HK1233495A1

HK1233495A1 - Syk inhibitors

Info

Publication number: HK1233495A1
Application number: HK17107126.3A
Authority: HK
Inventors: Peter A. Blomgren; Astrid Clarke; Kevin S. Currie; Julie Di Paolo; Jeffrey E. Kropf; Seung H. Lee; Jennifer R. LO; Scott A. Mitchell; Aaron C. Schmitt; Sundaramoorthi Swaminathan; Jin-Ming Xiong; Jianjun Xu; Zhongdong Zhao
Original assignee: 吉利德科学公司
Priority date: 2014-07-14
Filing date: 2015-07-09
Publication date: 2018-02-02

Description

SYK inhibitors

Technical Field

The present application relates to compounds and their use in the treatment of various diseases, including cancer and inflammatory disorders. The application also relates to processes for preparing said compounds and to pharmaceutical compositions comprising said compounds.

Background

Protein kinases, the largest family of human enzymes, include over 500 proteins. Spleen tyrosine kinase (Syk) is a member of the Syk family of tyrosine kinases and is a regulator of early B cell development as well as mature B cell activation, signaling, and survival.

Inhibition of Syk activity is useful in the treatment of allergic, autoimmune and inflammatory diseases, such as: SLE, rheumatoid arthritis, multiple vasculitis (multiple vasculitides), Idiopathic Thrombocytopenic Purpura (ITP), myasthenia gravis, allergic rhinitis, Chronic Obstructive Pulmonary Disease (COPD), Adult Respiratory Distress Syndrome (ARDs), and asthma. Furthermore, Syk has been reported to play an important role in ligand-independent trophoblast signaling through B cell receptors, which is known to be an important survival signal in B cells. Thus, inhibition of Syk activity may also be useful in the treatment of certain types of cancer, including B-cell lymphomas and leukemias. U.S. Pat. Nos. 8,455,493 and 8,440,667 disclose Syk inhibitors, the disclosures of which are incorporated herein by reference in their entirety.

There is a continuing need to provide compounds that are effective Syk inhibitors, including compounds having desirable pharmacokinetic properties for use as therapeutic agents for the treatment of cancer and other diseases, including compounds for use in the treatment of hematological and solid tumors in potential combination with other drugs or chemotherapeutic agents.

Disclosure of Invention

Accordingly, the present application provides compounds useful as Syk inhibitors. In one embodiment, the present application provides a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹is selected fromWherein ＊ represents R in the benzene ring shown in formula I¹The carbon atom to which it is attached;

R²is H or 2-hydroxyethoxy;

R³is H or methyl; and

R⁴is H or methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromIn some embodiments, R¹Is composed ofIn some embodiments, R¹Is composed ofIn some embodiments, R¹Is composed of

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R²Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R²Is 2-hydroxyethoxy.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R³Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H or 2-hydroxyethoxy; r³Is H or methyl; and R is⁴Is H or methyl. In variations of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H; r³Is H or methyl; and R is⁴Is H or methyl. In another variation of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy; r³Is H; and R is⁴Is H or methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is²、R³And R⁴Any of which is H. In another variation of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is²、R³And R⁴Any two of which are H.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is²、R³And R⁴Any of which is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is²、R³And R⁴Any two of which are H.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²And R³Each is H, and R⁴Is H or methyl.

In some embodiments, the compound of formula I is selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula I is selected from: 2- (5- ((6- (6-amino-5-methylpyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol; 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine; 2- ((4- (4- ((6- (6-aminopyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) phenyl) piperazin-1-yl) methyl) propane-1, 3-diol; 2- (5- ((6- (6-aminopyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol; (R) - (4- (4- ((6- (6-aminopyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) phenyl) morpholin-2-yl) methanol; 6- (6-aminopyrazin-2-yl) -5-methyl-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine; and 6- (6-amino-5-methylpyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine; or a pharmaceutically acceptable salt thereof.

The present application also provides methods of treating a disease or disorder in a subject, such as a human, using a compound of formula I, or a pharmaceutically acceptable salt thereof. Such diseases and conditions include inflammatory diseases, allergic diseases, autoimmune diseases, or cancers (including carcinomas, sarcomas, melanomas, lymphomas, and leukemias).

In some cases, diseases and disorders treatable with compounds disclosed herein include cancers such as bladder cancer, breast cancer, colorectal cancer, endometrial cancer, renal/renal cell carcinoma, lung cancer, pancreatic cancer, prostate cancer, thyroid cancer, leukemia, melanoma, and non-hodgkin's lymphoma.

In some embodiments, the disease is cancer, including a hematologic malignancy or a solid tumor. In some embodiments, the cancer is lymphoma, multiple myeloma, or leukemia. In some embodiments, the hematologic malignancy is leukemia or lymphoma.

In some embodiments, the disease or disorder treatable is a hematologic malignancy selected from lymphoma (e.g., Small Lymphocytic Lymphoma (SLL), non-hodgkin's lymphoma (NHL), indolent non-hodgkin's lymphoma (iNHL), refractory non-hodgkin's lymphoma, Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), lymphoplasmacytic lymphoma (LPL), Marginal Zone Lymphoma (MZL), immunoblastic large cell lymphoma, lymphoblastic lymphoma, splenic marginal zone B-cell lymphoma (+/-villous lymphocytes), nodal marginal zone lymphoma (+/-monocyte-like B cells), extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type, T-cell lymphoma (e.g., cutaneous T-cell lymphoma, extranodal T-cell lymphoma, anaplastic large cell lymphoma, lymphomas, and combinations thereof, Angioimmunoblastic T-cell lymphoma, mycosis fungoides (mycosis fungoides)), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, small non-dividing cell lymphoma, Burkitt's lymphoma, multiple myeloma, plasmacytoma, and leukemia (e.g., Acute Lymphocytic Leukemia (ALL), acute T-cell lymphoblastic leukemia (T-ALL), acute B-cell lymphocytic leukemia (B-ALL), B-cell prolymphocytic leukemia, Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), juvenile myelomonocytic leukemia (JMML), minimal residual disease (minimal residual disease MRD), hairy cell leukemia, bone marrow, fibrosis (e.g., primary or secondary fibrosis), or Chronic Myeloid Leukemia (CML), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD) and Waldenstrom's Macroglobulinemia (WM).

In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a cancer selected from the group consisting of: pancreatic cancer, urinary organ cancer, bladder cancer, colorectal cancer, colon cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, thyroid cancer, gallbladder cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), ovarian cancer, cervical cancer, gastric cancer, endometrial cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumors (e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma), bone cancer, soft tissue sarcoma, retinoblastoma, neuroblastoma, peritoneal leakage, malignant pleural effusion, mesothelioma, wilms 'tumor, trophoblastic tumors, vascular pericellular tumors, kaposi's sarcoma, mucus cancer, round cell carcinoma, squamous cell carcinoma, esophageal squamous cell carcinoma, oral cancer, adrenal cortical cancer, ACTH producing tumors.

In some embodiments, the disease or disorder treatable is selected from Systemic Lupus Erythematosus (SLE), myasthenia gravis, Goodpasture's syndrome, glomerulonephritis, hemorrhage, pulmonary hemorrhage, atherosclerosis, Rheumatoid Arthritis (RA), psoriatic arthritis, monoarthritis, osteoarthritis, gouty arthritis, spondylitis, behcet's disease (r) (SLE)disease), autoimmune thyroiditis, Reynaud's syndrome, acute disseminated encephalomyelitis, chronic idiopathic thrombocytopenic purpura, Multiple Sclerosis (MS), Sjogren's syndrome (x: (R)syndrome), autoimmune hemolytic anemia, tissue transplant rejection, hyperacute rejection of transplanted organs, allograft rejection, graft-versus-host disease, diseases involving leukocyte extravasation, diseases due to leukocyte dyscrasia and metastasis (disease states are two to one leukocyte dyscrasia and metasta)sis), granulocyte-transfusion-associated syndrome (granulocytic-associated syndromes), cytokine-induced toxicity, scleroderma, vasculitis (vasculitis), asthma, psoriasis, inflammatory bowel disease (e.g., chronic inflammatory bowel disease, ulcerative colitis, crohn's disease, necrotizing enterocolitis), irritable bowel syndrome, dermatomyositis, addison's disease, parkinson's disease, alzheimer's disease, diabetes, type I diabetes, sepsis, septic shock, endotoxic shock, gram-negative sepsis, gram-positive sepsis and toxic shock syndrome, multiple organ injury syndrome (multiple organ injury syndrome to septicemia) secondary to septicemia, trauma, hypovolemic shock, allergic conjunctivitis, vernal conjunctivitis and thyroid-associated eye disease, gram-eosinophilic granuloma, eczema, chronic bronchitis, acute respiratory distress syndrome, chronic bronchitis, chronic inflammation, vasculitis, chronic inflammation, allergic rhinitis, pollinosis, bronchial asthma, silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, emphysema, pneumonia, bacterial pneumonia, bronchiectasis and pulmonary oxygen poisoning, reperfusion injury of the myocardium or brain or limbs, heat injury, cystic fibrosis, keloid formation or scar tissue formation, fever and myalgia due to infection, brain or spinal cord injury due to minimal trauma, diseases involving leukocyte exudation, acute hypersensitivity, delayed hypersensitivity, urticaria, food allergy, sunburn of the skin, pelvic inflammatory disease, urethritis, uveitis, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, alcoholic hepatitis, gastritis, enteritis, contact dermatitis, atopic dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis (cholestitis), Polycythemia vera, essential thrombocythemia and polycystic kidney disease.

In some embodiments, the disease is an autoimmune disease. In some embodiments, the autoimmune disease is Systemic Lupus Erythematosus (SLE), myasthenia gravis, Rheumatoid Arthritis (RA), acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, Multiple Sclerosis (MS), sjogren's syndrome, psoriasis, autoimmune hemolytic anemia, asthma, ulcerative colitis, crohn's disease, irritable bowel syndrome, or Chronic Obstructive Pulmonary Disease (COPD). In some embodiments, the disease is an excessive or destructive immune response, such as asthma, rheumatoid arthritis, multiple sclerosis, Chronic Obstructive Pulmonary Disease (COPD), or systemic lupus erythematosus.

In some embodiments, the present application provides a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable vehicle.

In some embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, a stereoisomer, a mixture of stereoisomers, or a tautomer thereof, and at least one pharmaceutically acceptable vehicle. Examples of pharmaceutically acceptable vehicles may be selected from carriers and other excipients, adjuvants, and the like.

Also provided are methods of treating a disease or disorder in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, a stereoisomer, a mixture of stereoisomers, or a tautomer thereof, or a pharmaceutical composition thereof. In one variation of a method of treating a disease or disorder in a subject in need thereof (e.g., a human in need thereof), the method comprises administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the disease or disorder is an inflammatory disease, an allergic disease, an autoimmune disease, or cancer.

Also provided are methods of inhibiting kinase activity of a Syk kinase polypeptide by contacting the Syk kinase polypeptide with a compound of formula I, or a pharmaceutically acceptable salt, pharmaceutically acceptable ester, stereoisomer, mixture of stereoisomers, or tautomer thereof. In one aspect, methods of inhibiting kinase activity of a Syk kinase polypeptide by contacting the Syk kinase polypeptide with a compound of formula I or a pharmaceutically acceptable salt thereof are provided.

Also provided are methods of treating cancer in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof. In some embodiments, the subject is a human with a tumor that is responsive to Syk activity. In another embodiment, the subject is a human having a solid tumor that expresses Syk. In some embodiments, the subject is a human having a 17p deletion, a TP53 mutation, a NOTCH1, a SF3B1 mutation, an 11q deletion, or any combination thereof. In one embodiment, the subject is a human having a 17p deletion, a TP53 mutation, or any combination thereof. In another embodiment, the subject is a human having NOTCH1, a SF3B1 mutation, a 11q deletion, or any combination thereof.

In some embodiments, the vinca alkaloid is selected from: vincristine, vinblastine, vindesine, vinorelbine, desoxyvincaminol, vincaminol, vinblastine, vinspraine and vinorelbine (vineridine) and pharmaceutically acceptable salts thereof. In some embodiments, the at least one vinca alkaloid is selected from: vincristine, vinblastine, vindesine, vinorelbine, desoxyvincaminol, vincaminol, vinblastine, vinspraine and vindoline and pharmaceutically acceptable salts thereof. In some embodiments, the vinca alkaloid is selected from: vincristine, vinblastine, vindesine and vinorelbine, and pharmaceutically acceptable salts thereof. In other embodiments, the vinca alkaloid is selected from: vincristine and vinblastine, and pharmaceutically acceptable salts thereof. In one embodiment, the vinca alkaloid is vincristine and pharmaceutically acceptable salts thereof. In another embodiment, the vinca alkaloid is vinblastine and pharmaceutically acceptable salts thereof.

The present application also provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula I is:

and the vinca alkaloid is selected from vincristine and vinblastine. In one embodiment, the compound of formula I is:

and the vinca alkaloid is vincristine. In another embodiment, the compounds of formula I are

And the vinca alkaloid is vinblastine.

The present application also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, wherein the subject is a human who has not received any other anti-cancer therapy; and the subject is (i) refractory to at least one anti-cancer therapy, or (ii) relapsed after treatment with at least one anti-cancer therapy, or a combination of (i) and (ii). In some embodiments, the compound of formula I is

And the vinca alkaloid is selected from vincristine or vinblastine. In one embodiment, the compounds of formula I are

And the vinca alkaloid is vinblastine.

The present application also provides figures and examples to illustrate the unexpected improvement in the combination of a compound of formula I or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a vinca alkaloid or a pharmaceutically acceptable salt thereof compared to the effect of the compound of formula I or the vinca alkaloid administered as a single agent or used alone in monotherapy in the treatment of certain cancers and their respective cell lines. In some embodiments, the vinca alkaloid is selected from vincristine and vinblastine.

In some embodiments, the subject is refractory to at least one anti-cancer therapy. In other embodiments, the subject relapses after treatment with at least one anti-cancer therapy.

In some embodiments, about 50mg to 300mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered to a subject once daily. In other embodiments, about 100mg to 250mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered to a subject once daily. In one embodiment, about 180-200mg of a compound of formula I or a pharmaceutically acceptable salt thereof is administered to a subject once daily.

In one variation, the subject is a human having a 17p deletion, a TP53 mutation, or a combination thereof; and administering to the subject about 50mg to 300mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, once daily. In further variations, the subject is a human having a 17p deletion, a TP53 mutation, or a combination thereof; and administering to the subject about 100mg to 250mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, once daily. In further variations, the subject is a human having a 17p deletion, a TP53 mutation, or a combination thereof; and administering to the subject about 180 to about 200mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, once daily.

In other embodiments, the vinca alkaloid or a pharmaceutically acceptable salt thereof is administered to the subject once a week at a clinically approved dose or a sub-clinically approved dose. In some embodiments, the vinca alkaloid is administered to the subject once a week at a subclinical approved dose. In other embodiments, the vinca alkaloid is vincristine and is present at 0.1mg-M²To 1.5mg-M²Administering vincristine or a pharmaceutically acceptable salt thereof. In other embodiments, the vinca alkaloid is administered to the subject once a week at a dose of 0.25mg-M2 to 1.0mg-M2, and the vinca alkaloid is selected from the group consisting of vincristine and vinblastine. In other embodiments, the vinca alkaloid is administered to the subject once a day at a dose of 0.1mg-M2 to 0.2mg-M2, and the vinca alkaloid is selected from the group consisting of vincristine and vinblastine.

