The present application relates to therapeutic organic compounds of formula (I),wherein Q, R1, R2, R3, and R4 are defined herein. The invention also relates to pharmaceutical compositions comprising an effective amount of a therapeutic organic compound; and methods for treating and preventing disease such as cancer comprising administering and effective amount of a therapeutic organic compound to a subject in need thereof.

RELATED APPLICATIONS

This application is a §371 filing based on International Application No. PCT/US2010/038518, filed Jun. 14, 2010, which claims the benefit of Provisional U.S. Patent Application Ser. No. 61/186,584, filed Jun. 12, 2009. The entire contents of these patent applications are hereby incorporated herein by reference.

BACKGROUND

According to data collected by the American Cancer Society, more than 1.43 million people in the United States were diagnosed with cancer in 2008. Although earlier diagnoses and improved treatments have allowed for modest increases in five-year survival rates, the overall mortality rate per 100,000 people has gone down only 5 percent since 1950 due to the increased incidence of several types of cancer over the same period (SEER Cancer Statistics Review 1975-2004, NCI “55-Year Trends in U.S. Cancer Death Rates”).

Attending to this ongoing medical need, research directed toward the mechanisms by which cancer cells proliferate and survive has implicated the deregulation of protein kinases. Therefore, methods of modulating or inhibiting kinase activity, including the use of small molecule agents, represent a promising direction in oncology drug development.

Thus, there remains a need for compounds that inhibit the activity of one or more protein kinases, as they can be expected to be useful in the treatment of cancer.

SUMMARY OF THE INVENTION

The invention provides compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with abnormal or deregulated kinase activity, particularly diseases or disorders that involve abnormal activation of the Abl, BCR-Abl, c-kit, PDGFR and Src kinases. Such diseases include, for example, cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia.

Thus, in one aspect provided herein is a compound of Formula I. In one embodiment, Formula I is represented as Formula II.

In another aspect, provided herein is a method of treating cancer, comprising administering to a subject in need thereof a compound of Formula I. The cancer can be selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic malignancies, childhood leukemia, childhood lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS. In another embodiment, the cancer is pancreatic cancer or non-small cell lung cancer. In still another embodiment, the cancer is gastrointestinal stromal tumor or chronic myelogenous leukemia. In yet another embodiment, the cancer is acute myeloid leukemia. The cancer can be resistant to treatment with GLEEVEC®(imatinib), wherein treatment-resistance can be due to one or more point-mutations in an Abl kinase, a BCR-Abl kinase domain, a c-kit kinase, an Src kinase or a PDGFR kinase.

In another aspect, provided herein is the use of a compound of Formula I for the manufacture of a medicament for treating cancer in a subject.

In another aspect, provided herein is the use of a compound of Formula III for the manufacture of a medicament for treating cancer in a subject.

In yet another aspect, provided herein is a method of inhibiting the activity of a kinase, comprising utilizing a compound of Formula I. In one embodiment, the kinase is selected from Abl, Abl (T315I), BCR-Abl, BRAF, CDK11, CDK5, CDK2, CDK3, CDK7, DDR1, FLT1, FLT3, FLT4, HIPK1, kit, LOK, p38-gamma, PDGFRA, PDGFRB, or Src comprising utilizing a compound of Formula I.

In still another aspect, provided herein is a method of inhibiting the activity of a kinase, comprising utilizing a compound of Formula III. In one embodiment, the kinase is selected from Abl, Abl (T315I), BCR-Abl, BRAF, CDK11, CDK5, CDK2, CDK3, CDK7, DDR1, FLT1, FLT3, FLT4, HIPK1, kit, LOK, p38-gamma, PDGFRA, PDGFRB, or Src comprising utilizing a compound of Formula III.

In another aspect, provided herein is a method of inhibiting the activity of Abl kinase, BCR-Abl kinase, c-kit kinase, PDGFRA or PDGFRB kinase, or Src kinase comprising utilizing a compound of Formula I. In yet another embodiment, provided herein is a method of treating a disease in a subject, wherein the disease etiology or progression is at least partially mediated by the activity of Abl kinase, BCR-Abl kinase, c-kit kinase, Src kinase, or PDGFR kinase, comprising administering to the subject a compound of Formula I. In still another embodiment, provided herein is a method of treating cancer, comprising administering to a subject in need thereof a compound of Formula I in combination with a pharmaceutically effective amount of an additional anti-cancer agent. The additional anti-cancer agent can be imatinib or nilotinib.

In still another aspect, provided herein is a method of inhibiting the activity of Abl kinase, BCR-Abl kinase, c-kit kinase, PDGFRA or PDGFRB kinase, or Src kinase comprising utilizing a compound of Formula III. In yet another embodiment, provided herein is a method of treating a disease in a subject, wherein the disease etiology or progression is at least partially mediated by the activity of Abl kinase, BCR-Abl kinase, c-kit kinase, Src kinase, or PDGFR kinase, comprising administering to the subject a compound of Formula III. In still another embodiment, provided herein is a method of treating cancer, comprising administering to a subject in need thereof a compound of Formula III in combination with a pharmaceutically effective amount of an additional anti-cancer agent. The additional anti-cancer agent can be imatinib or nilotinib.

In another aspect, provided herein is a method of inhibiting the activity of FLT3 kinase comprising utilizing a compound of Formula III. In yet another embodiment, provided herein is a method of treating a disease in a subject, wherein the disease etiology or progression is at least partially mediated by the activity of FLT3 kinase, comprising administering to the subject a compound of Formula III. In still another embodiment, provided herein is a method of treating cancer, comprising administering to a subject in need thereof a compound of Formula III in combination with a pharmaceutically effective amount of an additional anti-cancer agent.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the Invention

This invention is directed toward compounds, intermediates thereto and derivatives thereof, as well as pharmaceutical compositions containing the compounds for use in treatment of protein kinase-associated disorders. The compounds of the invention or compositions thereof are useful as inhibitors of the kinase enzymes c-Abl, c-kit, BCR-Abl, PDGFR and combinations thereof. Furthermore, the compounds of the invention or compositions thereof can be used in the treatment of cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia. The present invention is also directed to methods of inhibiting protein kinase activity in cells, or for treating, preventing or ameliorating one or more symptoms of cancer using the compounds of the invention or pharmaceutical compositions, in combination with an additional anti-cancer agent.

In one aspect, the invention provides compounds of the Formula I:

Q is CH or N;

R3is H, C(O)—N(H)-aryl, C(O)—N(H)—C1-6-alkyl-heterocycle, C(O)—N(H)—C1-6-alkyl-heteroaryl, wherein the aryl, heteroaryl or heterocycle groups can be substituted or unsubstituted; and

In another embodiment, the aryl, heteroaryl and heterocyclic groups of R1, R3and R4can optionally be independently substituted one or more times with C1-6-alkyl, C1-6-alkoxy, SO2-heterocycle, C1-6-alkyl-heterocycle, heterocycle, CF3, or heteroaryl;

wherein the substituent heterocycle and heteroaryl groups can be optionally further independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C1-6-alkyl-OH, or C(O)—C1-6-alkyl.

In yet another embodiment of Formula I, R3is H, C(O)—N(H)-phenyl, C(O)—N(H)—(CH2)2-morpholino, C(O)—N(H)—C1-6-alkyl-morpholino, or C(O)—N(H)—C1-6-alkyl-imidazole, wherein the morpholino, imidazole, and phenyl groups of R3can optionally be independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C(O)—C1-6-alkyl, CO2—C1-6-alkyl, aryl, heteroaryl, heterocycle, SO2-heterocycle, SO2-aryl, SO2-heteroaryl, C1-6-alkyl-heterocycle, C1-6-alkyl-aryl, C1-6-alkyl-heteroaryl, CF3, or halogen and wherein the substituent aryl, heteroaryl and heterocyclic groups can optionally be further independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C1-6-alkyl-OH, or C(O)—C1-6-alkyl.

In another embodiment, at least one of R3and R4are not H. In yet another embodiment, R1is C(O)—C3-6-cycloalkyl, pyrimidine, C(O)N(H)-piperidine, C(O)-piperidine, C(O)C1-6-alkyl, H, C3-6-cycloalkyl, pyridine, Ph-SO2-piperazine, C(O)-PhCH2-piperazine-C1-6-alkyl, C(O)—C1-6-alkyl-piperazine, Ph-piperazine-C1-6-alkyl, C(O)-oxazolidinone-C1-6-alkyl-morpholino, wherein the pyrimidine group is optionally independently substituted one or more times with C1-6-alkyl or piperazine, wherein the piperazine is optionally substituted with C1-6-alkyl-OH; and wherein C(O)—C1-6-alkyl-piperazine is optionally substituted with C(O)C1-6-alkyl.

