethanol (40 mL) was heated to reflux for 6 h. After cooling and precipitating, the precipitate was collected by filtrate and washed with ethanol to give compound 4.

(673) 3. Reaction Scheme:

(674) ##STR00395##

(675) Experimental Details: A solution of compound 4 (48.7 mg, 2 mmol) and compound 5 (63 mg, 0.2 mmol) in anhydrous CH.sub.2C12 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

Example 32

(676) ##STR00396##

(677) 1. Reaction Scheme:

(678) ##STR00397##

(679) Experimental Details: To a solution of compound 2 (0.83 g, 5 mmol) in ethanol (100 mL) was added compound 2 (0.35 g, 5 mmol) dropwise. After stirring for 2 h, the reaction mixture was diluted with water. The resulting mixture was extracted with ethyl acetate (50 mL×3). The combined organic layers were dried over Na.sub.2SO.sub.4. The solvent was concentrated to give compound 3.

(680) 2. Reaction Scheme:

(681) ##STR00398##

(682) Experimental Details: A solution of 3 (1.0 g, 5 mmol) and hydrazine (5 mL) in ethanol (40 mL) was heated to reflux for 6 h. After cooling and precipitating, the precipitate was collected by filtrate and washed with ethanol to give compound 4.

(683) 3. Reaction Scheme:

(684) ##STR00399##

(685) Experimental Details: A solution of compound 4 (480 mg, 2 mmol) and compound 5 (60 mg, 0.2 mmol) in anhydrous CH.sub.2Cl.sub.2 (300 mL) was stirred under reflux for 6 h. The solvent was removed under reduced pressure. The residue was separated by prep-TLC to give the desired compound.

(686) All references to any publication, patent, or other citation are hereby incorporated by reference.

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