In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered prior to the administration of the vinca alkaloid or a pharmaceutically acceptable salt thereof. In other embodiments, the vinca alkaloid or a pharmaceutically acceptable salt thereof is administered prior to the administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the vinca alkaloid or a pharmaceutically acceptable salt thereof are administered simultaneously, wherein the vinca alkaloid is administered IV and the compound of formula I is administered via a tablet. In other embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the vinca alkaloid or a pharmaceutically acceptable salt thereof are administered once a day. In other embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the vinca alkaloid or a pharmaceutically acceptable salt thereof are administered once a week. In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once a day and the vinca alkaloid or a pharmaceutically acceptable salt thereof is administered once a week.

In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is leukemia. In one embodiment, the leukemia is Chronic Lymphocytic Leukemia (CLL). In some embodiments, the cancer is lymphoma. In one embodiment, the lymphoma is non-hodgkin's lymphoma (NHL). In one variation, the NHL is diffuse large B-cell lymphoma (DLBCL), Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Small Lymphocytic Lymphoma (SLL), lymphoplasmacytic lymphoma (LPL), and/or Marginal Zone Lymphoma (MZL). Thus, it is understood that in one aspect, the subject is a human having a hematological malignancy, such as leukemia or lymphoma.

In some embodiments, the cancer is selected from Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), Chronic Myeloid Leukemia (CML), Multiple Myeloma (MM), non-hodgkin's lymphoma (NHL), indolent non-hodgkin's lymphoma (iNHL), refractory non-hodgkin's lymphoma, Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Waldenstrom's Macroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma (LPL), and Marginal Zone Lymphoma (MZL).

In some embodiments, the cancer is a solid tumor cancer (or solid cancer tumor). In some embodiments, the cancer is a solid tumor and expresses spleen tyrosine kinase (Syk) activity. In other embodiments, the solid tumor cancer is selected from: pancreatic cancer, lung cancer, colorectal cancer, ovarian cancer, and hepatocellular carcinoma.

Also provided are kits comprising a compound of formula I, or a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, a stereoisomer, a mixture of stereoisomers, or a tautomer thereof. In one aspect, the kit comprises a compound of formula I or a pharmaceutically acceptable salt thereof. The kit can comprise a label and/or instructions for indicating that the compound is in treating a disease or disorder in a subject (e.g., a human) in need thereof. In some embodiments, the disease or disorder may be associated with or mediated by Syk activity. In other embodiments, the kit can further comprise instructions for indicating that the combination of vinca alkaloid and the compound of formula I is for use in treating a disease or disorder in a subject (e.g., a human) in need thereof. In some embodiments, the disease or disorder is a cancer (e.g., hematological malignancy, solid tumor cancer) that can be associated with or mediated by Syk activity.

Also provided are articles of manufacture comprising a compound of formula I, or a pharmaceutically acceptable salt, pharmaceutically acceptable ester, stereoisomer, mixture of stereoisomers, or tautomer thereof; and a container. In one aspect, the article of manufacture comprises a compound of formula I or a pharmaceutically acceptable salt thereof. In one embodiment, the container may be a vial (visual), a canister (jar), an ampoule, a preloaded syringe (pre-loaded system), or an intravenous bag (intravenous bag).

Other aspects and embodiments of the present application are described throughout the specification.

Drawings

Figures 1A and 1B show and summarize the inhibitory effect of the compound 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine of figure 1, when the compounds are co-administered, in combination with one of two vinca alkaloids, vincristine (figure 1A) and vinblastine (figure 1B), respectively, in the DLBCL cell line DHL-10.

FIGS. 2A and 2B summarize the inhibitory effect of the combination of the compound of formula I, 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine, and vincristine on cell viability in the malignant colon cell line MiaPaca expressing Syk and in the malignant colon cell line HepG2 not expressing Syk.

Figure 3 shows Syk expression levels in MiaPaca and HepG2 malignant colon cell lines.

Figure 4 summarizes Syk expression levels in malignant cell lines of the lung, pancreas and colon.

Detailed Description

It has been unexpectedly found that compounds of formula I, or pharmaceutically acceptable salts thereof, have advantageous properties, making them attractive compounds for the uses described herein. In addition to being Syk inhibitors, the compounds also have desirable solubility and pharmacokinetic properties. These findings are particularly surprising given the comparable parametric properties of compounds with similar base structures.

The following description sets forth exemplary compositions, methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present application, but is instead provided as a description of exemplary embodiments.

Also described for compounds of formula I are pharmaceutically acceptable salts, pharmaceutically acceptable esters, pharmaceutically acceptable solvates, hydrates, isomers (including optical isomers, racemates or other mixtures thereof), tautomers, isotopes, polymorphs and pharmaceutically acceptable prodrugs of such compounds.

The compounds of the present application may have asymmetric centers and may be produced as a racemic mixture or as a single enantiomer. The single enantiomers may be obtained by asymmetric synthesis or by resolution of racemic or non-racemic mixtures of intermediates at some suitable stage of the synthesis. The single enantiomers may also be obtained by resolution of the compounds by conventional means, such as crystallization in the presence of a resolving agent, or chromatography using, for example, a chiral High Pressure Liquid Chromatography (HPLC) column. The individual enantiomers as well as racemic and non-racemic mixtures of enantiomers are within the scope of the present application, all of which are intended to be included within the structures described in this specification unless explicitly stated otherwise.

The present application also provides methods of treating cancer in certain populations of subjects (e.g., humans) in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof in combination with a vinca alkaloid or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula I is

It may also be mentioned as the compound name: 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine. It is understood by those skilled in the art that the compound structures may be named or identified using other well known naming systems and symbols, including CAS and IUPAC.

The present application also provides figures and examples to illustrate the unexpected improvement in the combination of a compound of formula I or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a vinca alkaloid or a pharmaceutically acceptable salt thereof compared to the effect of the compound of formula I or the vinca alkaloid administered as a single agent or used alone in monotherapy in the treatment of certain cancers and their respective cell lines. In some embodiments, the compound of formula I is 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and the vinca alkaloid is selected from vincristine and vinblastine.

Definition of

As used in this disclosure, the following words and phrases are generally intended to have the meanings as set forth below, unless the context in which they are used indicates otherwise.

"isomers" are different compounds having the same molecular formula. Isomers include stereoisomers, enantiomers and diastereomers.

"stereoisomers" are isomers that differ only in the arrangement of atoms in space.

"enantiomers" are a pair of stereoisomers that are nonoverlapping mirror images of each other. A1: 1 mixture of a pair of enantiomers is a "racemic" mixture. The term "(±)" is used to designate a racemic mixture, where appropriate.

Absolute stereochemistry was determined according to Cahn Ingold Prelog R S system. When the compounds are pure enantiomers, the stereochemistry at each chiral carbon atom may be specified by R or S. Resolved compounds in which the absolute configuration is unknown are designated (+) or (-), depending on the direction (dextro-or levorotatory) of their plane of rotating polarized light at the wavelength of the sodium D line.

The term "therapeutically effective amount" refers to an amount sufficient to be therapeutically effective when administered to a subject (e.g., a mammal such as a human) in need of treatment, as defined below. The therapeutically effective amount will vary depending on the subject and disease being treated, the weight and age of the subject, the severity of the disease, the mode of administration, and the like, which can be readily determined by one of skill in the art. For example, a "therapeutically effective amount" of a compound of formula I, or a pharmaceutically acceptable salt thereof, is an amount that: it is sufficient to modulate Syk expression or activity and thereby treat a subject (e.g., a human) with the indication or ameliorate or alleviate an existing symptom of the indication. For example, a therapeutically effective amount may be an amount of: which is sufficient to reduce the symptoms of a disease or disorder responsive to inhibition of Syk activity.

The term "polymorph" refers to different crystal structures of a crystalline compound. Different polymorphs may result from differences in the crystallographic stack (stacked polymorphism) or from different stacking between different conformers of the same molecule (conformational polymorphism). It is to be understood that any polymorph of a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in treating a disease or condition described herein, while potentially offering different properties (including pharmacokinetic properties), will nevertheless result in a compound of formula I upon absorption into a subject, such that use of the compound of formula I encompasses use of any polymorph of a compound of formula I, or a pharmaceutically acceptable salt thereof.

The term "solvate" refers to a complex formed by combining a compound of formula I and a solvent. It is to be understood that any solvate of a compound of formula I used in the treatment of a disease or condition described herein, while potentially offering different properties (including pharmacokinetic properties), upon absorption into a subject, results in a compound of formula I such that use of the compound of formula I encompasses use of any solvate of the compound of formula I.

The term "hydrate" refers to a complex formed by combining a compound of formula I and water. It will be appreciated that any hydrate of a compound of formula I used in the treatment of a disease or condition described herein, while potentially offering different properties (including pharmacokinetic properties), will result in a compound of formula I once absorbed into a subject, such that use of a compound of formula I encompasses use of any hydrate of a compound of formula I.

The term "prodrug" refers to a compound derived from or readily convertible into a compound of formula I, which includes chemical groups that are convertible in vivo and/or separable from the remainder of the molecule to provide an active portion of a compound of formula I or a drug, or a pharmaceutically acceptable salt thereof or a biologically active metabolite thereof. It is to be understood that any prodrug of a compound of formula I used in the treatment of a disease or condition described herein, while potentially offering different properties (including pharmacokinetic properties), upon absorption into a subject, results in a compound of formula I such that the use of the compound of formula I encompasses the use of any prodrug of the compound of formula I.

The present application also provides isotopically labeled forms of the compounds detailed herein. Isotopically-labeled compounds have the structure depicted by the general formulae given herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to²H (deuterium, D),³H (tritium),¹¹C、¹³C、¹⁴C、¹⁵N、¹⁸F、³¹P、³²P、³⁵S、³⁶Cl and¹²⁵I. a number of isotopically-labelled compounds of the present application are provided, for example radioisotopes such as³H、¹³C and¹⁴c those into which it is introduced. Such isotopically labeled compounds are useful in metabolic studies, reaction kinetic studies, detection, or imaging techniques, such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or substrate tissue distribution assays, or for the radiotherapy of subjects (e.g., humans). Also provided for the isotopically labeled compounds described herein are any pharmaceutically acceptable salts, pharmaceutically acceptable esters, pharmaceutically acceptable solvates, hydrates, enantiomers, enantiomeric mixtures, tautomers, polymorphs and pharmaceutically acceptable prodrugs thereof.

The present application also includes compounds of formula I, or pharmaceutically acceptable salts thereof, wherein 1 to n hydrogens attached to carbon atoms are replaced with deuterium, wherein n is the number of hydrogens in the molecule. Such compounds may exhibit enhanced resistance to metabolism and are therefore useful for increasing the half-life (when administered to a mammal) of a compound of formula I or a pharmaceutically acceptable salt thereof. See, for example, Foster, "Deuterium Isotrope Effects in students of Drug Metabolism", TrendsPharmacol. Sci.5(12): 524-. Such compounds are synthesized by methods known in the art, for example, by using starting materials in which one or more hydrogen atoms have been replaced by deuterium.

Deuterium labeled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and/or improved therapeutic index.¹⁸The F-labelled compound may be used in PET or SPECT studies. Isotopically labeled compounds of the present invention and prodrugs thereof are generally prepared by carrying out the synthetic routes or examples described hereinafter and the steps disclosed in the preparations in which a non-isotopically labeled reagent is replaced by a conveniently available isotopically labeled reagent. It is understood that deuterium in this context may be considered as a substituent in the compounds of formula I.

The concentration of such heavier isotopes, in particular deuterium, can be defined by the isotopic enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise indicated, when a position is specifically designated as "H" or "hydrogen," it is understood that the position has an isotopically composed hydrogen in its natural abundance. Accordingly, in the compounds of the present invention, any atom specifically designated as deuterium (D) is meant to represent deuterium.

The term "inhibit" indicates a decrease, e.g., a significant decrease, in the baseline activity of a biological activity or process. By "inhibition of Syk activity" is meant a decrease in Syk activity in the presence of a compound of formula I, or a pharmaceutically acceptable salt thereof, relative to the activity of Syk in the absence of a compound of formula I, or a pharmaceutically acceptable salt thereof. The reduction in activity may be due to direct interaction of the compound with Syk, or due to interaction of the compound described herein with one or more other factors that in turn affect Syk activity. For example, the presence of the compound may reduce Syk activity by: by binding directly to Syk, by causing (directly or indirectly) another factor to reduce Syk activity, or by reducing (directly or indirectly) the amount of Syk present in the cell or organism. In some embodiments, inhibition of Syk activity may be compared in the same subject prior to treatment, or in other subjects not receiving treatment.

Inhibition of Syk activity also refers to inhibition of Syk activity that is observed in standard biochemical assay of Syk activity (e.g., ATP hydrolysis assay described below in example 8).

In some embodiments, a compound described herein (e.g., a compound of formula I or a pharmaceutically acceptable salt thereof) inhibits Syk kinase activity, IC₅₀Values less than or equal to 1. mu.M, for example 0.1nM to 1. mu.M or 1nM to 1. mu.M. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to less than 500nM, e.g., 0.1nM to 500nM or 1nM to 500 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to less than 200nM, e.g., 0.1nM to 200nM or 1nM to 200 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to less than 100nM, e.g., 0.1nM to 100nM or 1nM to 100 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 50nM, e.g., 0.1nM to 50nM or 1nM to 50 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 20nM, e.g., 0.1nM to 20nM or 1nM to 20 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 10nM, e.g., 0.1nM to 10nM or 1nM to 10 nM. In some embodiments, the IC₅₀Values were measured as described in the test of example 8.

"inhibition of B cell activity" refers to a decrease in B cell activity in response, directly or indirectly, to the presence of a compound of formula I, or a pharmaceutically acceptable salt thereof, relative to B cell activity in the absence of the compound, or pharmaceutically acceptable salt thereof. The reduction in activity may be due to direct interaction of the compound with Syk or with one or more other factors that in turn affect B cell activity.

Inhibition of B cell activity also refers to inhibition of CD86 expression that is observed in standard assays. In some embodiments, the IC of a compound described herein₅₀Values less than or equal to 10. mu.M, for example from 1nM to 10. mu.M or from 10nM to 10. mu.M. In some embodiments, the IC of the compound₅₀Values less than or equal to less than 1. mu.M, for example from 1nM to 1. mu.M or from 10nM to 1. mu.M. In some embodiments, the IC of the compound₅₀Values less than or equal to 500nM, e.g., 1nM to 500nM or 10nM to 500 nM.

"B cell activity" also includes activation, redistribution, recombination, or capping (bridging) of one or more different B cell membrane receptors or membrane-bound immunoglobulins (e.g., IgM, IgG, and IgD). Most B cells also have membrane receptors for the Fc region of IgG, either in the form of antigen-antibody complexes or aggregated IgG. B cells also carry membrane receptors for the activating components of complement (e.g., C3B, C3d, C4, and Clq). These different membrane receptors and membrane-bound immunoglobulins have membrane fluidity and are capable of redistribution and capping (which can trigger signaling).

B cell activity also includes the synthesis or production of antibodies or immunoglobulins. Immunoglobulins are synthesized by B cell lines and have common structural features and structural units. Based on the structural differences of the heavy chains (including the amino acid sequences and lengths of the polypeptide chains), five classes of immunoglobulins, namely IgG, IgA, IgM, IgD and IgE, are recognized. Antibodies to a given antigen may be detected in all or several classes of immunoglobulins or may be restricted to a single class or subclass of immunoglobulin. Autoantibodies or autoimmune antibodies can be similarly classified as one or several classes of immunoglobulins. For example, rheumatoid factor (an antibody to IgG) is generally considered an IgM immunoglobulin, but may also consist of IgG or IgA.