In another embodiment of the invention, Formula I is represented by the Formula II:

In one embodiment of Formula II, R1is C(O)—C3-6-cycloalkyl; Q is C(H); R2and R3are H; and R4is C(O)N(H)Ph, wherein the Ph group is optionally independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C(O)—C1-6-alkyl, CO2—C1-6-alkyl, aryl, heteroaryl, heterocycle, SO2-heterocycle, SO2-aryl, SO2-heteroaryl, C1-6-alkyl-heterocycle, C1-6-alkyl-aryl, C1-6-alkyl-heteroaryl, CF3, or halogen, wherein the substituent aryl, heteroaryl and heterocyclic groups can optionally be further independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C1-6-alkyl-OH, or C(O)—C1-6-alkyl.

In another embodiment of Formula II, Ph is optionally independently substituted one or more times with CF3, piperazine, C1-6-alkyl-piperazine, C1-6-alkyl-piperazine-C1-6-alkyl, CH2CH3, imidazole, or imidazole-C1-6-alkyl.

In still another embodiment of Formula II, R1is C(O)—C3-6-cycloalkyl; Q is C(H); R2and R3are H; and R4is C(O)N(H)Ph, wherein the Ph group is optionally independently substituted one or more times with CF3, C1-6-alkyl-piperazine, or imidazole, and wherein the substituent piperazine or imidazole is optionally independently substituted one or more times with C1-6-alkyl.

In yet another embodiment of Formula II, Ph is optionally independently substituted one or more times with CF3, piperazine, C1-6-alkyl-piperazine, C1-6-alkyl-piperazine-C1-6-alkyl, CH2CH3, imidazole, or imidazole-C1-6-alkyl.

In yet another embodiment, compounds of Formula (III) are included in the present invention:

wherein G is CR10or N;

wherein the substituent aryl, heteroaryl and heterocyclic groups can optionally be further independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C1-6-alkyl-OH, or C(O)—C1-6-alkyl.

R10is H or C1-3-alkyl;

wherein one of R13and R14is not H;

In another embodiment of Formula III, R13is H, C(O)—N(R28)-phenyl, C(O)—N(R29)—(CH2)2-morpholino, C(O)—N(R30)—C1-6-alkyl-morpholino, or C(O)—N(R30)—C1-6-alkyl-imidazole, wherein the morpholino, imidazole, and phenyl groups of R13can optionally be independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C(O)—C1-6-alkyl, CO2—C1-6-alkyl, aryl, heteroaryl, heterocycle, SO2-heterocycle, SO2-aryl, SO2-heteroaryl, C1-6-alkyl-heterocycle, C1-6-alkyl-aryl, C1-6-alkyl-heteroaryl, CF3, or halogen and wherein the substituent aryl, heteroaryl and heterocyclic groups can optionally be further independently substituted one or more times with OH, C1-6-alkyl, C1-6-alkoxy, C1-6-alkyl-OH, or C(O)—C1-6-alkyl.

In yet another embodiment of Formula III, at least one of R13and R14are not H.

In yet another embodiment, R1is C(O)—C3-6-cycloalkyl, pyrimidine, C(O)N(H)-piperidine, C(O)-piperidine, C(O)C1-6-alkyl, H, C3-6-cycloalkyl, pyridine, Ph-SO2-piperazine, C(O)-PhCH2-piperazine-C1-6-alkyl, C(O)—C1-6-alkyl-piperazine, Ph-piperazine-C1-6-alkyl, C(O)-oxazolidinone-C1-6-alkyl-morpholino, wherein the pyrimidine group is optionally independently substituted one or more times with C1-6-alkyl or piperazine, wherein the piperazine is optionally substituted with C1-6-alkyl-OH; and wherein C(O)—C1-6-alkyl-piperazine is optionally substituted with C(O)C1-6-alkyl.

Preferred embodiments of Formula I are equivalent to preferred embodiments of Formula III (including pharmaceutically acceptable salts thereof, as well as enantiomers, stereoisomers, rotamers, tautomers, diastereomers, atropisomers or racemates thereof) and are shown below in Table A and are also considered to be “compounds of the invention.” The compounds of the invention are also referred to herein as “protein kinase inhibitors.”

Compounds of the present invention are useful for the treatment of protein kinase-associated disorders.

As used herein, the term “protein kinase-associated disorder” includes disorders and states (e.g., a disease state) that are associated with the activity of a protein kinase. Non-limiting examples of protein kinase-associated disorders include abnormal cell proliferation, including protein kinase-associated cancers, viral infections, fungal infections, autoimmune diseases and neurodegenerative disorders.

Protein kinase-associated disorders also include diseases associated with apoptosis, including, but not limited to, cancer, viral infections, autoimmune diseases and neurodegenerative disorders.

Examples of protein kinase-associated cancers include carcinomas, hematopoietic tumors of lymphoid lineage, hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma.

Additional, non-limiting examples of protein-kinase associated disorders include tumor angiogenesis and metastasis. Non-limiting examples of protein-kinase associated disorders also include vascular smooth muscle proliferation associated with atherosclerosis, postsurgical vascular stenosis and restenosis, and endometriosis.

Non-limiting examples of protein-kinase associated disorders include viral infections in a patient in need thereof, wherein the viral infections include, but are not limited to, HIV, human papilloma virus, herpes virus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus.

Further non-limiting examples of protein-kinase associated disorders include those associated with infectious agents, including yeast, fungi, protozoan parasites such asPlasitiodium falciparum, and DNA and RNA viruses.

Compounds of the present invention are useful for the treatment of cancer, wherein the cancer is selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic malignancies, childhood leukemia, childhood lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS.

As used herein, the term “modulating” or “modulation” refers to the alteration of the catalytic activity of a protein kinase. In particular, modulating refers to the activation or inhibition of the catalytic activity of a protein kinase, depending on the concentration of the compound or salt to which the protein kinase is exposed or, more preferably, the inhibition of the catalytic activity of a protein kinase. The term “catalytic activity” as used herein refers to the rate of phosphorylation of tyrosine, serine or threonine under the influence, direct or indirect, of a protein kinase.

The three main classes that pharmacological inhibitors of kinase activity are categorized by are (1) Type I, or “DFG-in” ATP competitive inhibitors, which directly compete with ATP in the ATP binding site (i.e. dual SRrc ABL inhibitor dasatinib, (2) Type II, or “DFG-out” ATP competitive inhibitors, which, in addition to binding the ATP binding site also engage an adjacent hydrophobic binding site that is only accessible when the kinase is in an inactivated configuration (i.e. the activation loop is oriented in a conformation that would block substrate binding) (i.e. imatinib, nilotinib), and (3) non-ATP competitive inhibitors that binds at sites outside the ATP binding site that affect the activity of the kinase (i.e. GNF-2).

Second generation Abl inhibitors, such as dasatinib, and nilotinib, are highly active against imatinib-resistant leukemia. Both agents are significantly more potent against Bcr-Abl than imatinib, and are active against many imatinib-resistant Bcr-Abl mutants. However, neither agent is able to override imatinib resistance due to the mutation of a threonine to an isoleucine at residue 315 (T315I, the “gatekeeper” position). This highly prevalent and highly imatinib-resistant mutation is centrally located in the nucleotide binding cleft of Abl. Both dasatinib and nilotinib make a hydrogen bonding interaction to the side-chain hydroxyl group of T315, which is resistant to these compounds due to direct steric intrusion of the isobutyl side chain and a loss in the middle of the ATP-cleft of a hydrogen-bonding interaction.

In addition to BCR-Abl T315I, other gatekeeper residues that play an integral role in imatinib-resistant disease include c-kit-T670I, which is associated with imatinib-resistant gastrointestinal stromal tumor characterized by early metastasis and shorter progression-free survival; this mutation substantially modifies the binding pocket of c-Kit, and occurs only under the selective pressure of imatinib therapy, PDGFRA-T674M/I, which is found in the FIP1LI-PDGFRA kinase domain and gives rise to imatinib resistance in idiopathic hypereosinophilic syndrome (HES), and PDGFRB-T681M/I.

The compounds of the invention are type II class kinase inhibitors that traverse the gatekeeper position in a manner that accommodates amino acid side chains of a variety of sizes. A co-crystal structure of compound 2 (Table A) with Src demonstrates that compound 2 does indeed bind as a type II inhibitor (FIG. 1).

The above-listed protein kinases may exhibit one or more point mutations, including, but not limited to mutations of the hinge region, mutations of the P-loop, and mutations of the A-loop.

In a preferred embodiment, the protein kinase is selected from the group consisting of mutated or non-mutated Abl, mutated or non-mutated c-kit, mutated or non-mutated BCR-Abl, mutated or non-mutated PDGFR, mutated or non-mutated Src and any combination thereof. In a particularly preferred embodiment, the protein kinase is selected from the group consisting of mutated or non-mutated c-kit, mutated or non-mutated BCR-Abl, mutated or non-mutated PDGFR, and mutated or non-mutated Src.