In addition, B cell activity is also intended to include a series of events that result in clonal expansion (proliferation) of B cells from precursor B lymphocytes and differentiation into antibody-synthesized plasma cells that occur in tandem with antigen-binding and cytokine signaling from other cells.

"inhibition of B cell proliferation" refers to inhibition of proliferation of an abnormal B cell (e.g., a cancerous B cell, e.g., a lymphoma B cell) and/or inhibition of a normal, non-diseased B cell. The term "inhibition of B cell proliferation" indicates a significant reduction in the number of B cells in vivo or in vitro. Thus, inhibiting B cell proliferation in vitro refers to any significant reduction in the number of B cells in an in vitro sample contacted with a compound of formula I, or a pharmaceutically acceptable salt thereof, as compared to a corresponding sample not contacted with a compound of formula I, or a pharmaceutically acceptable salt thereof.

Inhibition of B cell proliferation also refers to the inhibition of B cell proliferation that is observable in a standard B cell proliferation thymidine incorporation assay (e.g., an assay as known in the art). In some embodiments, the IC of a compound described herein (e.g., a compound of formula I or a pharmaceutically acceptable salt thereof)₅₀Values less than or equal to 10. mu.M, for example from 1nM to 10. mu.M or from 10nM to 10. mu.M. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to less than 1. mu.M, for example from 1nM to 1. mu.M or from 10nM to 1. mu.M. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 500nM, e.g., 1nM to 500nM or 10nM to 500 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 200nM, e.g., 1nM to 200nM or 10nM to 200 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 100nM, e.g., 1nM to 100nM or 10nM to 100 nM.

By "basophil activation is meant the ability of a compound described herein to reduce the activation of basophils. Basophil activation is, for example, implicated in the inflammatory and autoimmune diseases described herein, and a reduction in basophil activation is desirable for the compounds described herein (e.g., a compound of formula I or a pharmaceutically acceptable salt thereof). Activation of basophils can be evaluated by the following method: CD63 expression was measured by basophils, as by the CD63 human whole blood basophil test (25% blood), e.g., the test described below in example 9.

In some embodiments, the EC of a compound described herein, e.g., a compound of formula I or a pharmaceutically acceptable salt thereof, in a suitable CD63 test₅₀Values less than or equal to 10. mu.M, for example from 1nM to 10. mu.M or from 10nM to 10. mu.M. In some embodiments, the EC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to less than 1. mu.M, for example from 1nM to 1. mu.M or from 10nM to 1. mu.M. In some embodiments, the EC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 500nM, e.g., 1nM to 500nM or 10nM to 500 nM. In some embodiments, the EC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 200nM, e.g., 1nM to 200nM or 10nM to 200 nM. In some embodiments, the EC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 150nM, e.g., 1nM to 150nM or 10nM to 150 nM. In some embodiments, the IC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 100nM, e.g., 1nM to 100nM or 10nM to 100 nM. In some embodiments, the EC of the compound or a pharmaceutically acceptable salt thereof₅₀Values less than or equal to 75nM, e.g., 1nM to 75nM or 10nM to 75 nM. In some embodiments, the EC is₅₀Values were measured as described in the test of example 9.

"kinetic solubility" refers to the evaluation of the solubility of a compound in a suitable buffer (e.g., phosphate buffer at pH 7.4) at a given temperature (e.g., at 37 ℃). In one instance, the kinetic solubility is measured at 37 ℃ in phosphate buffer at ph7.4, e.g., by the test described in example 10.

In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof as described herein has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 10 μ Μ, such as 10 μ Μ to 500 μ Μ or 10 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 20 μ Μ, such as 20 μ Μ to 500 μ Μ or 20 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 30 μ Μ, such as 30 μ Μ to 500 μ Μ or 30 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 40 μ Μ, such as 40 μ Μ to 500 μ Μ or 40 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 50 μ Μ, such as 50 μ Μ to 500 μ Μ or 50 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 60 μ Μ, such as 60 μ Μ to 500 μ Μ or 60 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 70 μ Μ, such as 70 μ Μ to 500 μ Μ or 70 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 80 μ Μ, such as 80 μ Μ to 500 μ Μ or 80 μ Μ to 250 μ Μ. In some embodiments, the compound or pharmaceutically acceptable salt thereof has a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 90 μ Μ, such as 90 μ Μ to 500 μ Μ or 90 μ Μ to 250 μ Μ. In some embodiments, the kinetic solubility is measured by the test described in example 10.

"human hepatocyte stability" is a measure of the stability of a compound to human hepatocyte metabolism and is evaluated as the predicted hepatic plasma clearance of the compound in L/hr/kg. The predicted hepatocyte clearance may be measured, for example, by the test described in example 11.

In some embodiments, the predicted liver plasma clearance of a compound described herein, e.g., a compound of formula I, is less than or equal to 0.50L/hr/kg, e.g., 0.005L/hr/kg to 0.50L/hr/kg or 0.01L/hr/kg to 0.50L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.40L/hr/kg, e.g., 0.005L/hr/kg to 0.40L/hr/kg or 0.01L/hr/kg to 0.40L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.30L/hr/kg, e.g., 0.005L/hr/kg to 0.30L/hr/kg or 0.01L/hr/kg to 0.30L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.20L/hr/kg, e.g., 0.005L/hr/kg to 0.20L/hr/kg or 0.01L/hr/kg to 0.20L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.10L/hr/kg, e.g., 0.005L/hr/kg to 0.10L/hr/kg or 0.01L/hr/kg to 0.10L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.09L/hr/kg, such as 0.005L/hr/kg to 0.09L/hr/kg or 0.01L/hr/kg to 0.09L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.08L/hr/kg, such as 0.005L/hr/kg to 0.08L/hr/kg or 0.01L/hr/kg to 0.08L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.07L/hr/kg, such as 0.005L/hr/kg to 0.07L/hr/kg or 0.01L/hr/kg to 0.07L/hr/kg. In some embodiments, the predicted liver plasma clearance of the compound is less than or equal to 0.06L/hr/kg, e.g., 0.005L/hr/kg to 0.06L/hr/kg or 0.01L/hr/kg to 0.06L/hr/kg. In some embodiments, the predicted hepatocyte clearance is measured by the test described in example 11.

"allergy" or "allergic disease" refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include eczema, allergic rhinitis (allergic rhinitis) or rhinitis (coroyza), pollinosis, bronchial asthma, urticaria (hives), and food allergies and other atopic conditions.

"asthma" refers to a respiratory disease characterized by inflammation, airway narrowing, and increased airway responsiveness to inhaled agents. Asthma is often, although not exclusively, associated with atopic or allergic symptoms.

"significant" refers to any detectable change of statistical significance in a standard parametric test in a statistical sense, such as the Student's T-test, where p < 0.05.

A "disease responsive to inhibition of Syk activity" is a disease in which inhibition of Syk kinase provides a therapeutic benefit, such as alleviation of symptoms, diminishment of disease progression, delay in disease onset, or inhibition of abnormal activity of certain cell types (monocytes, B cells, and mast cells).

"subject" refers to an animal (e.g., a mammal) that has been or is about to be the subject of treatment, observation or experiment. The methods described herein are useful for human therapy and veterinary applications. In some embodiments, the subject is a mammal; in some embodiments, the subject is a human; and in some embodiments, the subject is selected from a cat and a dog. By "subject in need thereof" or "human in need thereof" is meant a subject, such as a human, that may have or is suspected of having a disease or condition that would benefit from a treatment, such as treatment with a compound of formula I or a pharmaceutically acceptable salt thereof as described herein. This includes subjects who can be identified as being at risk for or susceptible to such a disease or condition, such that treatment will prevent the disease or condition from developing.

"treatment" is a means for obtaining a beneficial or desired result, including a clinical result. Beneficial or desired clinical results may include one or more of the following:

(i) inhibiting the disease or disorder (e.g., reducing one or more symptoms caused by the disease or disorder and/or alleviating the extent of the disease or disorder);

(ii) slowing or arresting the development of one or more clinical symptoms associated with the disease or disorder (e.g., stabilizing the disease or disorder, preventing or delaying the worsening or progression of the disease or disorder, and/or preventing or delaying the spread of the disease or disorder (e.g., metastasis)); and/or

(iii) Palliation of the disease, i.e., causing regression of clinical symptoms (e.g., ameliorating the disease, providing partial or complete remission of the disease or disorder, enhancing the efficacy of other medications, delaying disease progression, improving quality of life, and/or prolonging survival).

By "delaying" the development of a disease or condition is meant delaying, hindering, slowing, delaying, stabilizing and/or delaying the development of the disease or condition. Such delay may be a length of time that varies depending on the history of the disease or condition and/or the subject being treated. A method of "delaying" the progression of a disease or condition is a method that: which reduces the probability of disease or condition development within a given time frame and/or reduces the extent of disease or condition within a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects. Disease or condition development can be detected using standard methods, such as routine physical examination, mammography, imaging, or biopsy. Progression may also mean that the disease or condition may initially be undetectable and includes onset, recurrence and onset.

In many cases, the compounds of the present application are capable of forming acid and/or base salts through the presence of amino and/or carboxyl groups or similar groups.

"pharmaceutically acceptable salts" include, for example, salts with inorganic acids and salts with organic acids. Examples of salts may includeHydrochloride, phosphate, diphosphate, hydrobromide, sulfate, sulfinate, nitrate, malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate (methanesulfonate), phenylsulfonate (benzenesulfonate), p-tolylsulfonate (p-toluenesulfonate), 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoates (e.g. acetate, HOOC- (CH)₂)_n-COOH, wherein n is 0-4). Furthermore, if the compounds described herein are obtained as acid addition salts, the free base can be obtained by basifying the acid salt solution. Conversely, if the product is a free base, an addition salt (particularly a pharmaceutically acceptable addition salt) may be produced by: following the conventional procedure for preparing acid addition salts from base compounds, the free base is dissolved in a suitable organic solvent and the solution is treated with an acid. One skilled in the art will recognize various synthetic methods that may be used to prepare non-toxic pharmaceutically acceptable addition salts.

In some embodiments of the present disclosure, the pharmaceutically acceptable salt of the compound of formula I is a mesylate salt. In some embodiments of the present disclosure, the pharmaceutically acceptable salt of a vinca alkaloid is a sulfate salt. In some embodiments of the present disclosure, the pharmaceutically acceptable salt of the compound of formula I is a mesylate salt, and the pharmaceutically acceptable salt of the vinca alkaloid is a sulfate salt. In one embodiment of the disclosure, the pharmaceutically acceptable salt of the compound of formula I is mesylate and the vinca alkaloid is vincristine, wherein the pharmaceutically acceptable salt of vincristine is sulfate.

In some embodiments of the methods described herein, the compound of formula I or pharmaceutically acceptable salt is present in a pharmaceutical composition comprising the compound of formula I or pharmaceutically acceptable salt, and at least one pharmaceutically acceptable vehicle. A pharmaceutically acceptable vehicle may include a pharmaceutically acceptable carrier, adjuvant, and/or other excipient, and other ingredients may be considered pharmaceutically acceptable, as long as they are compatible with the other ingredients of the formulation and not toxic to the recipient thereof.

The pharmaceutical compositions of the compounds of formula I described herein may be prepared using any conventional method, for example, mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping, melt-spinning, spray-drying, or lyophilizing processes. Optimal pharmaceutical formulations can be determined by those skilled in the art based on the route of administration and the desired dosage. Such formulations may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered drug. Depending on the condition to be treated, these pharmaceutical compositions may be formulated and administered systemically or locally.

As used herein, a "pharmaceutically acceptable excipient" is a pharmaceutically acceptable vehicle, which includes, but is not limited to, any and all carriers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents is well known in the art for pharmaceutically active substances. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

The term "carrier" refers to an excipient or vehicle used to administer a compound, including, but not limited to, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, and the like. Carriers are generally described in the present application and in "Remington's Pharmaceutical Sciences" of e.w. martin. Examples of carriers include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethylcellulose, sodium carboxymethylcellulose, crospovidone, glyceryl isostearate, glyceryl monostearate, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxyeicosateyl hydroxystearate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 188, poloxamer 237, poloxamer 407, povidone, silicon dioxide, colloidal silicon dioxide, silicone adhesive 4102, and silicone emulsions. However, it will be understood that the choice of a carrier for a pharmaceutical composition and the amount of such carrier in the composition may vary depending on the method of formulation (e.g., dry granulation formulation, solid dispersion formulation).

The term "diluent" generally refers to a substance used to dilute a compound of interest prior to delivery. Diluents may also be used to stabilize the compound. Examples of diluents may include starch, sugar, disaccharides, sucrose, lactose, polysaccharides, cellulose ethers, hydroxypropyl cellulose, sugar alcohols, xylitol, sorbitol, maltitol, microcrystalline cellulose, calcium or sodium carbonate, lactose monohydrate, dicalcium phosphate, cellulose, compressible sugars, calcium phosphate dibasic dehydrate, mannitol, microcrystalline cellulose, and tricalcium phosphate.

The term "disintegrant" generally refers to a substance that, upon addition to a solid formulation, promotes disintegration or disintegration of the solid formulation after administration and allows the active ingredient to be released as efficiently as possible to allow rapid dissolution of the active ingredient. Examples of disintegrants may include corn starch, sodium starch glycolate, croscarmellose sodium, crospovidone, microcrystalline cellulose, modified corn starch, sodium carboxymethyl starch, povidone, pregelatinized starch, and alginic acid.

The term "precipitation inhibitor" generally refers to a substance that prevents or inhibits the precipitation of an active agent from a supersaturated solution. One example of a precipitation inhibitor includes Hydroxypropylmethylcellulose (HPMC).

The term "surfactant" generally refers to a substance that reduces the surface tension between a liquid and a solid, improves the wettability of the active agent, or increases the solubility of the active agent. Examples of surfactants include poloxamers and sodium lauryl sulfate.

The term "glidant" generally refers to substances used in tablet and capsule formulations to improve flowability during compression and to produce an anti-caking effect. Examples of glidants may include colloidal silicon dioxide, talc, fumed silica, starch derivatives and bentonite.

The term "adhesive" generally refers to any pharmaceutically acceptable film that can be used to adhere the active and inert ingredients of a carrier together to maintain cohesive and discrete portions. Examples of the binder may include hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone, copovidone, and ethyl cellulose.

The term "lubricant" generally refers to a substance added to a powder blend to prevent the compacted powder mass from sticking to equipment during the tableting or encapsulation process. The lubricant may help eject the tablet from the die and improve powder flow. Examples of the lubricant may include magnesium stearate, stearic acid, silicon dioxide, fat, calcium stearate, polyethylene glycol, sodium stearyl fumarate, or talc; and solubilizing agents, e.g. fatty acids, including lauric acid, oleic acid and C₈/C₁₀A fatty acid.

The term "vinca alkaloids" refers to those compounds and their pharmaceutically acceptable salts, which are derived from the plant madagascar latrine and are useful in the treatment of diabetes, hypertension and various cancers. Examples of vinca alkaloids include vincristine, vinblastine, vindesine, vinorelbine, desoxyvincaminol, vincaminol, vinbutine, vingmatine and vindoline. Generally, four major vinca alkaloids are used clinically: vinblastine, vinorelbine, vincristine, and vindesine. All vinca alkaloids are administered by intravenous Injection (IV).

The vinca alkaloids and their pharmaceutically acceptable salts disclosed in this invention are cytotoxic drugs-they stop cell division and cause cell death. During cell division, vinca alkaloid molecules bind to building molecules (building blocks) of proteins called tubulins, inhibiting their formation. Tubulin usually produces microtubules in the cell. These microtubules provide the cells with the structure and flexibility required for division and replication. Without microtubules, cells cannot divide. Unlike Syk inhibitors, which inhibit spleen tyrosine kinase, vinca alkaloids mechanistically occupy the building molecular structure of tubulin, thus theoretically preventing the formation of microtubules, thereby interfering with the ability of cancer cells to divide.

One vinca alkaloid, vinblastine, of the present invention inhibits angiogenesis or the process of new blood vessel growth from pre-existing blood vessels. Angiogenesis is a key step in the transformation of tumors to malignancy. Vinblastine is commonly used to treat hodgkin's disease, non-hodgkin's lymphoma, breast cancer and germ cell tumors. The side effects of vinblastine include: toxicity to white blood cells, nausea, vomiting, constipation, dyspnea, chest or tumor pain, wheezing and fever. Vinblastine is also sometimes associated with antidiuretic hormone secretion and angina pectoris.

Another vinca alkaloid of the present invention is vinorelbine, which has similar effect to vinblastine. Vinorelbine shows significant antitumor activity in patients with breast cancer and has an antiproliferative effect on osteosarcoma (bone tumor cells). Vinorelbine treatment can lead to side effects including decreased resistance to infection, bruising or bleeding, anemia, constipation, diarrhea, nausea, numbness or tingling of the hands and feet, fatigue (also known as peripheral neuropathy), and inflammation at the injection site. Less common side effects include alopecia and allergic reactions.

Another example or embodiment of a vinca alkaloid of the present invention is vincristine or a pharmaceutically acceptable salt thereof. Vincristine has a high affinity for tubulin dimers, which are building molecule dimers of proteins with only two backbone (blocks) lengths, and can be rapidly ligated and re-ligated at different sites, thus theoretically preventing the dimers from recombining (building) tubulin, thereby destroying tubulin and inhibiting microtubule formation. Vincristine is FDA approved for the treatment of acute leukemia, rhabdomyosarcoma, neuroblastoma, wilms 'tumor, hodgkin's disease, and other lymphomas. The most common side effects of vincristine are peripheral neuropathy, suppression of bone marrow activity, constipation, nervous system toxicity, nausea and vomiting, with neuropathy being the most common and most severe side effect. Thus, it has been reported that some patients with vincristine for tumor treatment had to stop the vincristine treatment.

The fourth common vinca alkaloid is vindesine or a pharmaceutically acceptable salt thereof. Vindesine has only a 24-hour serum half-life, but it has similar effects (expected effect and side effects) as vinblastine. In the treatment of melanoma, lung cancer and (in combination with other drugs) uterine cancer, vindesine is usually administered at a dose of 3mg per square meter of body surface area. Other side effects of vindesine include anemia, hemocytotoxicity, fatigue, the sensation of tingling or stinging on the skin, and skin toxicity.

Compound (I)

The compounds are provided here and elsewhere, for example in the summary and in the examples.

The compounds provided herein are named using ChemBioDraw Ultra12.0, and it is understood by those skilled in the art that the compound structures may be named or identified using other well known naming systems and symbols (including CAS and IUPAC).

Accordingly, provided herein are compounds useful as Syk inhibitors. In one aspect, the invention provides a compound of formula I:

wherein:

R²is H or 2-hydroxyAn ethoxy group;

R³is H or methyl; and is

R⁴Is H or methyl.

In the formula IIn some embodiments of the compound or pharmaceutically acceptable salt thereof, R¹Is selected fromAnd R is²Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromAnd R is²Is 2-hydroxyethoxy.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromAnd R is³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromAnd R is³Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromAnd R is⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromAnd R is⁴Is H. A compound of formula I or a pharmaceutically acceptable salt thereofIn some embodiments of the salts of (I), R¹Is selected fromAnd R is⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, and R³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, and R³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, and R³Is methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, and R³Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, and R⁴Is methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR³Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR³Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR³Is H, and R⁴Is methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR³Is methyl, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, R³Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, R³Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, R³Is H, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, R³Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, R³Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, R³Is H, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, R³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, R³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is H, R³Is methyl, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is selected fromR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is²Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is²Is 2-hydroxyethoxy.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is³Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof，R¹Is composed ofAnd R is⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, and R³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, and R³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, and R³Is methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, and R³Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, and R⁴Is methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR³Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR³Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR³Is H, and R⁴Is methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR³Is methyl, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is H, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is H, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is methyl, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof,R¹is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is methyl.

In the compounds of formula I or medicaments thereofIn some embodiments of the pharmaceutically acceptable salts, R is¹Is composed ofAnd R is⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, and R³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, and R³Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, and R³Is methyl. In some embodiments of the compounds of formula I, R¹Is composed ofR²Is 2-hydroxyethoxy, and R³Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is H, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is H, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is H, R³Is H, andR⁴is methyl.

In certain embodiments of the compound of formula I or a pharmaceutically acceptable salt thereofIn the embodiment, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is methyl.

In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is²Is H. In some embodiments of the compounds of formula I, R¹Is composed ofAnd R is²Is 2-hydroxyethoxy.

A compound of formula I or a pharmaceutically acceptable salt thereofIn some embodiments of the salts, R¹Is composed ofAnd R is⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofAnd R is⁴Is methyl.

A compound of formula I or a pharmaceutically acceptable salt thereofIn some embodiments of the salts of (I), R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H or methyl. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is H. In some embodiments of the compound of formula I or a pharmaceutically acceptable salt thereof, R¹Is composed ofR²Is 2-hydroxyethoxy, R³Is methyl, and R⁴Is methyl.

In one aspect, embodiments of the present application that relate to compounds of formula I also relate to pharmaceutically acceptable salts of compounds of formula I, even if not explicitly stated herein.

In some embodiments, the compound of formula I is selected from:

or a pharmaceutically acceptable salt thereof.

The present application also provides a compound of formula II, or a pharmaceutically acceptable salt thereof:

wherein:

R¹⁰is selected fromWherein ＊ represents R in the benzene ring shown in formula I¹The carbon atom to which it is attached;

R²⁰is H or 2-hydroxyethoxy;

R³⁰is H or methyl; and is

R⁴Is H, halogen (i.e., F, Cl, Br or I), methyl or halogen-substituted methyl (i.e., methyl in which 1 to 3 hydrogen atoms are substituted by 1 to 3 halogen atoms which may be the same or different, e.g., fluoromethyl, chloromethyl, difluoromethyl, dichloromethyl, chlorofluoromethyl, trifluoromethyl, etc.).

Representative compounds of the invention are listed in table a below. The compounds in table a are named using ChemBioDraw ultra12.0, it being understood that other names can be used to identify the same structure of a compound. Other compounds or groups may be named using common names or systematic or non-systematic names. The compounds may also be named using other naming systems and symbols announced in the chemical arts, including, for example, Chemical Abstracts Service (CAS) and international union of theory and applied chemistry (IUPAC). Where the nomenclature of compounds is ambiguous, the structures can be resolved as provided herein.

Table a. representative compounds

The compounds described herein (e.g., compounds of formula I or pharmaceutically acceptable salts thereof) provide unique advantages as Syk inhibitors. The compounds described herein are inhibitors of Syk kinase activity, e.g., as measured by inhibition of Syk kinase activity in biochemical assays, or as measured by reduction of basophil activation as measured by CD63 expression, as described in the examples. The compounds described herein also have desirable properties for use as pharmaceuticals, including kinetic solubility in phosphate buffer at pH7.4 at 37 ℃ and low levels of hepatocyte clearance. These features result in Syk inhibitors with pharmacokinetic profiles that provide a therapeutic window enabling the compounds to be effective at lower doses than currently known compounds for the treatment of diseases. As such, the compounds provide effective doses with minimal off-target activity, which can mitigate unwanted side effects, reduce the chance of drug-drug interactions, and increase subject compliance for a given treatment regimen.

In some embodiments, the compounds described herein (e.g., a compound of formula I or a pharmaceutically acceptable salt thereof) are effective in one or more of inhibition of Syk kinase activity or reduction in basophil activation as measured by CD63 expression, e.g., the compoundsInhibition of Syk kinase activity, IC₅₀A value of less than or equal to 1 μ M, less than or equal to 500nM, less than or equal to 200nM, less than or equal to 100nM, less than or equal to 50nM, less than or equal to 20nM, or less than or equal to 10nM, as demonstrated by an assay suitable for Syk kinase activity, e.g., the assay described in example 8; and/or reducing CD63 expression activity, EC₅₀Values less than or equal to 1 μ M, less than or equal to 500nM, less than or equal to 200nM, less than or equal to 150nM, less than or equal to 100nM, or less than or equal to 75nM, as demonstrated by an assay suitable for measuring CD63 expression in basophils, such as the assay described in example 9.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is effective in both Syk kinase inhibition and reduction of CD63 expression, e.g., the compound has Syk kinase activity, IC₅₀A value of less than or equal to 1 μ M, less than or equal to 500nM, less than or equal to 200nM, less than or equal to 100nM, less than or equal to 50nM, less than or equal to 20nM, or less than or equal to 10nM, as demonstrated by an assay suitable for Syk kinase activity, e.g., the assay described in example 8; and having reduced expression of CD63, EC₅₀Values less than or equal to 1 μ M, less than or equal to 500nM, less than or equal to 200nM, less than or equal to 150nM, less than or equal to 100nM, or less than or equal to 75nM, as demonstrated by an assay suitable for measuring CD63 expression in basophils, such as the assay described in example 9.

In some embodiments, in addition to having one or more of Syk kinase inhibition or a reduction in basophil activation as measured by CD63 expression (including both Syk kinase inhibition and a reduction in basophil activation as measured by CD63 expression), a compound of formula I or a pharmaceutically acceptable salt thereof has one or more of the properties desirable for use as a medicament, including kinetic solubility and low levels of hepatocyte clearance. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, has desirable properties for one or more of kinetic solubility and low levels of hepatocyte clearance, including kinetic solubility in phosphate buffer at pH7.4 at 37 ℃ of greater than or equal to 10 μ Μ, greater than or equal to 20 μ Μ, greater than or equal to 30 μ Μ, greater than or equal to 40 μ Μ, greater than or equal to 50 μ Μ, greater than or equal to 60 μ Μ, greater than or equal to 70 μ Μ, greater than or equal to 80 μ Μ or greater than or equal to 90 μ Μ, as evidenced by a suitable measurement of kinetic solubility, e.g., the test described in example 10; and/or a predicted hepatocyte clearance of less than or equal to 0.50L/hr/kg, less than or equal to 0.40L/hr/kg, less than or equal to 0.30L/hr/kg, less than or equal to 0.20L/hr/kg, less than or equal to 0.10L/hr/kg, less than or equal to 0.09L/hr/kg, less than or equal to 0.08L/hr/kg, less than or equal to 0.07L/hr/kg, or less than or equal to 0.06L/hr/kg, as evidenced by a suitable measurement of predicted hepatocyte clearance, e.g., the test described in example 11.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, has desirable properties of kinetic solubility and low levels of hepatocyte clearance, including kinetic solubility in phosphate buffer at pH7.4 at 37 ℃ of greater than or equal to 10 μ Μ, greater than or equal to 20 μ Μ, greater than or equal to 30 μ Μ, greater than or equal to 40 μ Μ, greater than or equal to 50 μ Μ, greater than or equal to 60 μ Μ, greater than or equal to 70 μ Μ, greater than or equal to 80 μ Μ or greater than or equal to 90 μ Μ, as demonstrated by suitable measurements of kinetic solubility, e.g., the test described in example 10; and a predicted hepatocyte clearance of less than or equal to 0.50L/hr/kg, less than or equal to 0.40L/hr/kg, less than or equal to 0.30L/hr/kg, less than or equal to 0.20L/hr/kg, less than or equal to 0.10L/hr/kg, less than or equal to 0.09L/hr/kg, less than or equal to 0.08L/hr/kg, less than or equal to 0.07L/hr/kg, or less than or equal to 0.06L/hr/kg, as evidenced by a suitable measurement of predicted hepatocyte clearance, e.g., the test described in example 11.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is effective in both Syk kinase inhibition and reduction of CD63 expression, and has desirable properties of kinetic solubility and low level of hepatocyte clearance, e.g., the compound hasSyk kinase Activity, IC₅₀A value of less than or equal to 1 μ M, less than or equal to 500nM, less than or equal to 200nM, less than or equal to 100nM, less than or equal to 50nM, less than or equal to 20nM, or less than or equal to 10nM, as demonstrated by an assay suitable for Syk kinase activity, e.g., the assay described in example 8; and has reduced expression, EC, of CD63₅₀A value of less than or equal to 1 μ Μ, less than or equal to 500nM, less than or equal to 200nM, less than or equal to 150nM, less than or equal to 100nM or less than or equal to 75nM, as demonstrated by an assay suitable for measuring CD63 expression in basophils, e.g., the assay described in example 9; and a kinetic solubility in phosphate buffer at 37 ℃ at pH7.4 of greater than or equal to 10 μ Μ, greater than or equal to 20 μ Μ, greater than or equal to 30 μ Μ, greater than or equal to 40 μ Μ, greater than or equal to 50 μ Μ, greater than or equal to 60 μ Μ, greater than or equal to 70 μ Μ, greater than or equal to 80 μ Μ or greater than or equal to 90 μ Μ, as demonstrated by suitable measurements of kinetic solubility, such as the test described in example 10; and the predicted hepatocyte clearance is less than or equal to 0.50L/hr/kg, less than or equal to 0.40L/hr/kg, less than or equal to 0.30L/hr/kg, less than or equal to 0.20L/hr/kg, less than or equal to 0.10L/hr/kg, less than or equal to 0.09L/hr/kg, less than or equal to 0.08L/hr/kg, less than or equal to 0.07L/hr/kg, or less than or equal to 0.06L/hr/kg, as evidenced by a suitable measurement of the predicted hepatocyte clearance, such as the test described in example 11.

Application method

The present invention provides methods of treating a subject (e.g., a mammal, such as a human) having a disease responsive to inhibition of Syk activity, comprising administering to a subject having or suspected of having such a disease an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. In one aspect, the subject, such as a human, is administered a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof can be administered to a subject (e.g., a human) at risk of, or having a family history of, the disease or disorder.

In some embodiments, the compounds of formula I, or pharmaceutically acceptable salts thereof, may also inhibit other kinases, such that diseases, disease symptoms, and conditions associated with these kinases may also be treated.

The method of treatment also includes inhibiting Syk activity and/or inhibiting B cell activity by inhibiting ATP binding or hydrolysis via Syk or other mechanism in vivo in a subject having a disease responsive to inhibition of Syk activity by administering an effective concentration of a compound of formula I or a pharmaceutically acceptable salt thereof. An example of an effective concentration is a concentration sufficient to inhibit Syk activity in vitro. Effective concentrations can be determined experimentally (e.g., by testing the blood concentration of the compound after administration to a human) or theoretically by calculating bioavailability.

In some embodiments, the disorder responsive to inhibition of Syk activity and/or B cell activity is cancer, an allergic disease and/or an autoimmune disease and/or an inflammatory disease, and/or an acute inflammatory response.