In one embodiment, a compound of the present invention is characterized as an inhibitor of a combination of protein kinases, e.g., BCR-Abl and/or c-kit and/or PDGFR.

In certain embodiments, a compound of the present invention is used for protein kinase-associated diseases, and/or as an inhibitor of any one or more protein kinases. It is envisioned that a use can involve the inhibition one or more isoforms of the protein kinase.

In one embodiment, the compounds of the present invention selectively inhibit FLT3 kinase activity and the proliferation, viability, and cell cycle progression of leukemic cells harboring mutant FLT3.

In another embodiment, the compounds of the present invention selectively inhibit FLT3 kinase activity and the proliferation, viability, and cell cycle progression of leukemic cells harboring mutant FLT3, with no apparent effect on cells harboring wild-type FLT3.

The efficacy of therapeutic kinase inhibitors such as imatinib, dasatinib and nilotinib is typically correlated with their affinity toward one or more kinase targets that are associated with a particular disease state. Point-mutations, which can occur naturally or under the selective pressure of chemotherapy, can decrease the affinity of the chemotherapeutic for its kinase target, thereby conferring resistance to these therapies.

The compounds of the present invention are useful in overcoming drug resistance due to acquired point mutations in the molecular targets.

The compounds of the invention are also inhibitors of mutated or non-mutated forms of the kinase enzymes Abl, BCR-Abl, c-kit PDGFR and Src, which are implicated in certain disease states related to cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia. BCR-Abl activates a number of cell cycle-controlling proteins and enzymes, speeding up cell division and inhibiting DNA repair, thus resulting in genomic instability and, potentially, blast crisis in CML. Aberrant activation of c-kit is observed in most gastrointestinal stromal tumors, while the effects of PDGFR include cell proliferation and angiogenesis.

Without being bound by theory, it is believed that inhibition of the kinase enzymes Abl, BCR-Abl, c-kit, PDGFR and Src will promote apoptosis, inhibit cancer cell proliferation and inhibit tumor growth.

The present invention also includes treatment of one or more symptoms of cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia, as well as protein kinase-associated disorders as described above, but the invention is not intended to be limited to the manner by which the compound performs its intended function for treatment of a disease. The present invention includes treatment of diseases described herein in any manner that allows treatment to occur.

In certain embodiments, the compounds of the invention are used alone or in combination with other therapeutic agents, e.g. imatinib, nilotinib or dasatinib.

In another embodiment, the invention provides a pharmaceutical composition of any of the compounds of the present invention. In a related embodiment, the invention provides a pharmaceutical composition of any of the compounds of the present invention and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes the compounds as novel chemical entities.

In other embodiments, the present invention provides a method for inhibiting the activity of a protein kinase. The method includes contacting a cell with any of the compounds of the present invention. In a related embodiment, the method further provides that the compound is present in an amount effective to selectively inhibit the activity of a protein kinase.

Additionally, a method of the invention includes administering to a subject an effective amount of a protein kinase-modulating compound of the invention, e.g., protein kinase-modulating compounds of Formula I, Formula II or Formula III, as well as Table A (including pharmaceutically acceptable salts thereof, as well as enantiomers, stereoisomers, rotamers, tautomers, diastereomers, atropisomers or racemates thereof).

In certain embodiments, the compounds of the invention are used for the treatment of cancer. In one embodiment, compound 2 is used for the treatment of cancer. In another embodiment, compound 3 is used for the treatment of cancer.

In other embodiments, the present invention provides a use of any of the compounds of the invention for manufacture of a medicament to treat cancer.

In other embodiments, the invention provides a method of manufacture of a medicament, including formulating any of the compounds of the present invention for treatment of a subject.

One embodiment provided herein is a method of treating pancreatic cancer, comprising administering compound 2 to a subject in need thereof, such that the pancreatic cancer is treated.

Another embodiment provided herein is a method of treating non-small cell lung cancer, comprising administering compound 2 to a subject in need thereof, such that the non-small cell lung cancer is treated.

In yet another embodiment provided herein is a method of treating gastrointestinal stromal tumor, comprising administering compound 2 to a subject in need thereof, such that the gastrointestinal stromal tumor is treated.

In still another embodiment provided herein is a method of treating chronic myelogenous leukemia, comprising administering compound 2 to a subject in need thereof, such that the chronic myelogenous leukemia is treated.

In another embodiment provided herein is a method of treating acute myeloid leukemia, comprising administering compound 2 to a subject in need thereof, such that the acute myeloid leukemia is treated.

One other embodiment provided herein is a method of treating pancreatic cancer, comprising administering compound 3 to a subject in need thereof, such that the pancreatic cancer is treated.

Another embodiment provided herein is a method of treating non-small cell lung cancer, comprising administering compound 3 to a subject in need thereof, such that the non-small cell lung cancer is treated.

In yet another embodiment provided herein is a method of treating gastrointestinal stromal tumor, comprising administering compound 3 to a subject in need thereof, such that the gastrointestinal stromal tumor is treated.

In still another embodiment provided herein is a method of treating chronic myelogenous leukemia, comprising administering compound 3 to a subject in need thereof, such that the chronic myelogenous leukemia is treated.

In another embodiment provided herein is a method of treating acute myeloid leukemia, comprising administering compound 3 to a subject in need thereof, such that the acute myeloid leukemia is treated.

In certain embodiments, the compounds of the invention are used as medicaments. In one embodiment, compound 2 is used as a medicament. In another embodiment, compound 3 is used as a medicament.

One embodiment, provided herein is the use of compound 2 for the manufacture of a medicament for the treatment of pancreatic cancer in a subject in need thereof.

In another embodiment, provided herein is the use of compound 2 for the manufacture of a medicament for the treatment of non-small cell lung cancer in a subject in need thereof.

In yet another embodiment, provided herein is the use of compound 2 for the manufacture of a medicament for the treatment of gastrointestinal stromal tumor in a subject in need thereof.

In still another embodiment, provided herein is the use of compound 2 for the manufacture of a medicament for the treatment of chronic myelogenous leukemia in a subject in need thereof.

In one embodiment, provided herein is the use of compound 2 for the manufacture of a medicament for the treatment of acute myeloid leukemia in a subject in need thereof.

One other embodiment, provided herein is the use of compound 3 for the manufacture of a medicament for the treatment of pancreatic cancer in a subject in need thereof.

In another embodiment, provided herein is the use of compound 3 for the manufacture of a medicament for the treatment of non-small cell lung cancer in a subject in need thereof.

In yet another embodiment, provided herein is the use of compound 3 for the manufacture of a medicament for the treatment of gastrointestinal stromal tumor in a subject in need thereof.

In still another embodiment, provided herein is the use of compound 3 for the manufacture of a medicament for the treatment of chronic myelogenous leukemia in a subject in need thereof.

In another embodiment, provided herein is the use of compound 3 for the manufacture of a medicament for the treatment of acute myeloid leukemia in a subject in need thereof.

DEFINITIONS

The term “treat,” “treated,” “treating” or “treatment” includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. In certain embodiments, the treatment comprises the induction of a protein kinase-associated disorder, followed by the activation of the compound of the invention, which would in turn diminish or alleviate at least one symptom associated or caused by the protein kinase-associated disorder being treated. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.

The term “use” includes any one or more of the following embodiments of the invention, respectively: the use in the treatment of protein kinase-associated disorders; the use for the manufacture of pharmaceutical compositions for use in the treatment of these diseases, e.g., in the manufacture of a medicament; methods of use of compounds of the invention in the treatment of these diseases; pharmaceutical preparations having compounds of the invention for the treatment of these diseases; and compounds of the invention for use in the treatment of these diseases; as appropriate and expedient, if not stated otherwise. In particular, diseases to be treated and are thus preferred for use of a compound of the present invention are selected from cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia, or inflammation, cardiac hypertrophy, and HIV infection, as well as those diseases that depend on the activity of protein kinases. The term “use” further includes embodiments of compositions herein which bind to a protein kinase sufficiently to serve as tracers or labels, so that when coupled to a fluor or tag, or made radioactive, can be used as a research reagent or as a diagnostic or an imaging agent.

The term “subject” is intended to include organisms, e.g., prokaryotes and eukaryotes, which are capable of suffering from or afflicted with a disease, disorder or condition associated with the activity of a protein kinase. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia, or inflammation, cardiac hypertrophy, and HIV infection, and other diseases or conditions described herein (e.g., a protein kinase-associated disorder). In another embodiment, the subject is a cell.

The language “protein kinase-modulating compound,” “modulator of protein kinase” or “protein kinase inhibitor” refers to compounds that modulate, e.g., inhibit, or otherwise alter, the activity of a protein kinase. Examples of protein kinase-modulating compounds include compounds of the invention, i.e., Formula I, as well as the compounds of Table A (including pharmaceutically acceptable salts thereof, as well as enantiomers, stereoisomers, rotamers, tautomers, diastereomers, atropisomers or racemates thereof).