Also provided are methods of inhibiting B cell activity in a subject in need thereof comprising administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

Also provided are methods of inhibiting B cell proliferation in a subject in need thereof comprising administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

Also provided are methods of treating a subject having cancer, an allergic and/or autoimmune disease and/or an inflammatory disease and/or an acute inflammatory response by administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, conditions and diseases that can be treated using a compound of formula I or a pharmaceutically acceptable salt thereof include, but are not limited to, lymphoma (e.g., Small Lymphocytic Lymphoma (SLL), non-hodgkin's lymphoma (NHL), indolent non-hodgkin's lymphoma (iNHL), refractory non-hodgkin's lymphoma, Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), lymphoplasmacytic lymphoma (LPL), Marginal Zone Lymphoma (MZL), immunoblastic large cell lymphoma, lymphoblastic lymphoma, splenic marginal zone B cell lymphoma (+/-villous lymphoma), nodal marginal zone lymphoma (+/-monocyte-like B cells), extranodal marginal zone B cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type, T cell lymphoma (e.g., cutaneous T cell lymphoma, extranodal T cell lymphoma, lymphomatosis, lymphomas, etc.) Anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, mycosis fungoides), B cell lymphoma, Diffuse Large B Cell Lymphoma (DLBCL), mediastinal large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, small non-dividing cell lymphoma, Burkitt's lymphoma, multiple myeloma, plasmacytoma, leukemias (e.g., Acute Lymphocytic Leukemia (ALL), acute T cell lymphoblastic leukemia (T-ALL), acute B cell lymphocytic leukemia (B-ALL), B cell prolymphocytic leukemia, Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), juvenile myelomonocytic leukemia (JMML), Minimal Residual Disease (MRD), hairy cell leukemia, myelofibrosis (e.g., primary or secondary myelofibrosis), or Chronic Myeloid Leukemia (CML), myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), Waldenstrom's Macroglobulinemia (WM), polycythemia vera, essential thrombocythemia, pancreatic cancer, urinary carcinoma, bladder cancer, colorectal cancer, colon cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, thyroid cancer, gallbladder cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), ovarian cancer, cervical cancer, gastric cancer, endometrial cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumors (e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, adult anaplastic astrocytoma), bone cancer, soft tissue sarcoma, retinoblastoma, neuroblastoma, peritoneal leakage, Malignant pleural effusion, mesothelioma, Wilms 'tumor (Wilms tumor), trophoblastic neoplasms (trophoblastic neoplasms), vascular involucrima, Kaposi's sarcoma, mucous cancer (myxoid carcinoma), round cell carcinoma, squamous cell carcinoma, esophageal squamous cell carcinoma, oral cancer, adrenocortical carcinoma, ACTH-producing tumors (ACTH-producingtumors), Systemic Lupus Erythematosus (SLE), myasthenia gravis, Goodpasture's syndrome, glomerulonephritis, hemorrhage, pulmonary hemorrhage, atherosclerosis, Rheumatoid Arthritis (RA), psoriatic arthritis, monoarthritis, osteoarthritis, gouty arthritis, spondylitis, Behcet's disease, autoimmune thyroiditis, Raynaud's syndrome, acute disseminated encephalomyelitis, chronic idiopathic thrombocytopenic purpura, Multiple Sclerosis (MS), Sjogren's syndrome, autoimmune hemolytic anemia, hemolytic anemia, Tissue transplant rejection, hyperacute rejection of transplanted organs, allograft rejection, graft-versus-host disease, diseases involving leukocyte extravasation, diseases due to leukocyte dyscrasia and metastasis, granulocyte transfusion-related syndrome, cytokine-induced toxicity, scleroderma, vasculitis, asthma, psoriasis, inflammatory bowel disease (e.g., chronic inflammatory bowel disease, ulcerative colitis, Crohn's disease, necrotizing enterocolitis), irritable bowel syndrome, dermatomyositis, Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes, type I diabetes, sepsis, septic shock, endotoxic shock, gram-negative sepsis, gram-positive sepsis, toxic shock syndrome, multiple organ injury syndrome secondary to sepsis, trauma, hypovolemic shock, allergic conjunctivitis, vernal conjunctivitis, chronic inflammatory bowel disease, Thyroid-related eye disease, eosinophilic granuloma, eczema, chronic bronchitis, acute respiratory distress syndrome, allergic rhinitis, pollinosis, bronchial asthma, silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, emphysema, pneumonia, bacterial pneumonia, bronchiectasis, pulmonary oxygen poisoning, reperfusion injury of the myocardium or brain or limbs, heat injury, cystic fibrosis, keloid formation or scar tissue formation, fever and myalgia due to infection, brain or spinal cord injury due to minimal trauma, diseases involving leukocyte exudation, acute hypersensitivity, delayed hypersensitivity, urticaria, food allergy, sunburn of the skin, pelvic inflammatory disease, urethritis, uveitis, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, alcoholic hepatitis, Gastritis, enteritis, contact dermatitis, atopic dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis, and polycystic kidney disease.

In some embodiments, there is provided a method of treating a subject suffering from an allergic disease and/or an autoimmune disease and/or an inflammatory disease and/or an acute inflammatory response by administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the disease is selected from systemic lupus erythematosus, myasthenia gravis, goodpasture's syndrome, glomerulonephritis, hemorrhage, pulmonary hemorrhage, atherosclerosis, rheumatoid arthritis, psoriatic arthritis, monoarthritis, osteoarthritis, gouty arthritis, spondylitis, behcet's disease, autoimmune thyroiditis, raynaud's syndrome, acute disseminated encephalomyelitis, chronic idiopathic thrombocytopenic purpura, multiple sclerosis, sjogren's syndrome, autoimmune hemolytic anemia, tissue transplant rejection, hyperacute rejection of transplanted organs, allograft rejection, graft-versus-host disease, diseases involving leukocyte extravasation, diseases due to leukocyte dyscrasia and metastasis, syndrome associated with granulocyte infusion, cytokine-induced toxicity, scleroderma, vasculitis, autoimmune diseases, and the like, Asthma, psoriasis, chronic inflammatory bowel disease, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, irritable bowel syndrome, dermatomyositis, Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes, type I diabetes, sepsis, septic shock, endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shock syndrome, multiple organ injury syndrome secondary to sepsis, trauma, hypovolemic shock, allergic conjunctivitis, vernal conjunctivitis, thyroid-related eye disease, eosinophilic granuloma, eczema, chronic bronchitis, acute respiratory distress syndrome, allergic rhinitis, pollinosis, bronchial asthma, silicosis, pulmonary sarcoidosis, pleuritis, alveolitis, emphysema, pneumonia, bacterial pneumonia, bronchiectasis, pulmonary oxygen poisoning, reperfusion injury of myocardium or brain or limbs, Thermal injury, cystic fibrosis, keloid formation or scar tissue formation, fever and myalgia due to infection, brain or spinal cord injury due to minimal trauma, diseases involving leukocyte exudation, acute hypersensitivity, delayed-type hypersensitivity, urticaria, food allergy, sunburn of the skin, pelvic inflammatory disease, urethritis, uveitis, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, alcoholic hepatitis, gastritis, enteritis, contact dermatitis, atopic dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis, and polycystic kidney disease.

In some embodiments, there is provided a method of treating a subject having an autoimmune disease selected from systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, sjogren's syndrome, psoriasis, autoimmune hemolytic anemia, asthma, ulcerative colitis, crohn's disease, irritable bowel disease, and chronic obstructive pulmonary disease by administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the autoimmune disease has an excessive or destructive immune response, such as asthma, rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, or systemic lupus erythematosus.

In some embodiments, there is provided a method of treating a subject having rheumatoid arthritis by administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.

Syk is a known inhibitor of apoptosis in lymphoma B cells. Defective apoptosis contributes to the pathogenesis and drug resistance of human leukemias and lymphomas. Accordingly, there is also provided a method of promoting or inducing apoptosis in a cell expressing Syk, comprising contacting the cell with a compound of formula I, or a pharmaceutically acceptable salt thereof.

In some embodiments, there is provided a method of treating a subject having a cancer selected from the group consisting of a carcinoma, a sarcoma, a melanoma, a lymphoma, and a leukemia. In some embodiments, the cancer is a solid tumor or a hematologic malignancy.

In some embodiments, there is provided a method of treating a subject having a hematologic malignancy selected from small lymphocytic lymphoma, non-hodgkin's lymphoma, indolent non-hodgkin's lymphoma, refractory non-hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, marginal zone lymphoma, immunoblastic large cell lymphoma, lymphoblastic lymphoma, splenic marginal zone B-cell lymphoma (+/-villous lymphocytes), nodal marginal zone lymphoma (+/-mononuclear-like B cells), extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type, cutaneous T-cell lymphoma, extranodal T-cell lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, mycosis fungoides, B-cell lymphoma, lymphomatosis, neuroblastoma, lymphomatosis, lympho, Diffuse large B-cell lymphoma, mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, small non-dividing cell lymphoma, burkitt's lymphoma, multiple myeloma, plasmacytoma, acute lymphocytic leukemia, acute T-cell lymphoblastic leukemia, acute B-cell lymphocytic leukemia, B-cell prolymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, juvenile myelomonocytic leukemia, minimal residual disease, hairy cell leukemia, primary myelofibrosis, secondary myelofibrosis, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorders, and waldenstrom's macroglobulinemia.

In some embodiments, there is provided a method of treating a subject having a cancer by administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein the cancer is leukemia or lymphoma. In some embodiments, the cancer is selected from acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, myelodysplastic syndrome, myeloproliferative disease, chronic myeloid leukemia, multiple myeloma, indolent non-hodgkin's lymphoma, refractory non-hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, waldenstrom's macroglobulinemia, T-cell lymphoma, B-cell lymphoma, and diffuse large B-cell lymphoma. In one embodiment, the cancer is acute lymphoblastic leukemia of T cell type, or acute lymphocytic leukemia of B cell type. The non-hodgkin's lymphomas encompass indolent B cell diseases including, for example, follicular lymphoma, lymphoplasmacytic lymphoma, waldenstrom's macroglobulinemia, and marginal zone lymphoma, as well as aggressive lymphomas including, for example, burkitt's lymphoma, diffuse large B cell lymphoma, and mantle cell lymphoma. In one embodiment, the cancer is indolent non-hodgkin's lymphoma.

In some embodiments, there is provided a method of treating a subject having a hematological malignancy by administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. In particular embodiments, the hematological malignancy is a leukemia (e.g., chronic lymphocytic leukemia) or a lymphoma (e.g., non-hodgkin's lymphoma).

In some embodiments, there is provided a method of treating a subject having chronic lymphocytic leukemia by administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.

In some embodiments, methods of treating a subject having a solid tumor by administering an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, are provided. In some embodiments, the solid tumor is a cancer selected from pancreatic cancer, urinary bladder cancer, colorectal cancer, colon cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, thyroid cancer, gallbladder cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), ovarian cancer, cervical cancer, gastric cancer, endometrial cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumors (e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma), bone cancer, soft tissue sarcoma, retinoblastoma, neuroblastoma, peritoneal leakage, malignant pleural effusion, mesothelioma, wilms 'tumor, trophoblastic tumors, hemangiothecal tumors, kaposi's sarcoma, mucous cancer, round cell carcinoma, squamous cell carcinoma, colorectal carcinoma, colon carcinoma, breast cancer, prostate cancer, bladder cancer, lung, Esophageal squamous cell carcinoma, oral cancer, adrenocortical carcinoma, and ACTH-producing tumors. In some embodiments, the solid tumor is non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.

The present application also provides a compound described herein, e.g., a compound of formula I or a pharmaceutically acceptable salt thereof, for use in treating a disease or disorder described herein, e.g., cancer (including carcinomas, sarcomas, melanomas, lymphomas, and leukemias), allergic diseases, and/or autoimmune diseases, and/or inflammatory diseases and/or acute inflammatory responses.

The present application also provides a compound described herein, for example a compound of formula I or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of a disease or disorder described herein, for example cancer (including carcinomas, sarcomas, melanomas, lymphomas and leukemias), allergic diseases and/or autoimmune diseases and/or inflammatory diseases and/or acute inflammatory responses.

The present application also provides methods of using a compound of formula I or a pharmaceutically acceptable salt thereof (for therapeutic or prophylactic treatment with selective or specific inhibition of Syk activity) in combination with a vinca alkaloid or a pharmaceutically acceptable salt thereof (for therapeutic or prophylactic treatment with selective or specific inhibition of tubulin or microtubule formation). The methods comprise administering to a subject (e.g., a human) in need of treatment a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, in an amount sufficient to inhibit Syk activity and/or inhibit tubulin or microtubule formation. The method can be used to treat a subject (e.g., a human) having or suffering from a disorder whose symptoms or pathology are mediated by Syk expression or activity.

"Treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following:

(i) reducing one or more symptoms resulting from the disease;

(ii) reducing the severity of the disease and/or stabilizing the disease (e.g., delaying the progression of the disease);

(iii) delay of disease transmission (e.g., metastasis);

(iv) delay or slow the recurrence of the disease and/or progression of the disease;

(v) ameliorating the disease and/or providing relief (partial or total) of the disease and/or reducing the dosage of one or more other drugs required to treat the disease;

(vi) improve quality of life, and/or

(vii) Prolonging survival.

By "delaying" the development of a disease or condition is meant delaying, hindering, slowing, delaying, stabilizing and/or delaying the development of the disease or condition. Such delay may be of varying lengths of time depending on the history of the disease or condition and/or the subject being treated. A method of "delaying" the progression of a disease or condition is a method that: which reduces the probability of development of a disease or condition over a given time frame and/or reduces the severity of a disease or condition over a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects. Disease or condition development can be detected using standard methods, e.g., routine physical examination,

Mammography, imaging, or biopsy. Progression may also mean that the disease or condition may initially be undetectable and includes onset, recurrence and onset.

In some embodiments, a combination of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a vinca alkaloid, or a pharmaceutically acceptable salt thereof, can be administered to a subject (e.g., a human) at risk of, or with a family history of, the disease or disorder.

The term "inhibit" indicates a decrease in the baseline activity of a biological activity or process. By "inhibition of Syk activity" is meant a decrease in Syk activity in the presence of a compound of formula I, or a pharmaceutically acceptable salt thereof, relative to the activity of Syk in the absence of the compound, or a pharmaceutically acceptable salt thereof. In some embodiments, inhibition of Syk activity may be compared to the same subject prior to treatment, or to other subjects not receiving treatment. By "inhibition of tubulin forming activity" is meant a decrease in tubulin forming activity in the presence of vinca alkaloid, or a pharmaceutically acceptable salt thereof, in direct or indirect response to tubulin forming activity in the absence of said vinca alkaloid, or a pharmaceutically acceptable salt thereof, relative to tubulin forming activity in the absence of said vinca alkaloid. In some embodiments, the inhibition of tubulin formation may be compared to the same subject prior to treatment, or to other subjects not receiving treatment.

Disease and disorder

In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is used in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, for treating cancer. In some embodiments, the combination of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a vinca alkaloid, or a pharmaceutically acceptable salt thereof, is used to treat a hematologic malignancy. In some embodiments, the combination of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a vinca alkaloid, or a pharmaceutically acceptable salt thereof, inhibits the growth or proliferation of cancer cells of hematopoietic origin. In some embodiments, the cancer cell is a cell of lymphoid origin, and in some embodiments, the cancer cell is involved in or derived from a B lymphocyte or a B lymphocyte progenitor.