As used herein, the term “alkyl” refers to a fully saturated branched or unbranched hydrocarbon moiety. Preferably the alkyl comprises 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbons, 1 to 4 carbons, or 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like. Furthermore, the expression “Cx-Cy-alkyl”, wherein x is 1-5 and y is 2-10 indicates a particular alkyl group (straight- or branched-chain) of a particular range of carbons. For example, the expression C1-C4-alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.

The term “alkenyl,” alone or in combination refers to a straight-chain, cyclic or branched hydrocarbon residue comprising at least one olefinic bond and the indicated number of carbon atoms. Preferred alkenyl groups have up to 8, preferably up to 6, particularly preferred up to 4 carbon atoms. Examples of alkenyl groups are ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, isobutenyl, 1-cyclohexenyl, 1-cyclopentenyl.

The term “alkynyl” includes unsaturated aliphatic groups analogous in length to the alkyls described above, but which contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. The term alkynyl further includes alkynyl groups that include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6for straight chain, C3-C6for branched chain). The term C2-C6includes alkynyl groups containing 2 to 6 carbon atoms.

As used herein, the term “cycloalkyl” refers to saturated or unsaturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbon atoms, preferably 3-9, or 3-7 carbon atoms. Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl and the like. Exemplary bicyclic hydrocarbon groups include bornyl, indyl, hexahydroindyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl and the like. Exemplary tricyclic hydrocarbon groups include adamantyl and the like.

The term “cycloalkenyl” refers to a partially unsaturated cyclic hydrocarbon group containing 1 to 3 rings and 4 to 8 carbons per ring. Exemplary groups include cyclobutenyl, cyclopentenyl, and cyclohexenyl. The term “cycloalkenyl” also includes bicyclic and tricyclic groups in which at least one of the rings is a partially unsaturated, carbon-containing ring and the second or third ring may be carbocyclic or heterocyclic, provided that the point of attachment is to the cycloalkenyl group.

“Alkoxy” refers to those alkyl groups, having from 1 to 10 carbon atoms, attached to the remainder of the molecule via an oxygen atom. Alkoxy groups with 1-8 carbon atoms are preferred. The alkyl portion of an alkoxy may be linear, cyclic, or branched, or a combination thereof. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, butoxy, cyclopentyloxy, and the like. An alkoxy group can also be represented by the following formula: —OR′, where R′ is the “alkyl portion” of an alkoxy group.

The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of the stated number of carbon atoms and from one to five heteroatoms, more preferably from one to three heteroatoms, selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroalkyl group is attached to the remainder of the molecule through a carbon atom or a heteroatom.

The term “alkylcarbonyl” refers to a group having the formula —C(O)—Rii, wherein Riiis an alkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties. An “alkylcarbonyl” group can be attached to the remainder of the molecule via an alkyl group (i.e., -alkyl-C(O)—Rii).

The term “alkoxycarbonyl” refers to a group having the formula —C(O)O—Riii, wherein Riiiis an alkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties. An “alkoxycarbonyl” group can be attached to the remainder of the molecule via an alkyl group (i.e., -alkyl-C(O)O—Riii).

The term “heteroalkylcarbonyl” refers to a group having the formula —C(O)Riv, wherein Rivis a heteroalkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties. A “heteroalkylcarbonyl” group can be attached to the remainder of the molecule via an alkyl or heteroalkyl group (i.e., -alkyl-C(O)O—Rivor -heteroalkyl-C(O)O—Riv).

The term “aryl” includes aromatic monocyclic or multicyclic e.g., tricyclic, bicyclic, hydrocarbon ring systems consisting only of hydrogen and carbon and containing from six to nineteen carbon atoms, or six to ten carbon atoms, where the ring systems may be partially saturated. Aryl groups include, but are not limited to, groups such as phenyl, tolyl, xylyl, anthryl, naphthyl and phenanthryl. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).

The term “heteroaryl,” as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition of heterocycle below, “heteroaryl” is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl” refers to a five-member to ten-member, fully saturated or partially unsaturated nonaromatic heterocylic groups containing at least one heteroatom such as O, S or N. The most frequent examples are piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl or pirazinyl. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom.

The term “amine” or “amino” should be understood as being broadly applied to both a molecule, or a moiety or functional group, as generally understood in the art, and may be primary, secondary, or tertiary. The term “amine” or “amino” includes compounds where a nitrogen atom is covalently bonded to at least one carbon, hydrogen or heteroatom. The terms include, for example, but are not limited to, “alkyl amino,” “arylamino,” “diarylamino,” “alkylarylamino,” “alkylaminoaryl,” “arylaminoalkyl,” “alkaminoalkyl,” “amide,” “amido,” and “aminocarbonyl.” The term “alkyl amino” comprises groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term “dialkyl amino” includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups. The term “arylamino” and “diarylamino” include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively. The term “alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl” refers to an amino group which is bound to at least one alkyl group and at least one aryl group. The term “alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.

The term “amide,” “amido” or “aminocarbonyl” includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl” or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl or alkynyl groups bound to an amino group bound to a carbonyl group. It includes arylaminocarbonyl and arylcarbonylamino groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group. The terms “alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,” “arylaminocarbonyl,” “alkylcarbonylamino,” “alkenylcarbonylamino,” “alkynylcarbonylamino,” and “arylcarbonylamino” are included in term “amide.” Amides also include urea groups (aminocarbonylamino) and carbamates (oxycarbonylamino).

In a particular embodiment of the invention, the term “amine” or “amino” refers to substituents of the formulas N(R8)R9, CH2N(R8)R9and CH(CH3)N(R8)R9, wherein R8and R9are each, independently, selected from the group consisting of H and (C1-C4-alkyl)0-1G, wherein G is selected from the group consisting of COOH, H, PO3H, SO3H, Br, Cl, F, O—C1-4-alkyl, S—C1-4-alkyl, aryl, C(O)OC1-C6-alkyl, C(O)C1-C4-alkyl-COOH, C(O)C1-C4-alkyl and C(O)-aryl.

The description of the disclosure herein should be construed in congruity with the laws and principals of chemical bonding. For example, it may be necessary to remove a hydrogen atom in order accommodate a substitutent at any given location. Furthermore, it is to be understood that definitions of the variables (i.e., “R groups”), as well as the bond locations of the generic formulae of the invention (e.g., Formulas I, II or III), will be consistent with the laws of chemical bonding known in the art. It is also to be understood that all of the compounds of the invention described above will further include bonds between adjacent atoms and/or hydrogens as required to satisfy the valence of each atom. That is, bonds and/or hydrogen atoms are added to provide the following number of total bonds to each of the following types of atoms: carbon: four bonds; nitrogen: three bonds; oxygen: two bonds; and sulfur: two-six bonds.

The compounds of this invention may include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates) are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof. Compounds described herein may be obtained through art recognized synthesis strategies.

It will also be noted that the substituents of some of the compounds of this invention include isomeric cyclic structures. It is to be understood accordingly that constitutional isomers of particular substituents are included within the scope of this invention, unless indicated otherwise. For example, the term “tetrazole” includes tetrazole, 2H-tetrazole, 3H-tetrazole, 4H-tetrazole and 5H-tetrazole.

Isotopes

The present invention includes all pharmaceutically acceptable isotopically-labeled compounds of the invention, i.e. compounds of Formulas (I), (II) and (III), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

Certain isotopically-labelled compounds of Formulas (I), (II) and (III), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.3H, and carbon-14, i.e.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Isotopically-labeled compounds of Formulas (I), (II) and (III) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.

Combinations

The compounds of this invention are also useful in combination with known anti-cancer agents. Such known anti-cancer agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, and other angiogenesis inhibitors.

“Estrogen receptor modulators” refers to compounds, which interfere or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds, which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, .alpha.-difluoromethylornithine, ILX23-7553, trans-N-(4′-hydroxyphenyl) retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic agents” refer to compounds which cause cell death primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, microtubulin inhibitors, and topoisomerase inhibitors.

“Antiproliferative agents” also includes monoclonal antibodies to growth factors, other than those listed under “angiogenesis inhibitors”, such as trastuzumab, and tumor suppressor genes, such as p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134, for example).

“HMG-CoA reductase inhibitors” refers to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase. Compounds which have inhibitory activity for HMG-CoA reductase can be readily identified by using assays well-known in the art. For example, see the assays described or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131 at pp. 30-33. The terms “HMG-CoA reductase inhibitor” and “inhibitor of HMG-CoA reductase” have the same meaning when used herein.

Examples of HMG-CoA reductase inhibitors that may be used include, but are not limited to lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and cerivastatin. The structural formulae of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, “Cholesterol Lowering Drugs”, Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.

Assays

The inhibition of protein kinase activity by the compounds of the invention may be measured using a number of assays available in the art. Examples of such assays are described in the Exemplification section below.

Pharmaceutical Compositions

The compounds of the present invention are suitable as active agents in pharmaceutical compositions that are efficacious particularly for treating protein kinase-associated disorders and cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia. The pharmaceutical composition in various embodiments has a pharmaceutically effective amount of the present active agent along with other pharmaceutically acceptable excipients, carriers, fillers, diluents and the like.

The language “pharmaceutically effective amount” or “pharmaceutically acceptable amount” of the compound is that amount necessary or sufficient to treat or prevent a protein kinase-associated disorder, e.g. prevent the various morphological and somatic symptoms of a protein kinase-associated disorder, and/or a disease or condition described herein. In an example, an effective amount of a compound of the invention is the amount sufficient to treat a protein kinase-associated disorder in a subject. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular compound of the invention. For example, the choice of the compound of the invention can affect what constitutes an “effective amount.” One of ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the compounds of the invention without undue experimentation.

The regimen of administration can affect what constitutes a pharmaceutically effective amount. A compound of the invention can be administered to the subject either prior to or after the onset of a protein kinase-associated disorder. Further, several divided dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the compound(s) of the invention can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

In one non-limiting embodiment, the phrase “pharmaceutically effective amount” refers to the amount of a compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by kinase enzymes c-Abl, BCR-Abl, c-kit and/or PDGFR, or (ii) associated with kinase enzymes c-Abl, BCR-Abl, c-kit and/or PDGFR activity, or (iii) characterized by abnormal activity of kinase enzymes c-Abl, BCR-Abl, c-kit and/or PDGFR; or (2) reduce or inhibit the activity of kinase enzymes c-Abl, BCR-Abl, c-kit and/or PDGFR; or (3) reduce or inhibit the expression of kinase enzymes c-Abl, BCR-Abl, c-kit and/or PDGFR. In another non-limiting embodiment, the phrase “pharmaceutically effective amount” refers to the amount of a compound of the present invention that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and/or ameliorate cancer, e.g. pancreatic cancer, non-small cell lung cancer, gastrointestinal stromal tumor, or chronic myelogenous leukemia. In still another non-limiting embodiment, the term “pharmaceutically effective amount” refers to the amount of a compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reduce or inhibit the activity of kinase enzymes c-Abl, BCR-Abl, c-kit and/or PDGFR; or at least partially reduce or inhibit the expression of kinase enzymes c-Abl, BCR-Abl, c-kit and/or PDGFR.

The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular organic compound. For example, the choice of the organic compound can affect what constitutes an “effective amount.” One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the acceptable amount of the organic compound without undue experimentation.

Compounds of the invention may be used in the treatment of states, disorders or diseases as described herein, or for the manufacture of pharmaceutical compositions for use in the treatment of these diseases. Methods of use of compounds of the present invention in the treatment of these diseases, or pharmaceutical preparations having compounds of the present invention for the treatment of these diseases.

The language “pharmaceutical composition” includes preparations suitable for administration to mammals, e.g., humans. When the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount treats a protein kinase-associated disorder.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.

Synthetic Procedure

Compounds of the present invention are prepared from commonly available compounds using procedures known to those skilled in the art, including any one or more of the following conditions without limitation:

Acid addition salts of the compounds of the invention are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric or phosphoric acids and organic acids e.g. succinic, malaeic, acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalates can be used for example in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.

Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulphonate derived from methane sulphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid, the toluene-p-sulphonate derived from p-toluene sulphonic acid, and the like. Particularly preferred salts are sodium, lysine and arginine salts of the compounds of the invention. Such salts can be formed by procedures well known and described in the art.

Other acids such as oxalic acid, which can not be considered pharmaceutically acceptable, can be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the invention and its pharmaceutically acceptable acid addition salt.

Metal salts of a chemical compound of the invention include alkali metal salts, such as the sodium salt of a chemical compound of the invention containing a carboxy group.

In vivo hydrolyzable esters or amides of certain compounds of the invention can be formed by treating those compounds having a free hydroxy or amino functionality with the acid chloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform. Suitable bases include triethylamine or pyridine. Conversely, compounds of the invention having a free carboxy group can be esterified using standard conditions which can include activation followed by treatment with the desired alcohol in the presence of a suitable base.

Mixtures of isomers obtainable according to the invention can be separated in a manner known per se into the individual isomers; diastereoisomers can be separated, for example, by partitioning between polyphasic solvent mixtures, recrystallisation and/or chromatographic separation, for example over silica gel or by, e.g., medium pressure liquid chromatography over a reversed phase column, and racemates can be separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by means of fractional crystallisation, or by chromatography over optically active column materials.

Intermediates and final products can be worked up and/or purified according to standard methods, e.g., using chromatographic methods, distribution methods, (re-) crystallization, and the like.

At all stages of the reactions, mixtures of isomers that are formed can be separated into the individual isomers, for example diastereoisomers or enantiomers, or into any desired mixtures of isomers, for example racemates or mixtures of diastereoisomers, for example analogously to the methods described in Science of Synthesis: Houben-Weyl Methods of Molecular Transformation. Georg Thieme Verlag, Stuttgart, Germany, 2005.

The solvents from which those solvents that are suitable for any particular reaction may be selected include those mentioned specifically or, for example, water, esters, such as lower alkyl-lower alkanoates, for example ethyl acetate, ethers, such as aliphatic ethers, for example diethyl ether, or cyclic ethers, for example tetrahydrofuran or dioxane, liquid aromatic hydrocarbons, such as benzene or toluene, alcohols, such as methanol, ethanol or 1- or 2-propanol, nitriles, such as acetonitrile, halogenated hydrocarbons, such as methylene chloride or chloroform, acid amides, such as dimethylformamide or dimethyl acetamide, bases, such as heterocyclic nitrogen bases, for example pyridine or N-methylpyrrolidin-2-one, carboxylic acid anhydrides, such as lower alkanoic acid anhydrides, for example acetic anhydride, cyclic, linear or branched hydrocarbons, such as cyclohexane, hexane or isopentane, or mixtures of those solvents, for example aqueous solutions, unless otherwise indicated in the description of the processes. Such solvent mixtures may also be used in working up, for example by chromatography or partitioning.

The compounds, including their salts, may also be obtained in the form of hydrates, or their crystals can, for example, include the solvent used for crystallization. Different crystalline forms may be present.

The invention relates also to those forms of the process in which a compound obtainable as an intermediate at any stage of the process is used as starting material and the remaining process steps are carried out, or in which a starting material is formed under the reaction conditions or is used in the form of a derivative, for example in a protected form or in the form of a salt, or a compound obtainable by the process according to the invention is produced under the process conditions and processed further in situ.

This invention also encompasses pharmaceutical compositions containing, and methods of treating protein kinase-associated disorders through administering, pharmaceutically acceptable prodrugs of compounds of the compounds of the invention. For example, compounds of the invention having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of compounds of the invention. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in J. Med. Chem. 1996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities.

Any reference to a compound of the present invention is therefore to be understood as referring also to the corresponding pro-drugs of the compound of the present invention, as appropriate and expedient.

Advantageously, the present invention also provides kits for use by a consumer for treating disease. The kits comprise a) a pharmaceutical composition comprising an antibiotic and a pharmaceutically acceptable carrier, vehicle or diluent; and, optionally, b) instructions describing a method of using the pharmaceutical composition for treating the specific disease. The instructions may also indicate that the kit is for treating disease while substantially reducing the concomitant liability of adverse effects associated with antibiotic administration.

A “kit” as used in the instant application includes a container for containing the separate unit dosage forms such as a divided bottle or a divided foil packet. The container can be in any conventional shape or form as known in the art which is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag (for example, to hold a “refill” of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle which is in turn contained within a box.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be packed. Next, the tablets or capsules are placed in the recesses accordingly and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

It may be desirable to provide a written memory aid, where the written memory aid is of the type containing information and/or instructions for the physician, pharmacist or subject, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested or a card which contains the same type of information. Another example of such a memory aid is a calendar printed on the card e.g., as follows “First Week, Monday, Tuesday,” . . . etc. . . . “Second Week, Monday, Tuesday, . . . ” etc. Other variations of memory aids will be readily apparent. A “daily dose” can be a single tablet or capsule or several tablets or capsules to be taken on a given day.

Another specific embodiment of a kit is a dispenser designed to dispense the daily doses one at a time. Preferably, the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a mechanical counter, which indicates the number of daily doses that, has been dispensed. Another example of such a memory-aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.

Exemplification of the Invention

The invention is further illustrated by the following examples, which should not be construed as further limiting. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology and immunology, which are within the skill of the art.

General Synthesis Processes

General Synthesis Methods

Non-limiting general synthesis procedures are depicted in Schemes 1-3. A variety of alternative conditions are known to those of skill in the art.