Hematologic malignancies that can be treated using the disclosed methods include, but are not limited to, lymphomas (e.g., malignancies of lymphoid and reticuloendothelial tissues, such as burkitt lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, lymphocytic lymphoma); multiple myeloma; leukemia (e.g., lymphocytic leukemia, chronic myeloid (granulocytic) leukemia). Other cancer cells of hematopoietic origin or otherwise that express Syk can also be treated by administering the polymorphs and compositions described herein.

In specific embodiments, the hematological malignancy is a leukemia or lymphoma. In some embodiments, the hematologic malignancy is Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), Chronic Myeloid Leukemia (CML), Multiple Myeloma (MM), non-hodgkin's lymphoma (NHL), indolent non-hodgkin's lymphoma (iNHL), refractory non-hodgkin's lymphoma, Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Waldenstrom's Macroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma (LPL), and Marginal Zone Lymphoma (MZL).

In one embodiment, the cancer is acute lymphoblastic leukemia of the T-cell type (T-ALL) or B-cell acute lymphoblastic leukemia (B-ALL). In another embodiment, the cancer is Chronic Lymphocytic Leukemia (CLL). In a further embodiment, the cancer is non-hodgkin's lymphoma (NHL). In one embodiment, the NHL is diffuse large B-cell lymphoma (DLBCL), Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Small Lymphocytic Lymphoma (SLL), lymphoplasmacytic lymphoma (LPL), and Marginal Zone Lymphoma (MZL). In one embodiment, the cancer is indolent non-hodgkin's lymphoma (iNHL).

In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is used in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, for treating a solid tumor. In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, is used in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, for the treatment of certain solid tumors, such as pancreatic cancer, lung cancer, colon cancer, colorectal cancer, breast cancer, esophageal cancer, adenocarcinoma, hepatocellular carcinoma. In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, is used in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, for treating certain solid tumors in which expression of Syk activity is possessed, or in which Syk is expressed. Other solid tumor cancer cells that express Syk can also be treated by administering the polymorphic forms and compositions described herein.

In another aspect, methods of treating a subject (e.g., a human) having a Syk-mediated disease by administering to the subject a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, are provided. Also provided are methods of modulating Syk in a subject (e.g., a human) by administering to the subject a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof.

In any of the methods described herein, a compound of formula I, or a pharmaceutically acceptable salt thereof, can be administered to the subject in a unit dose (e.g., in the form of a tablet). In any of the methods described herein, the vinca alkaloid or a pharmaceutically acceptable salt thereof may be administered to the subject by IV (intravenous) delivery.

Any of the therapeutic methods provided herein can be used to treat advanced cancer. Any of the therapeutic methods provided herein can be used to treat locally advanced cancer. Any of the therapeutic methods provided herein can be used to treat primary cancer. Any of the therapeutic methods provided herein can be used to treat cancer in remission. In some embodiments of any of the methods of treatment provided herein, the cancer relapses after a remission period. In some embodiments of any of the methods of treatment provided herein, the cancer is a progressive cancer.

Test subject

Any of the provided methods of treatment may be used to treat a subject who has been diagnosed with or is suspected of having an allergic disease and/or an autoimmune disease and/or an inflammatory disease and/or an acute inflammatory response or cancer.

In some embodiments of any of the methods provided herein, the subject is a human at risk of developing cancer (e.g., a human who is genetically or otherwise predisposed to developing cancer) and who has been diagnosed with or has not been diagnosed with cancer. As used herein, a "at risk" subject is a subject at risk of developing cancer (e.g., a hematological malignancy). The subject may or may not have a detectable disease and may or may not have exhibited a detectable disease prior to treatment with the methods of the invention. A subject at risk may have one or more so-called risk factors, which are measurable parameters associated with the development of cancer, such as described herein. Subjects with one or more of these risk factors have a higher probability of developing cancer than individuals without these risk factors.

These risk factors may include, for example, age, sex, race, diet, previous medical history, presence of precursor disease, genetic (e.g., genetic) causes, and environmental exposure. In some embodiments, subjects at risk for cancer include, for example, subjects in which relatives already have the disease, and those at risk as determined by analysis of genetic or biochemical markers. The history of having cancer may also be a risk factor, such as cancer recurrence.

The invention also provides methods of treating a subject (e.g., a human) exhibiting one or more symptoms associated with cancer (e.g., a hematological malignancy). In some embodiments, the subject is in an early stage of cancer. In other embodiments, the subject is at an advanced stage of cancer.

The invention also provides methods of treating a subject (e.g., a human) being treated for cancer (e.g., a hematological malignancy) with one or more standard therapies, such as chemotherapy, radiation therapy, immunotherapy and/or surgery. Thus, in some of the above embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, may be administered before, during, or after chemotherapy, radiation therapy, immunotherapy, and/or surgery.

In another aspect, provided herein are methods of treating a subject (e.g., a human) that is "refractory" to a cancer treatment or "relapsed" following a cancer treatment (e.g., a hematologic malignancy). A subject that is "refractory" to an anticancer therapy is one that is not responsive to the particular treatment, also referred to as resistant. The cancer may develop resistance to the treatment at the beginning of the treatment, or may develop resistance during the course of the treatment (e.g., after the treatment has shown some effect on the cancer but not enough to be considered remission or partial remission). A subject who "relapses" is one in which the cancer, or signs and symptoms of cancer, recover after a period of improvement (e.g., after treatment has been shown to effectively reduce the cancer, e.g., after the subject has remission or partial remission).

In some embodiments, the subject may be a human that is (i) refractory to at least one anti-cancer therapy, or (ii) relapsed after treatment with at least one anti-cancer therapy, or both (i) and (ii). In some embodiments, the subject is refractory to at least two, at least three, or at least four anti-cancer therapies (including, e.g., standard or experimental chemotherapies).

In some embodiments, the subject is at least one, at least two, at least three, or more than one,Or at least four anti-cancer therapies (including, e.g., standard or experimental chemotherapies) selected from: fludarabine, rituximab, obituzumab (obinutuzumab), alkylating agents, alemtuzumab and other chemotherapeutic treatments, such as CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone); R-CHOP (rituximab-CHOP); hypercladia (hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, cytarabine); R-hyperCVAD (rituximab-hyperCVAD); FCM (fludarabine, cyclophosphamide, mitoxantrone); R-FCM (Rituximab, fludarabine, cyclophosphamide, mitoxantrone); bortezomib and rituximab; temsirolimus and rituximab; temsirolimus andiodine-131 tositumomabAnd CHOP; CVP (cyclophosphamide, vincristine, prednisone); R-CVP (rituximab-CVP); ICE (ifosfamide, carboplatin, etoposide); R-ICE (Rituximab-ICE); FCR (fludarabine, cyclophosphamide, rituximab); FR (fludarabine, rituximab); pace (dexamethasone, thalidomide, cisplatin),Cyclophosphamide, etoposide).

Other examples of chemotherapeutic treatments, including standard or experimental chemotherapies, are described below. In addition, some lymphomas are treated by Cheson, B.D., Leonard, J.P., "Monoclonal Antibody Therapy for B-CellNon-Hodgkin's Lymphoma" The New England and Journal of Medicine 2008,359(6), "P.613-626 and Wierda, W.G.," Current and Investigational therapeutics for Patents with CLL "Hematology 2006, p.285-294. In the United States, the pattern of Lymphoma development is analyzed in Morton, L.M., et al, "Lymphoma Inc. Pattern by WHO Subtype in the United States, 1992-.

For example, treatment of non-hodgkin's lymphoma (NHL), especially of B cell origin, includes the use of monoclonal antibodies, standard chemotherapeutic methods (e.g., CHOP, CVP, FCM, MCP, etc.), radioimmunotherapy, and combinations thereof, especially the integration of antibody therapy with chemotherapy. Examples of non-conjugated monoclonal antibodies for non-hodgkin's lymphoma/B cell cancers include rituximab, alemtuzumab, human or humanized anti-CD 20 antibody, lumiximab (lumiximab), anti-TRAIL, bevacizumab, galiximab (galiximab), epratuzumab, SGN-40, and anti-CD 74. Examples of experimental antibody drugs for the treatment of non-hodgkin lymphoma/B cell cancers include ofatumumab (ofatumumab), ha20, PRO131921, alemtuzumab, galiximab, SGN-40, CHIR-12.12, epratuzumab, luximab, aprezumab (apolizumab), milatuzumab (milatuzumab), and bevacizumab. Examples of standard chemotherapeutic regimens for non-hodgkin's lymphoma/B cell cancers include CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), FCM (fludarabine, cyclophosphamide, mitoxantrone), CVP (cyclophosphamide, vincristine, and prednisone), MCP (mitoxantrone, chlorambucil, and prednisolone), R-CHOP (rituximab plus CHOP), R-FCM (rituximab plus FCM), R-CVP (rituximab plus CVP), and R MCP (R MCP). Examples of radioimmunotherapy for non-hodgkin lymphoma/B cell cancers include yttrium-90-labeled ibritumomab tiuxetan and iodine-131-labeled tositumomab tiuxetan.

In another example, therapeutic treatments for Mantle Cell Lymphoma (MCL) include combination chemotherapy, such as CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), hypercvidad (hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, cytarabine), and FCM (fludarabine, cyclophosphamide, mitoxantrone). In addition, these protocols can be supplemented with the monoclonal antibody rituximab (Rituxan) to form combination therapies R-CHOP, hyperCVAD-R and R-FCM. Other methods include combining any of the above therapies with stem cell transplantation or treatment with ICE (ifosfamide, carboplatin, and etoposide). Other treatmentsMethods of treating mantle cell lymphoma include immunotherapy, for example with monoclonal antibodies, such as rituximab (Rituxan). Rituximab is useful in the treatment of indolent B-cell cancers, including marginal zone lymphoma, WM, CLL and small lymphocytic lymphomas. Combinations of rituximab and chemotherapeutic agents are particularly effective. An improved method is radioimmunotherapy, in which monoclonal antibodies are combined with radioisotope particles (e.g., iodine-131 tositumomab)And yttrium-90 ibritumomab tiuxetan) And (4) combining. In another example of the above-described method,for sequential treatment with CHOP. Another example of immunotherapy involves the use of cancer vaccines, which are based on the genetic makeup of the tumor in the individual subject. An example of a lymphoma vaccine is GTOP-99Still other methods of treating mantle cell lymphoma include autologous stem cell transplantation plus high dose chemotherapy, or treatment of mantle cell lymphoma includes administration of proteasome inhibitors, e.g.(bortezomib or PS-341), or angiogenesis inhibitors, such as thalidomide, in particular in combination with Rituxan. Another therapeutic approach is the administration of drugs that cause degradation of Bcl-2 protein and increase the susceptibility of cancer cells to chemotherapy, such as oblimersen (gensense) in combination with other chemotherapeutic agents. Other methods of treatment include administration of mTOR inhibitors, which can result in inhibition of cell growth and even cell death; non-limiting examples are temsirolimus (CCI-779), and temsirolimus andor other chemotherapeutic agent combinations。

Other therapies for MCL have recently been disclosed (Nature Reviews; Jares, P.2007). Examples of such include flavididol (Flavopiridol), PD0332991, R-roscovitine (Sericiib, CYC202), styryl sulfone, Obatoclax (GX15-070), TRAIL, anti-TRAIL DR4 and DR5 antibodies, temsirolimus (CCl-779), everolimus (RAD001), BMS-345541, curcumin, vorinostat (SAHA), thalidomide, lenalidomide (lenalidomide,CC-5013) and geldanamycin (17-AAG).

Examples of other therapeutic agents for the treatment of Waldenstrom Macroglobulinemia (WM) include perifosine, bortezomibRituximab, sildenafil citrateCC-5103, thalidomide, epratuzumab (hLL 2-anti-CD 22 humanized antibody), simvastatin, enzastaurin, campath-1H, dexamethasone, DTPACE, oblimersen (oblimersen), anti-oncone A10, anti-oncone AS2-1, alemtuzumab, β -alethine, cyclophosphamide, doxorubicin hydrochloride, prednisone, vincristine sulfate, fludarabine, filgrastim, melphalan, recombinant interferon α, carmustine, cisplatin, cyclophosphamide, cytarabine, etoposide, melphalan, dolastatin 10, indium In 111 monoclonal antibody MN-14, yttrium Y90 human epratuzumab, anti-thymocyte globulin, busulfan, cyclosporine, methotrexate, mycopremol ethylester, therapeutic allogeneic lymphocyte, yttrium Y90, ibrinomycin, western, tacrolimus, thiepinol, interleukin, paclitaxel, recombinant interleukin-2, recombinant interleukin A-263, recombinant interleukin-2, docetaxel, recombinant interleukin-platinum inhibitor, recombinant interleukin A, docetaxel, recombinant interleukin-2, recombinant interleukin-platinum inhibitor, recombinant interleukin-platinum inhibitor, and recombinant interleukinInsulin (tanespimamycin), everolimus, pelvetine, vorinostat, alvocidib, recombinant flt3 ligand, recombinant human thrombopoietin, lymphokine activated killer cell, amifostine trihydrate, aminocamptothecin, irinotecan hydrochloride, caspofungin acetate, clofarabine, alfa-eptine, nelarabine, pentostatin, sargrastim, vinorelbine ditartrate, WT-1 analog peptide vaccine, WT 1126-134 peptide vaccine, fenretinide, ixabepilone (ixabepilone), oxaliplatin, monoclonal antibody CD19, monoclonal antibody CD20, omega-3 fatty acids, mitoxantrone hydrochloride, octreotide acetate, tositumomab and iodine I-131 tositumomab, motoxafen gadolinium, arsenic trioxide, tipifarnib (tipifarnib), autologous human tumor-derived HSPPC-96, visotuzumab, bryostatin 1, and polyethylene glycol liposomal doxorubicin hydrochloride, and any combination thereof.

Examples of therapeutic methods for the treatment of WM include peripheral blood stem cell transplantation, autologous hematopoietic stem cell transplantation, autologous bone marrow transplantation, antibody therapy, biological therapy, enzyme inhibitor therapy, systemic irradiation, infusion of stem cells, stem cell-supported bone marrow ablation (stem cell-supported bone marrow ablation), ex vivo processed peripheral blood stem cell transplantation, cord blood transplantation, immunoenzyme technology, pharmacological studies, low-LET cobalt-60 gamma ray therapy, bleomycin, routine surgery, radiotherapy, and allogeneic hematopoietic stem cell transplantation other than bone marrow eradication.

Examples of other therapeutic agents useful in drug therapy for the treatment of diffuse large B-cell lymphoma (DLBCL) (Blood2005Abramson, J.) include cyclophosphamide, doxorubicin, vincristine, prednisone, anti-CD 20 monoclonal antibody, etoposide, bleomycin, many of the drugs listed in waldenstrom, and any combination thereof, such as ICE and R-ICE.

Examples of other therapeutic agents (Spectrum, 2006, Fernandes, D.) for the treatment of Chronic Lymphocytic Leukemia (CLL) include chlorambucil (Cinchonin), cyclophosphamide (Cyloxan, Endoxan, Endoxana, Cyclostatin), fludarabine (Fudalua), pentostatin (Nipent), cladribine (Leust)arin), doxorubicin: (doxorubicin) ((ii)Doxorubicin), vincristine (ancepin), prednisone, prednisolone, alemtuzumab (Campath, MabCampath), many of the drugs listed in waldenstrom, and combinations of chemotherapy and chemoimmunotherapy, including common combination regimens: CVP (cyclophosphamide, vincristine, prednisone); R-CVP (rituximab-CVP); ICE (ifosfamide, carboplatin, etoposide); R-ICE (Rituximab-ICE); FCR (fludarabine, cyclophosphamide, rituximab); and FR (fludarabine, rituximab).