As shown in Scheme 1, the transformation of A to B involves coupling of the aryl compound A to an organometallic coupling partner (e.g., a boronic acid), using conditions well-known in the art (e.g., using a metal catalyst such as dichlorobis(triphenylphospine)palladium(II), a ligand such as 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl, a base such as Na2CO3, and a solvent such as dioxane). The transformation of B to C involves formation of a 2-aminothiazole moiety using conditions well-known in the art. For example, when Y is NH2and Z is a halogen, said compound may be treated with reagents such as cyclopropanecarbonyl chloride, ammonium thiocyanate, and potassium carbonate in solvents such as acetone and dioxane, at a temperature such as 50° C. The transformation of B to C also involves the replacement of the substituent R (e.g., C1-C6alkyl) with the substituent R′ (e.g., H) using conditions well-known in the art (e.g., using solvents such as water, methanol, and tetrahydrofuran, and reagents such as lithium hydroxide monohydrate and hydrochloric acid).

As shown in Scheme 2, the transformation of C to D involves the reaction of the carboxylic acid or carboxylic acid derivative C with a compound RR—NH2using conditions well-known in the art (e.g., using solvents such as DMF and reagents such as N,N-diisopropylethylamine and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium at a temperature such as room temperature).

As shown in Scheme 3, the transformation of C to E involves conditions well-known in the art (e.g., using solvents such as toluene and reagents such as triethylamine and diphenylphosphorylazide), followed by reaction with a compound such as RR—NH2, or a compound such as RR—CO2H using conditions well-known in the art (e.g., using solvents such as DMF and reagents such as N,N-diisopropylethylamine and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium at a temperature such as room temperature).

All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesis the compounds of the present invention are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art (Houben-Weyl 4th Ed. 1952, Methods of Organic Synthesis, Thieme, Volume 21). Further, the compounds of the present invention can be produced by organic synthesis methods known to one of ordinary skill in the art as shown in the following examples.

Referring to the examples that follow, compounds of the preferred embodiments were synthesized using the methods described herein, or other methods, which are known in the art.

SYNTHESIS EXAMPLES

Part A

To a solution of 2-chloro-5-nitropyridine (5.23 g, 32.99 mmol) in dioxane (170 mL) was added 3-(ethoxycarbonyl)phenylboronic acid (6.28 g, 32.99 mmol) and 1N Na2CO3aqueous solution (82.5 mL, 82.5 mmol). The reaction mixture was degassed using Argon gas for 20 min followed by the addition of dichlorobis(triphenylphospine)palladium(II) (1.38 g, 1.99 mmol) and 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (1.26 g, 2.97 mmol). The reaction flask was put into the preheated oil-bath at 90° C. The reaction mixture was further stirred at 90° C. for a period of 10 h after which it was filtered and partitioned between ethyl acetate and water. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated. The crude product was purified by flash column chromatography using a 95:5 v/v hexane:ethyl acetate as solvent to afford ethyl 3-(5-nitropyridin-2-yl)benzoate (8.0 g, 89% yield) as a bright yellow solid.

Part B

To a solution of ethyl 3-(5-nitropyridin-2-yl)benzoate (8.0 g, 29.40 mmol) in ethanol (150 mL) was added 5% Pd/C (800 mg). The reaction mixture was stirred under H2balloon pressure for 16 h. The reaction mixture was filtered and concentrated to give 6.9 g (97%, yield) of ethyl 3-(5-aminopyridin-2-yl)benzoate as a tan solid.

Part C

To a solution of ethyl 3-(5-aminopyridin-2-yl)benzoate (7.52 g, 31.06 mmol) in DMF (150 mL) was added N-bromosuccinimide (5.58 g, 31.37 mmol) at 0° C. for 5 min. The reaction mixture was quenched with satd. NaHCO3solution (150 mL) at 0° C. The mixture was partitioned between ethyl acetate and water. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated. The crude product was purified by flash column chromatography using a 85:15 v/v hexane:ethyl acetate as solvent to afford ethyl 3-(5-amino-6-bromopyridin-2-yl)benzoate (8.6 g, 86% yield) as a reddish brown solid.

Part D

To a solution of ethyl 3-(5-amino-6-bromopyridin-2-yl)benzoate (10.44 g, 32.60 mmol) in NMP (150 mL) was added potassium ethyl xanthogenate (26.14 g, 163.11 mmol) and acetic acid (9.4 mL, 163.11 mmol). The reaction mixture was heated at 150° C. for 16 hours. The mixture was cooled to 50° C. and iodomethane (20.3 mL, 326 mmol) was added. The reaction mixture was further stirred for 30 minutes and partitioned between ethyl acetate and water. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated. The crude product was purified by flash column chromatography using a 85:15 v/v hexane:ethyl acetate as solvent to afford ethyl 3-(2-(methylthio)thiazolo[5,4-b]pyridin-5-yl)benzoate (7.6 g, 70% yield) as a bright brown solid.

Part E

To a solution was ethyl 3-(2-(methylthio)thiazolo[5,4-b]pyridin-5-yl)benzoate (5.6 g, 16.9 mmol) in THF (25 mL) and methanol (25 mL) was added Oxone (41.55 g, 67.67 mmol) in water (25 mL). The reaction mixture was stirred for 16 hours at room temperature. The reaction mixture was filtered and concentrated to give 5.8 g (94%, yield) of ethyl 3-(2-(methylsulfonyl)thiazolo[5,4-b]pyridin-5-yl)benzoate as a bright brown solid.

Part F

Ethyl 3-(2-(methylsulfonyl)thiazolo[5,4-b]pyridin-5-yl)benzoate (3.6 g, 9.91 mmol) was added to a 2N ammonia solution in IPA (24.8 mL, 49.57 mmol). The reaction mixture was heated at 90° C. and stirred for 30 h. The solvent was removed with in vacuo to give 2.7 g (91%, crude yield) ethyl 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)benzoate as a yellow solid.

Part G

To a solution of ethyl 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)benzoate (800 mg, 2.67 mmol) in dichloromethane (13 mL) was added pyridine (0.74 mL, 4.05 mmol) and cyclopropanecarbonyl chloride (0.27 mL, 2.94 mmol). The reaction mixture was stirred for 4 hours. The reaction mixture was diluted with dichloromethane (20 mL) and washed with 1N aqueous HCl solution and brine, dried over MgSO4, filtered and concentrated. The crude product was used for the next step without further purification.

Part H

Ethyl 3-(2-(cyclopropanecarboxamido)thiazolo[5,4-b]pyridin-5-yl)benzoate (870 mg, 2.37 mmol) was dissolved in the mixture of water (3.0 mL), THF (3.0 mL) and methanol (3.0 mL) followed by the addition of lithium hydroxide monohydrate (497 mg, 11.85 mmol). The reaction mixture was stirred at room temperature for 16 h and was neutralized with 1N aqueous HCl until pH=6 (monitored with pH paper). Upon removal of the organic solvent in vacuo the resulting brown solid was collected and dried to give 620 mg (87% yield) of 3-(2-(cyclopropanecarboxamido)thiazolo[5,4-b]pyridin-5-yl)benzoic acid.

Part I

Part A

To a solution of ethyl 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)benzoate (400 mg, 1.34 mmol) in a mixture of water (2.0 mL), THF (2.0 mL) and methanol (2.0 mL) was added lithium hydroxide monohydrate (281 mg, 11.85 mmol). The reaction mixture was stirred at room temperature for 16 h and was neutralized with 1N aqueous HCl until pH=6 (monitored with pH paper). The organic solvent was removed in vacuo and the resulting brown solid was collected and dried to give 310 mg (85% yield) of 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)benzoic acid.

Part B

To a solution 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)benzoic acid (310 mg, 1.14 mmol) in DMF (5.0 mL) was added N,N-diisopropylethylamine (0.56 mL, 3.42 mmol), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (649 mg, 1.71 mmol) and 3-(trifluoromethyl) aniline (0.28 mL, 2.28 mmol). The reaction mixture was stirred at room temperature for 16 h. The crude product was diluted with satd. NH4Cl solution (10 mL). The resulting brown solid was collected and washed with water and dried to give 320 mg (67% yield) of the 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)-N-(3-(trifluoromethyl)phenyl)benzamide.

Part C

To a solution of 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)-N-(3-(trifluoromethyl)phenyl)benzamide (33 mg, 0.079 mmol) in 2-butanol (1 mL) was added tert-butyl 4-(6-chloro-2-methylpyrimidin-4-yl)piperazine-1-carboxylate (24 mg, 0.079 mmol) and K2CO3(82.5 mL). The reaction mixture was degassed using Argon gas for 20 min to which was added tris(dibenzylideneacetone)dipalladium(0) (4 mg, 7.1 μmol) and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (4 mg, 4.7 μmol). The reaction flask was put into the preheated oil-bath at 90° C. The reaction mixture was further stirred at 90° C. for a period of 4 hours after which, it was filtered and concentrated. The crude reaction mixture was dissolved in dichloromethane (1 mL) to which was added trifluoroacetic acid (30 μL, 0.40 mmol). The reaction mixture was stirred for 4 h. The majority of the organic solvent was removed in vacuo and the crude product was diluted with DMSO (1 mL) and purified by preparative HPLC to give the title compound as a TFA salt.

To a solution of 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)-N-(3-(trifluoromethyl)phenyl)benzamide (50 mg, 0.12 mmol) and triethylamine (25 μL, 0.18 mmol) in dichloromethane (1.0 mL) was added 4-nitrophenyl carbonochloridate (26 mg, 0.13 mmol) at 0° C. The reaction mixture was stirred at room temperature for 1 h. Tert-butyl 4-aminopiperidine-1-carboxylate (36 mg, 0.18 mmole) and triethylamine (25 μL, 0.18 mmol) were added to the reaction mixture. The reaction mixture was stirred for 4 h after which time the solvent was removed in vacuo. The reaction mixture was diluted with dichloromethane (5 mL) and washed with 1N aqueous HCl and brine. The organic layer was dried over MgSO4and concentrated in vacuo. To a solution of the crude mixture in dichloromethane (1 mL) was added trifluoroacetic acid (40 μL, 0.60 mmol). The reaction mixture was stirred for 4 h. The majority of the organic solvent was removed in vacuo and the crude product was diluted with DMSO (1 mL) and purified by preparative HPLC to give title compound in a TFA salt form.

To a solution of 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (15 mg, 0.07 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (26 mg, 0.14 mmol), 4-dimethylaminopyridine (8 mg, 0.07 mmol) in DMF was added 3-(2-aminothiazolo[5,4-b]pyridin-5-yl)-N-(3-(trifluoromethyl)phenyl)benzamide (28 mg, 0.07 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with ethyl acetate (5 mL) and washed 1N aqueous HCl and brine. The organic layer was dried over MgSO4and the solvent removed in vacuo. To a solution of the crude mixture in dichloromethane (1 mL) was added trifluoroacetic acid (30 μL, 0.34 mmol). The reaction mixture was stirred for 4 h. The majority of the organic solvent was removed in vacuo and the crude product was diluted with DMSO (1 mL) and purified by preparative HPLC to give title compound in a TFA salt form.

To a solution of 3-(2-(cyclopropanecarboxamido)thiazolo[5,4-b]pyridin-5-yl)benzoic acid (150 mg, 0.44 mmol) and triethylamine (90 μL, 0.66 mmol) in toluene (2 mL) was added diphenylphosphoryl azide (0.11 mL, 0.49 mmol). The resulting mixture was stirred at room temperature for 30 minutes and heated at 80° C. for 1 h. To a reaction mixture was added 4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl) aniline (121 mg, 0.42 mmol) and triethylamine (90 μL, 0.66 mmol). The reaction mixture was stirred at 80° C. for 2 h and most of organic solvent was removed in vacuo. The crude product was diluted with DMSO (3 mL) and purified by preparative HPLC to give the title compound as a TFA salt.

Part A

To a solution of 5-bromo-3-nitropyridin-2-amine (5.00 g, 23.97 mmol) in dioxane (115 mL) was added 3-(ethoxycarbonyl)phenylboronic acid (4.65 g, 23.97 mmol) and 1N Na2CO3aqueous solution (92.2 mL, 92.2 mmol). The reaction mixture was degassed using Argon gas for 20 min followed by the addition of dichlorobis(triphenylphospine) palladium(II) (971 mg, 1.38 mmol) and 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (881 mg, 2.07 mmol). The reaction flask was put into the preheated oil-bath at 90° C. The reaction mixture was further stirred at 90° C. for a period of 10 h after which it was filtered and partitioned between ethyl acetate and water. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated. The crude product was purified by flash column chromatography using a 90:10 v/v hexane:ethyl acetate as solvent to afford ethyl 3-(6-amino-5-nitropyridin-3-yl)benzoate (5.2 g, 78% yield) as a tan solid.

Part B

A solution of tert-butylnitrite (3.23 mL, 27.17 mmol), anhydrous copper(II) chloride (2.9 g, 21.74 mmol) and anhydrous acetonitrile (90 mL) was warmed to 70° C. Ethyl 3-(6-amino-5-nitropyridin-3-yl)benzoate (5.2 g, 18.11 mmol) was then added portionwise over a period 10 minutes to the reaction solution. The reaction mixture was maintained at 70° C. for 2 hours and then allowed to cool to room temperature. The reaction mixture was then poured into 110 mL of 20% aqueous HCl solution, followed by extraction with ethyl acetate. The organic extracts were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography using a 95:5 v/v hexane:ethyl acetate as solvent to afford ethyl 3-(6-chloro-5-nitropyridin-3-yl)benzoate (3.4 g, 61% yield) as a tan solid.

Part C

To a solution of ethyl 3-(6-chloro-5-nitropyridin-3-yl)benzoate (3.0 g, 9.08 mmol) in ethyl acetate (50 mL) was added Tin(II) chloride dihydrate (11.06 g, 49.01 mmol). The reaction mixture was stirred for 10 hours at room temperature. The reaction mixture was cooled to 0° C. and then NH4OH solution was added to the reaction mixture and it was allowed to reach around pH=5 (monitored with pH paper). The reaction mixture was neutralized with Na2CO3and the resulting white solid was filtered and washed with ether (100 mL) three times. The organic layer was concentrated to give 2.3 g (84%, yield) of ethyl 3-(5-amino-6-chloropyridin-3-yl)benzoate as a tan solid.

Part D

Ammonium thiocyanate (303 mg, 3.98 mmol) was dissolved in acetone (10 mL) and heated to 50° C. and which point a clear solution was obtained. Cyclopropanecarbonyl chloride (0.37 mL, 3.98 mmol) was added dropwise and the resulting white suspension was refluxed for 20 minutes. Ethyl 3-(5-amino-6-chloropyridin-3-yl)benzoate (1.0 g, 3.62 mmol) dissolved in dioxane (18 mL) and K2CO3(2.0 g, 14.48 mmol) were added to the reaction mixture. The reaction mixture was refluxed for 8 hours at 120° C. After cooling to room temperature, the reaction solution was poured into ice water and the resulting tan solid was collected by filtration, washed with water and ether and dried. The crude product was used for the next step without further purification.

Part E

Ethyl 3-(2-(cyclopropanecarboxamido)thiazolo[5,4-b]pyridin-6-yl)benzoate (900 mg, 2.45 mmol) was dissolved in a mixture of water (4.0 mL), THF (4.0 mL) and methanol (4.0 mL) and lithium hydroxide monohydrate (513 mg, 12.23 mmol) was added. The reaction mixture was stirred at room temperature for 16 h and was neutralized with 1N aqueous HCl until pH 6 (monitored with pH paper). The organic solvent was removed in vacuo and the resulting brown solid was collected by filtration and dried to give 720 mg (86% yield) of 3-(2-(cyclopropanecarboxamido)thiazolo[5,4-b]pyridin-5-yl)benzoic acid.

Part F

Part A

A mixture of ammonium thiocyanate (668 mg, 8.77 mmol) and acetone (10 mL) was heated until a clear solution was obtained at 50° C. Cyclopropanecarbonyl chloride (0.80 mL, 8.77 mmol) was added dropwise and the resulting white suspension was refluxed for 20 minutes. 3,5-dibromopyrazin-2-amine (2.0 g, 7.97 mmol) dissolved in acetone (18 mL) and K2CO3(4.4 g, 31.88 mmol) were added to the reaction mixture. The reaction mixture was refluxed for 4 hours. After cooling to room temperature, the reaction solution was poured into ice water and the resulting tan solid was collected by filtration, washed with water and ether and dried. The crude product (1.6 g, 67% yield) was used for the next step without further purification.

Part B

To a solution of N-(6-bromothiazolo[4,5-b]pyrazin-2-yl)cyclopropanecarboxamide (1.00 g, 3.36 mmol) in dioxane (16 mL) was added 4-boronobenzoic acid (557 mg, 3.36 mmol) and 1N Na2CO3aqueous solution (13.2 mL, 13.2 mmol). The reaction mixture was degassed using Argon gas for 20 min followed by the addition of dichlorobis(triphenylphospine) palladium(II) (141 mg, 0.20 mmol) and 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (128 mg, 0.30 mmol). The reaction flask was put into the preheated oil-bath at 120° C. The reaction mixture was further stirred at 120° C. for a period of 10 h after which it was filtered. 2.0 N NaOH solution was added to the filtrate until pH 10 (monitored with pH paper) and then partitioned between ethyl acetate and water. A 6.0 N aqueous HCl solution was added to the water layer until pH 6 (monitored with pH paper). The resulting brown solid was collected by filtration and dried to give 740 mg (64% yield) of 4-(2-(cyclopropanecarboxamido)thiazolo[4,5-b]pyrazin-6-yl)benzoic acid.