In another aspect, there is provided a method of sensitizing a subject (e.g., a human) that is (I) refractory to at least one chemotherapy treatment, or (ii) relapsed after treatment with a chemotherapy, or both (I) and (ii), wherein the method comprises administering to the subject a compound of formula I, or a pharmaceutically acceptable salt or pharmaceutical composition thereof. Sensitized subjects are subjects that respond to a treatment involving administration of a compound of formula I or a pharmaceutically acceptable salt thereof, or subjects that are not resistant to such a treatment.

In another aspect, the present invention provides a method of treating cancer with a complication (complication) in a subject (e.g., a human), wherein the treatment is also effective to treat the complication. A "complication" of cancer is a disease that occurs with the cancer at the same time.

In some embodiments, the invention provides methods of treating Chronic Lymphocytic Leukemia (CLL) with complications in a subject (e.g., a human), wherein the treatment is also effective in treating the complications. Many subjects with CLL will have one or more other diseases, such as diseases affecting the blood pressure system, the vascular and cardiac systems, the endocrine and metabolic systems, the genitourinary system, the musculoskeletal system, the respiratory system, the nervous system, the upper and lower digestive systems, the mental system, the ear, the nasal and laryngeal systems, the renal system, or the hepatic system. Specific complications of CLL include, but are not limited to, one or more other cancers (e.g., breast Cancer, head and neck Cancer, lung Cancer, melanoma, non-Hodgkin's T-cell Lymphoma, prostate Cancer, colon Cancer, small bowel Cancer, gynecological and urinary tract cancers), hypertension, hyperlipidemia, coronary artery disease, peripheral vascular disease, cardiomyopathy, valvular heart disease, atrial fibrillation, cerebrovascular disease (e.g., transient ischemic attacks, stroke), chronic obstructive pulmonary disease, joint disease, peptic ulcer, inflammatory bowel disease, psychiatric diseases, thyroid diseases, benign prostatic hyperplasia, diabetes and osteoarthritis (Satram-Hoang et al, Journal of Cancer Therapy, 2013; 4: 1321-.

In some embodiments, a method of treating a complication of CLL in a subject (e.g., a human), wherein the method comprises administering to the subject a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the complication is selected from: one or more other cancers (e.g., breast cancer, head and neck cancer, lung cancer, melanoma, non-hodgkin's T cell lymphoma, prostate cancer, colon cancer, small bowel cancer, gynecological and urinary tract cancers), hypertension, hyperlipidemia, coronary artery disease, peripheral vascular disease, cardiomyopathy, valvular heart disease, atrial fibrillation, cerebrovascular disease (e.g., transient ischemic attack, stroke), chronic obstructive pulmonary disease, joint disease, peptic ulcer, inflammatory bowel disease, psychosis, thyroid disease, benign prostatic hyperplasia, diabetes and osteoarthritis.

Monotherapy and combination therapy

Also provided are methods of treatment wherein a compound of formula I, or a pharmaceutically acceptable salt thereof, is the only active agent administered to a subject, and methods of treatment wherein a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered to a subject in combination with one or more additional active agents. Both monotherapy and combination therapy are intended and described for use in the methods described herein, e.g., in methods of treating any disease or disorder detailed herein, and for use in any subject detailed herein.

Monotherapy

In some embodiments, a method of treating cancer, an allergic disease and/or an autoimmune disease and/or an inflammatory disease and/or an acute inflammatory response, comprising administering to a subject in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein the subject is not treated for the same disease or disorder with another drug or procedure.

In some embodiments, wherein a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered as a monotherapy to a subject, who has been diagnosed with, or is suspected of having, a cancer, the subject may be a human, who (I) is refractory to at least one anti-cancer therapy, or (ii) relapses after treatment with at least one anti-cancer therapy, or both (I) and (ii). In some embodiments, the subject is refractory to at least two, at least three, or at least four anti-cancer therapies (including, e.g., standard or experimental chemotherapies). For example, in some embodiments, the subject may be a human that is (i) refractory to therapy with an anti-CD 20 antibody, an alkylating agent (e.g., bendamustine), a purine analog (e.g., fludarabine), an anthracycline, or any combination thereof; (ii) relapse after treatment with an anti-CD 20 antibody, an alkylating agent (e.g., bendamustine), a purine analog (e.g., fludarabine), an anthracycline, or any combination thereof, or both (i) and (ii).

As described above, a human subject that is refractory to at least one anti-cancer therapy and/or relapsed after treatment with at least one anti-cancer therapy may have been subjected to one or more prior treatments. In some embodiments, such a subject has undergone one, two, three, or four, or at least one, at least two, at least three, at least four, or at least five, or between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, or between 1 and 4 anti-cancer treatments prior to treatment using the methods described herein (e.g., prior to administration of the compound of formula I or a pharmaceutically acceptable salt thereof as monotherapy).

It will be appreciated that when a subject (e.g., a human) is treated with a compound of formula I, or a pharmaceutically acceptable salt thereof, as a monotherapy, the subject may also be on one or more other therapies that are not anti-cancer therapies.

In some embodiments, there is provided a method of treating a complication of a cancer (including, but not limited to CLL) in a subject (e.g., a human) that has been diagnosed with the cancer (e.g., CLL), wherein the method comprises administering to the subject a therapeutic combination with a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, to treat the complication. In some embodiments, the complication is selected from: one or more other cancers (e.g., breast cancer, head and neck cancer, lung cancer, melanoma, non-hodgkin's T cell lymphoma, prostate cancer, colon cancer, small intestine cancer, gynecological and urinary tract cancers), hypertension, hyperlipidemia, coronary artery disease, peripheral vascular disease, cardiomyopathy, valvular heart disease, atrial fibrillation, cerebrovascular disease (e.g., transient ischemic attack, stroke), chronic obstructive pulmonary disease, joint disease, peptic ulcer, inflammatory bowel disease, psychosis, thyroid disease, benign prostatic hyperplasia, diabetes, and osteoarthritis.

Combination therapy

In some embodiments, a method of treating cancer, an allergic disease and/or an autoimmune disease and/or an inflammatory disease and/or an acute inflammatory response comprises administering to a subject in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a second active agent useful for treating cancer, an allergic disease and/or an autoimmune disease and/or an inflammatory disease and/or an acute inflammatory response. For example, the second agent can be an anti-inflammatory drug. Treatment with the second active agent can be performed prior to, concurrently with, or subsequent to treatment with a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, is combined with another active agent in a single dosage form.

The present application also provides methods of treatment in which a compound of formula I, or a pharmaceutically acceptable salt thereof, administered to a subject (e.g., a human) who has been diagnosed with, or is suspected of having, cancer is administered to the subject in combination with one or more additional therapies, including one or more of the anti-cancer therapies described above. Thus, in some embodiments, there is provided a method of treating cancer in a subject (e.g., a human) in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, together with one or more additional therapies useful for treating cancer. The one or more additional therapies may involve administration of one or more therapeutic agents. Suitable anti-cancer therapies that can be used in combination with a compound of formula I or a pharmaceutically acceptable salt thereof include, but are not limited to, one or more agents selected from the group consisting of: chemotherapeutic agents (e.g., mitomycin C, carboplatin, taxol, cisplatin, paclitaxel (paclitaxel), etoposide, doxorubicin), radiation therapy antineoplastic agents, topoisomerase I inhibitors (e.g., camptothecin or topotecan), topoisomerase II inhibitors (e.g., daunomycin and etoposide), alkylating agents (e.g., cyclophosphamide, melphalan, and BCNU), tubulin directing agents (e.g., taxol and vinblastine), PI3K inhibitors (e.g., compound A, B, and C below), inhibitors of lysyl oxidase-like protein 2, and biologicals (e.g., antibodies such as anti-CD 20 antibodies, IDEC8, immunotoxins, and cytokines).

In some embodiments, a method of treating cancer in a subject (e.g., a human) in need thereof, comprises administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, and one or more additional therapies selected from fludarabine, rituximab, obituzumab, alemtuzumab, cyclophosphamide, chlorambucil, doxorubicin hcl, vincristine sulfate, melphalan, busulfan, carmustine, prednisone, prednisolone, dexamethasone, methotrexate, cytarabine, mitoxantrone hcl, bortezomib, temsirolimus, carboplatin, etoposide, thalidomide, cisplatin, ruxizumab, anti-TRAIL, bevacizumab, galiximab, epratuzumab, SGN-40, bevacizumab, or a combination therapy, anti-CD 74, Aframucimumab, ha20, PRO131921, CHIR-12.12, apracluzumab, milnacumab, bevacizumab, yttrium-90-labeled ibritumomab tiuxetan, tositumomab, iodine-131 tositumomab, ifosfamide, GTOP-99 vaccine, Olimersen, fradapine, PD0332991, R-roscovitine, styrylsulfone, Obatoclax, TRAIL, anti-TRAIL DR4 and DR5 antibodies, everolimus, BMS-345541, curcumin, vorinostat, lenalidomide, geldanamycin, piperacillin, sildenafil citrate, CC-5103, simvastatin, enzastaurin, campath-1H, PACE, anti-keton A10, anti-ketoneas 2-1, beta-alethitine, filgrastimulin, interferon-10-alpha, thymocyte-14-albumin, MN 111-14-indium, Cyclosporin, mycophenolate mofetil, therapeutic allogeneic lymphocytes, tarolimus, thiotepa, paclitaxel, aldesleukin, docetaxel, ifosfamide, mesna, recombinant interleukin-12, recombinant interleukin-11, ABT-263, dinil-il, tanespimycin, everolimus, pegylated filgrastim, vorinostat, alvocidib, recombinant flt3 ligand, recombinant human thrombopoietin, lymphokine-activated killer cells, amifostine trihydrate, aminocamptothecin, irinotecan hydrochloride, caspofungin acetate, clofarabine, alfa ebergine, nelarabine, pentostatin, sargrastim, vinorelbine ditartrate, WT-1 analog peptide vaccine, WT 1126-134 peptide vaccine, fenvea amine, salpirone, oxaliplatin, monoclonal antibody CD19, Monoclonal antibodies CD20, omega-3 fatty acids, octreotide acetate, motoxafen gadolinium, arsenic trioxide, tipifarnib, autologous human tumor-derived HSPPC-96, vetuzumab, bryostatin 1, pegylated liposome hydrochloride, peripheral blood stem cell transplantation, autologous hematopoietic stem cell transplantation, autologous bone marrow transplantation, infused stem cells, bone marrow depletion of stem cell support, ex vivo processed peripheral blood stem cell transplantation, umbilical cord blood transplantation, low-LET cobalt-60 gamma ray therapy, bleomycin, conventional surgery, radiotherapy, and allogeneic hematopoietic stem cell transplantation without bone marrow eradication.

In some embodiments, the one or more additional therapies involve the use of a phosphatidylinositol 3-kinase (PI3K) inhibitor, including, for example, compound A, B or C, or a pharmaceutically acceptable salt of such compounds. The structures of compounds A, B and C are provided below.

In other embodiments of the methods described above involving the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with one or more additional therapies, the one or more additional therapies are different from the therapies with compound a, compound B, or compound C, or a pharmaceutically acceptable salt of these compounds. In one embodiment of the methods described above involving the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with one or more additional therapies, the one or more additional therapies are different from the therapy with compound a, or a pharmaceutically acceptable salt thereof. In yet another embodiment of the above methods involving the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with one or more additional therapies, the one or more additional therapies are different from the therapy with compound B, or a pharmaceutically acceptable salt thereof. In yet another embodiment of the methods described above involving the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with one or more additional therapies, the one or more additional therapies are different from the therapy with compound C, or a pharmaceutically acceptable salt thereof.

In other embodiments, the one or more additional therapeutic agents can be an inhibitor of lysyl oxidase-like protein 2(LOXL2) and a substance that binds LOXL2, including, for example, a humanized monoclonal antibody (mAb) of the immunoglobulin IgG4 isotype to human LOXL 2.

The compounds of formula I or pharmaceutically acceptable salts thereof are useful as chemosensitizers and, therefore, in combination with other chemotherapeutic agents, particularly agents that induce apoptosis.

The invention also provides a method of increasing the sensitivity of a cancer cell to chemotherapy, comprising administering to a subject (e.g., a human) receiving chemotherapy a chemotherapeutic agent and a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount sufficient to increase the sensitivity of the cancer cell to the chemotherapeutic agent. Examples of other chemotherapeutic agents that may be used in combination with the chemical entities described herein include topoisomerase I inhibitors (camptothecin or topotecan), topoisomerase II inhibitors (e.g., daunomycin and etoposide), alkylating agents (e.g., cyclophosphamide, melphalan, and BCNU), tubulin directing agents (e.g., taxol and vinblastine), and biologies (e.g., antibodies, such as anti-CD 20 antibodies, IDEC8, immunotoxins, and cytokines). In one embodiment of the method of treating cancer, the chemotherapeutic agent is different from compound a, or a pharmaceutically acceptable salt thereof. In another embodiment of the method for increasing the sensitivity of a cancer cell to chemotherapy, the chemotherapeutic agent is different from compound B or a pharmaceutically acceptable salt thereof. In yet another embodiment of the method for increasing the sensitivity of a cancer cell to chemotherapy, the chemotherapeutic agent is different from compound C or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof is combined with(rituximab) or other drugs that act by selectively depleting CD20+ B cells.

The present application includes a method of treating cancer, allergic and/or autoimmune diseases and/or inflammatory diseases and/or acute inflammatory responses comprising administering to a subject in need thereof an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in combination with an anti-inflammatory agent. Anti-inflammatory drugs include, but are not limited to, NSAIDs, non-specific and COX-2 specific cyclooxygenase inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptor antagonists, immunosuppressants, and methotrexate. Examples of NSAIDs include, but are not limited to, ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, a combination of diclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, nabumetone sodium, sulfasalazine, tolmetin sodium, and hydroxychloroquine. Examples of NSAIDs also include COX-2 specific inhibitors (i.e., compounds that have an IC50 that inhibits COX-2 that is at least 1/50-low to the IC50 that inhibits COX-1), such as celecoxib, valdecoxib, lumiracoxib, etoricoxib, and/or rofecoxib.

In other embodiments, the anti-inflammatory drug is a salicylate. Salicylates include, but are not limited to, acetylsalicylic acid or aspirin, sodium salicylate and choline salicylate and magnesium salicylate. The anti-inflammatory drug may also be a corticosteroid. For example, the corticosteroid may be selected from: cortisone, dexamethasone, methylprednisolone, prednisolone sodium phosphate, and prednisone. In some embodiments, the anti-inflammatory therapeutic agent is a gold compound, such as gold sodium thiomalate or auranofin. In some embodiments, the anti-inflammatory drug is a metabolic inhibitor, e.g., a dihydrofolate reductase inhibitor, e.g., methotrexate or a dihydroorotate dehydrogenase inhibitor, e.g., leflunomide.

In some embodiments, a combination is used wherein the at least one anti-inflammatory compound is an anti-C5 monoclonal antibody (e.g., eculizumab or pexelizumab), a TNF antagonist (e.g., etanercept), or infliximab, which is an anti-TNF α monoclonal antibody.

In some embodiments, a combination is used wherein at least one therapeutic agent is an immunosuppressive compound, such as methotrexate, leflunomide, cyclosporine, tacrolimus, azathioprine, or mycophenolate mofetil.