Part C

Chemical Compounds and Biologic Reagents

All compounds were initially dissolved in DMSO to make 10 mM stock solutions, and then were serially diluted to obtain final concentrations for in vitro experiments.

Cell Lines and Cell Culture

Ba/F3.p210 cells were obtained by transfecting the IL-3-dependent marine hematopoietic Ba/F3 cell line with a pGD vector containing p210BCR-ABL (B2A2) cDNA (Daley and Baltimore, 1988; Sattler et al., 1996; Okuda et al., 1996). Murine hematopoietic 32D cells were transduced with retrovirus to express p210 Bcr-ABL (32D.p210 cells) (Matulonis et al., 1993); the 32D-T315I cell line was transfected by electroporation with the imatinib resistant BCR-ABL construct (pCI-neo Mammalian Expression Vector; Promega (#E1841) harboring the point mutation T315I (Weisberg et al., 2005). Ba/F3 cells were stably transfected by electroporation with imatinib-resistant BCR-ABL constructs (pCI-neo Mammalian Expression Vector; Promega (#E1841) harboring the point mutations T315I, F317L, F486S, and M351T; transfectants were selected for neomycin resistance and IL-3-independent growth (Weisberg et al., 2005). Ba/F3 cells were stably transfected by electroporation with BCR-ABL point mutants identified in a random mutagenesis screen for BCR-ABL point mutants conferring resistance to nilotinib: Q252H, E292K, Y253C, Y253H, E255K, and V289L (Ray et al., 2007). Ba/F3 cells were made to express Tel-PDGFRβ as described by Golub et al., 1994, and Carroll et al., 1996. Constructs of D842V-PDGFRα and V561D-PDGFRα cDNA cloned into pcDNA3.1 were stably transfected into Ba/F3 cells by electroporation, and cells were selected for neomycin resistance and IL3-independent growth as described by Weisberg et al., 2006.

All cell lines were cultured with 5% CO2at 37° C. in RPMI (Mediatech, Inc., Herndon, Va.) with 10% fetal calf serum (FCS) and supplemented with 1% L-glutamine. Parental Ba/F3 cells were similarly cultured with 15% WEHI-conditioned medium as a source of IL-3. Transfected cell lines were cultured in media supplemented with 1 mg/ml G418.

Antibodies and Immunoblotting and Immunoprecipitation

Anti-p-Tyr (clone 4G10, Upstate Biotechnology, NY) was used at 1:1000 for immunoblotting. The ABL antibody (clone 24-21, Calbiochem, San Diego, Calif.) was used at 1:1000 for immunoblotting. The KIT antibody (C-19, Santa Cruz Biotechnology, CA) was used at 1:1000 for immunoblotting. The phospho-KIT antibody (Tyr719, Cell Signaling, Danvers, Mass.) was used at 1:1000 for immunoblotting. PDGFRA antibody (C-20, Santa Cruz Biotechnology, CA) was used at 1:200 for immunoblotting. The monoclonal anti-β-actin antibody (clone AC-15) (Sigma-Aldrich, St. Louis, Mo.) was used at a 1:2000 dilution. Cells were lysed in lysis buffer (0.02 M Tris [pH 8.0], 0.15 M NaCl, 10% glycerol, 1% NP-40 (wt/vol), 0.1 M NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 40 μg/ml leupeptin, and 20 μg/ml aprotinin). Protein lysates were incubated for 25 min on ice, with vortexing at 5 min intervals, and then centrifuged for 15 min at 12,000×g. Supernatants were saved, and the Bio-Rad Protein Assay was used to determine protein yields (Bio-Rad Laboratories, Hercules, Calif.). Equivalent amounts of protein were subsequently loaded directly onto a gel for immunoblotting experiments. For immunoprecipitation, cell lysates were incubated with FLT3/Flk-2 (C-20) antibody and protein A Sepharose overnight with rocking at 4° C. As a control, cell lysates were also incubated with protein A Sepharose beads alone. Following incubation, immune complexes were washed 2× with lysis buffer, 2× with 1×PBS, and were dissolved in Laemmeli's sample buffer by boiling for 5 min. For immunoblotting and immunoprecipitation, whole cell lysates and immune complexes, respectively, were resolved on a sodium dodecyl sulfate (SDS)-7.5% polyacrylamide gel. Following this, protein was electrophoretically transferred to a Protran nitrocellulose transfer and immobilization membrane (Schleicher and Schuell, Dassel, Germany). The membrane was then blocked overnight at 4° C. with 5% nonfat dry milk in 1×TBS (10 mM Tris-HCl [pH 8.0], 150 mM NaCl) and then probed for 2 hr at 25° C. with pTYR, antibody or overnight at 4 C with FLT3/Flk-2 (C-20) antibody in 1×TBST buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween20). Following 3 washes with 1×TBST, membranes were incubated for 1 hr at 25° C. with anti-mouse immunoglobulin (horseradish peroxidase-linked whole antibody from sheep) or anti-rabbit immunoglobulin (horseradish peroxidase linked whole antibody from donkey) (Amersham Life Science, Inc., Arlington Heights, Ill.). The membrane was washed 5× in 1×TBST buffer, with 5 min intervals between buffer changes, and bound antibodies were detected with enhanced luminol and oxidizing reagent as specified by the manufacturer (NEN Life Science Products, Boston, Mass.). Bound antibodies were removed with stripping buffer (2% SDS, 0.0625 mol/L Tris [pH 6.8], and 0.7% 2-mercaptoethanol) 50° C. for 30 min. The filter was then probed with additional antibodies.

Cell Cycle Analysis

Cell cycle analysis was performed using approximately 500,000 cells, which were centrifuged at 1500 rpm for 5 min, washed in PBS, and the pellet re-suspended in 500 μl of propidium iodide solution (50 μg/ml propidium iodide, 0.1% NP-40, 0.1% sodium citrate). The mixture was stored in the dark at 4° C. for a minimum of 15 min, and then analyzed by flow cytometry. Apoptosis of drug-treated cells was measured using the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis).

Apoptosis Assay

Cells cultured in the presence or absence of drug were washed 1× with phosphate-buffered saline (PBS) and centrifuged for 5 min at 1500 rpm. Washed cell pellets were re-suspended in 100 μl of 20% Annexin-V-fluorescein labeling reagent and 20% propidium iodide (PI) in HEPES buffer. Cells were incubated for 15 min at room temperature, followed by dilution with 0.8 ml of HEPES buffer. Samples were then analyzed by flow cytometry. As controls, cells were incubated for 15 min with PI alone, Annexin-V-fluorescein labeling reagent alone, or HEPES buffer, and then diluted with HEPES buffer and analyzed by flow cytometry.

Drug Combination Studies

For drug combination studies, compounds were added simultaneously at fixed ratios to cells, and cell viability was determined by trypan blue exclusion and expressed as the function of growth affected (FA) drug-treated versus control cells. Synergy was assessed by Calcusyn software (Biosoft, Ferguson, Mo. and Cambridge, UK), using the Chou-Talalay method (Chou and Talalay, 1984). The combination index=[D]1[Dx]1+[D]2/[Dx]2, where [D]1and [D]2are the concentrations required by each drug in combination to achieve the same effect as concentrations [Dx]1and [Dx]2of each drug alone. Values less than one indicate synergy, whereas values greater than one indicate antagonism.

Bioluminescent Imaging

Cells were transduced with a retrovirus encoding firefly luciferase (MSCV-Luc), and selected with puromycin at 2 μg/ml to generate 32D.p210-luc+ cells, as described by Weisberg et al., 2005. Ba/F3-KIT-T670I cells were transduced with MSCV-luc-neo vector following the same protocol as described by Weisberg et al., 2005.

Kinase Screen

KINOMEscan™ (Ambit Biosciences, San Diego, Calif.), a high-throughput method for screening small molecular agents against a large panel of human kinases, was utilized for compound 2. The technology is a competition binding assay that profiled the selectivity of compound 2 against 350 kinases, each fused to a proprietary tag. The quantity of each kinase bound to an immobilized, active site-directed ligand was measured in the presence and absence of compound 2.

FIG. 1depicts: A) The chemical structure of compound 2 (Table A) with substructure names indicated; B) Kinase selectivity of compound 2 based on screening 400 kinases. Kinases where significant binding affinity was detected at 10 μM were retested in dose-response format to determine a dissociation constant. The size of the red circle is proportion to Kd. Numerical Kd's are listed in Table IV; and C) Crystal structure of compound 2 with Src kinase domain showing the ATP-binding site. Compound 2 (blue sticks) and Src (green ribbon) are shown as are hydrogen-bonding interactions (orange hatched lines) with hinge residues (Y340, M341), αC-helix (E310), DFG-motif (D404) and V383. The compound binds in the “DFG-out” conformation as evident by the position of F405. Ample space is available adjacent to the gatekeeper residue T338 providing a rationale for the tolerance for a larger amino acid at this position.

EQUIVALENTS

INCORPORATION BY REFERENCE