The present application also provides a method of treatment wherein a compound of formula I or a pharmaceutically acceptable salt thereof, administered to a subject (e.g., a human) who has been diagnosed as having or is suspected of having an autoimmune disease, is administered to the subject in combination with one or more anti-inflammatory or immunosuppressive agents selected from ibuprofen, flurbiprofen, naproxen sodium, diclofenac sodium, misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, calcium fenoprofen, ketoprofen, nabumetone sodium, sulfasalazine, tolmetin sodium, hydroxychloroquine, celecoxib, valdecoxib, lumiracoxib, etoricoxib, acetylsalicylic acid, sodium salicylate, choline salicylate, magnesium salicylate, cortisone, dexamethasone, sodium, and mixtures thereof, Methylprednisolone, prednisolone sodium phosphate, prednisone, gold sodium thiomalate, auranofin, methotrexate, leflunomide dihydroorotate, leflunomide, cyclosporine, talomim, azathioprine, mycophenolate mofetil, eculizumab, pekelizumab, etanercept, and infliximab.

The present invention provides that a therapeutic method of administering a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, to a subject (e.g., a human) is the only anticancer therapeutic regimen administered to the subject. The present invention provides therapeutic methods for administering a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, to a subject (e.g., a human), wherein the subject is not undergoing any other anti-cancer therapy. In one variation, the subject is not undergoing any other anti-cancer treatment with one or more PI3K inhibitors. In some embodiments, the PI3K inhibitor may include compounds A, B and C, the structures of which are provided below.

In one variation, the subject is not undergoing any other anti-cancer treatment with compound a or a pharmaceutically acceptable salt thereof. In a further variation, the subject is not undergoing any other anti-cancer treatment with compound B or a pharmaceutically acceptable salt thereof. In a further variation, the subject is not undergoing any other anti-cancer treatment with compound C or a pharmaceutically acceptable salt thereof.

In some embodiments, wherein the compound of formula I or a pharmaceutically acceptable salt thereof and the vinca alkaloid or a pharmaceutically acceptable salt thereof are administered to the subject, which may be a human, in a monotherapy treatment regimen, that (I) is refractory to at least one anti-cancer therapy, or (ii) relapses after treatment with at least one anti-cancer therapy, or a combination of (I) and (ii). In some embodiments, the subject is refractory to at least two, at least three, or at least four anti-cancer therapies (including, e.g., standard or experimental chemotherapies).

It will be appreciated that when a subject (e.g. a human) is treated with a compound of formula I or a pharmaceutically acceptable salt thereof as described herein in combination with a vinca alkaloid or a pharmaceutically acceptable salt thereof as a monotherapy treatment regimen, the subject may also be on one or more other therapies than anticancer therapy.

In some embodiments, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, wherein: the vinca alkaloid is selected from the group consisting of vincristine, vindesine, vinorelbine, and vinblastine, and the subject is a human that (i) is refractory to at least one anti-cancer therapy, or (ii) relapses after treatment with at least one anti-cancer therapy, or a combination thereof. In some other embodiments, there is provided a method of treating cancer in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, wherein the vinca alkaloid is selected from the group consisting of vincristine, vindesine, vinorelbine and vinblastine, and wherein further the subject is a human not undergoing any other anti-cancer treatment; and the subject is (i) refractory to at least one anti-cancer therapy, or (ii) relapsed after treatment with at least one anti-cancer therapy, or a combination thereof.

In some embodiments, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, wherein: the vinca alkaloid is selected from vincristine and vinblastine, and the subject is a human that (i) is refractory to at least one anti-cancer therapy, or (ii) relapses after treatment with at least one anti-cancer therapy, or a combination thereof. In some other embodiments, there is provided a method of treating cancer in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, wherein the vinca alkaloid is selected from the group consisting of vincristine and vinblastine, and wherein further, the subject is a human who has not undergone any other anti-cancer treatment; and the subject is (i) refractory to at least one anti-cancer therapy, or (ii) relapsed after treatment with at least one anti-cancer therapy, or a combination thereof.

In one embodiment, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, wherein: the compound of formula I is 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine, the vinca alkaloid is vincristine, and the subject is a human, which is (I) refractory to at least one anti-cancer therapy, or (ii) relapses after treatment with at least one anti-cancer therapy, or a combination thereof. In one other embodiment, there is provided a method of treating cancer in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, which is vincristine, and wherein further, the subject is a human who has not undergone any other anti-cancer treatment; and the subject is (i) refractory to at least one anti-cancer therapy, or (ii) relapsed after treatment with at least one anti-cancer therapy, or a combination thereof.

In one embodiment, there is provided a method of treating cancer in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine, the vinca alkaloid is vinblastine, and the subject is a human that is (I) refractory to at least one anti-cancer therapy, or (ii) relapses after treatment with at least one anti-cancer therapy, or a combination thereof. In one other embodiment, there is provided a method of treating cancer in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, which is vinblastine, and wherein further the subject is a human who has not undergone any other anti-cancer treatment; and the subject is (i) refractory to at least one anti-cancer therapy, or (ii) relapsed after treatment with at least one anti-cancer therapy, or a combination thereof.

In other embodiments, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, and the vinca alkaloid, or a pharmaceutically acceptable salt thereof, are administered to the subject in a monotherapy treatment regimen, the subject may have a 17p deletion, a TP53 mutation, NOTCH1, a SF3B1 mutation, an 11q deletion, or any combination thereof. In some embodiments, wherein the compound of formula I or a pharmaceutically acceptable salt thereof and the vinca alkaloid or a pharmaceutically acceptable salt thereof are administered to the subject in a monotherapy treatment regimen, the subject has a 17p deletion, a TP53 mutation, or a combination thereof. In additional embodiments, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, and the vinca alkaloid, or a pharmaceutically acceptable salt thereof, are administered to the subject in a monotherapy treatment regimen, the subject has a NOTCH1, a SF3B1 mutation, an 11q deletion, or any combination thereof.

The invention also provides methods of treatment in which a combination of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a vinca alkaloid, or a pharmaceutically acceptable salt thereof, administered to a subject (e.g., a human) is administered to the subject (e.g., a human) in combination with one or more additional therapies, including one or more of the anti-cancer therapies described above. Thus, in some embodiments, a method of treating cancer in a subject (e.g., a human) in need of treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in combination with a vinca alkaloid, or a pharmaceutically acceptable salt thereof, along with one or more additional therapies useful for treating cancer. The one or more additional therapies may involve the administration of one or more therapeutic agents as described herein.

For example, in other embodiments, the one or more additional therapeutic agents can be an inhibitor of lysyl oxidase-like protein 2(LOXL2) and a substance that binds LOXL2, including, for example, a humanized monoclonal antibody (mAb) of the immunoglobulin IgG4 isotype that is directed against human LOXL 2.

In other embodiments, the one or more additional therapeutic agents may be anti-inflammatory drugs. Treatment with one or more additional therapeutic agents may be performed prior to, concurrently with, or subsequent to treatment with the pharmaceutical compositions described herein. In some embodiments, the pharmaceutical compositions described herein are combined with another therapeutic agent in a single dosage form and then administered prior to, simultaneously with, or after administration of a vinca alkaloid disclosed herein, or a pharmaceutically acceptable salt thereof. Suitable anti-cancer drugs that may be used in combination with at least one chemical entity described herein include, but are not limited to, chemotherapeutic agents such as mitomycin C, carboplatin, taxol, cisplatin, paclitaxel, etoposide, doxorubicin, or a combination comprising at least one of the foregoing chemotherapeutic agents. Radiation therapy antineoplastic agents may also be used alone or in combination with chemotherapeutic agents.

It is to be understood that any combination of the above additional therapeutic agents can be used, as if each and every combination were listed individually. For example, in some embodiments, the additional therapeutic agent comprises a PI3K inhibitor and a LOXL2 inhibitor.

Pharmaceutical compositions and administration

The compounds of formula I or pharmaceutically acceptable salts thereof are typically administered in the form of pharmaceutical compositions. Accordingly, the present invention provides pharmaceutical compositions comprising as an active ingredient one or more compounds described herein, or a pharmaceutically acceptable salt, or a pharmaceutically acceptable ester thereof, in combination with one or more pharmaceutically acceptable vehicles such as excipients, carriers (including inert solid diluents and fillers), diluents (including sterile aqueous solutions and various organic solvents), penetration enhancers, solubilizers, and adjuvants. The pharmaceutical composition may be administered alone or in combination with other therapeutic agents. Such compositions are prepared in a manner well known in the Pharmaceutical art (see, e.g., Remington's Pharmaceutical Sciences, machine publishing Co., Philadelphia, PA 17th Ed. (1985); and Modern pharmaceuticals, Marcel Dekker, Inc.3rd Ed. (G.S. Bank & C.T. Rhodes, Eds.).

The pharmaceutical compositions may be administered in single or multiple doses by any acceptable mode of administration of the agents having similar utility, such as described in those patents and patent applications incorporated herein by reference, including rectal, buccal, intranasal, and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, topical, as an inhalant, or by implantation or coating devices such as stents, for example, or arterial insertion of a pillared polymer.

One mode of administration is parenteral administration, especially by injection. The compounds of formula I or pharmaceutically-acceptable salts thereof may be incorporated into forms for administration by injection including aqueous or oily suspensions or emulsions (with sesame, corn, cottonseed, or peanut oil), as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles. Aqueous solutions in saline are also routinely used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycols and the like (and suitable mixtures thereof), cyclodextrin derivatives and vegetable oils may also be used. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating a compound of the invention in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which provide a powder of the active ingredient plus the other desired ingredient from a previously sterile-filtered solution thereof. In some embodiments, for parenteral administration, sterile injectable solutions are prepared which contain a therapeutically effective amount (e.g., 0.1-1000mg) of a compound of formula I or a pharmaceutically acceptable salt thereof. It will be understood, however, that the amount of the compound actually administered will be determined by a physician, based on relevant factors, including the condition to be treated, the chosen route of administration, the actual compound administered and its associated activity, the age and weight and response of the individual subject, the severity of the subject's symptoms, and the like.

Oral administration is another route of administration of a compound of formula I or a pharmaceutically acceptable salt thereof. Administration may be by capsule or enteric coated tablet, etc. In preparing pharmaceutical compositions comprising a compound of formula I or a pharmaceutically acceptable salt thereof, the active ingredient is typically diluted by an excipient and/or encapsulated within such a carrier, which may be in the form of a capsule, sachet (sachet), paper or other container. When the excipient serves as a diluent, it may be in the form of a solid, semi-solid, or liquid material (as above), which serves as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

Some examples of suitable excipients in oral formulations include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, povidone, cellulose, sterile water, syrup, and methyl cellulose. The formulation may additionally comprise: lubricants such as talc, magnesium stearate and mineral oil; a wetting agent; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoate; a sweetener; and a flavoring agent.

The pharmaceutical compositions described herein can be formulated to provide rapid, slow or delayed release of the active ingredient upon administration to a subject by means known in the art. Controlled release drug delivery systems for oral administration include isotonic pump systems and dissolution systems (depot containing polymer coatings or drug-polymer matrix formulations). Examples of controlled release systems are disclosed in U.S. Pat. nos. 3,845,770; 4,326,525; 4,902,514, respectively; and 5,616,345. Another formulation for use in the methods of the invention utilizes a transdermal delivery device ("patch"). Such transdermal patches may be used to provide continuous or intermittent infusion of a compound of the invention in controlled amounts. The construction and use of transdermal patches for delivering drugs is known in the art. See, for example, U.S. Pat. nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be configured for continuous, pulsed or on demand (on demand) delivery of drugs.

In some embodiments, the compositions described herein are formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage forms for human patients and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient (e.g., a tablet, capsule, ampoule). The compounds are typically administered in a pharmaceutically effective amount. In some embodiments, for oral administration, each dosage unit comprises from about 1mg to about 5000mg, from about 1mg to about 4000mg, from about 1mg to about 3000mg, from about 1mg to about 2000mg, from about 2mg to about 2000mg, from about 5mg to about 2000mg, from about 10mg to about 2000mg, from about 1mg to about 1000mg, from about 2mg to about 1000mg, from about 5mg to about 1000mg, from about 10mg to about 1000mg, from about 25mg to about 1000mg, from about 50mg to about 1000mg, from about 75mg to about 1000mg, from about 100mg to about 1000mg, from about 125mg to about 1000mg, from about 150mg to about 1000mg, from about 175mg to about 1000mg, from about 200mg to about 1000mg, from about 225mg to about 1000mg, from about 250mg to about 1000mg, from about 300mg to about 1000mg, from about 350mg to about 1000mg, from about 400mg to about 1000mg, from about 450mg to about 1000mg, from about 500mg to about 1000mg, from about 1000mg, About 750mg to about 1000mg, about 800mg to about 1000mg, about 850mg to about 1000mg, about 900mg to about 1000mg, about 950mg to about 1000mg, about 1mg to about 750mg, about 2mg to about 750mg, about 5mg to about 750mg, about 10mg to about 750mg, about 25mg to about 750mg, about 50mg to about 750mg, about 75mg to about 750mg, about 100mg to about 750mg, about 125mg to about 750mg, about 150mg to about 750mg, about 175mg to about 750mg, about 200mg to about 750mg, about 225mg to about 750mg, about 250mg to about 750mg, about 300mg to about 750mg, about 350mg to about 750mg, about 400mg to about 750mg, about 450mg to about 750mg, about 500mg to about 750mg, about 550mg to about 750mg, about 600mg to about 750mg, about 650mg to about 700mg, about 1mg to about 750mg, about 500mg to about 500mg, about 500mg to about 750mg, about 500mg to about 750mg, About 50mg to about 500mg, about 75mg to about 500mg, about 100mg to about 500mg, about 125mg to about 500mg, about 150mg to about 500mg, about 175mg to about 500mg, about 200mg to about 500mg, about 225mg to about 500mg, about 250mg to about 500mg, about 300mg to about 500mg, about 350mg to about 500mg, about 400mg to about 500mg, about 450mg to about 500mg, about 1mg to about 400mg, about 2mg to about 400mg, about 5mg to about 400mg, about 10mg to about 400mg, about 25mg to about 400mg, about 50mg to about 400mg, about 75mg to about 400mg, about 100mg to about 400mg, about 125mg to about 400mg, about 150mg to about 400mg, about 175mg to about 400mg, about 200mg to about 400mg, about 225mg to about 400mg, about 250mg to about 400mg, about 300mg to about 300mg, about 1mg to about 300mg, about 300mg to about 400mg, about 300mg to about 400mg, About 25mg to about 300mg, about 50mg to about 300mg, about 75mg to about 300mg, about 100mg to about 300mg, about 125mg to about 300mg, about 150mg to about 300mg, about 175mg to about 300mg, about 200mg to about 300mg, about 225mg to about 300mg, about 250mg to about 300mg, about 1mg to about 250mg, about 2mg to about 250mg, about 5mg to about 250mg, about 10mg to about 250mg, about 25mg to about 250mg, about 50mg to about 250mg, about 75mg to about 250mg, about 100mg to about 250mg, about 125mg to about 250mg, about 150mg to about 250mg, about 175mg to about 250mg, about 200mg to about 250mg, about 225mg to about 250mg, about 1mg to about 225mg, about 2mg to about 225mg, about 5mg to about 225mg, about 10mg to about 225mg, about 25mg to about 225mg, about 50mg to about 225mg, about 225mg to about 225mg, About 175mg to about 225mg, about 200mg to about 225mg, about 1mg to about 200mg, about 2mg to about 200mg, about 5mg to about 200mg, about 10mg to about 200mg, about 25mg to about 200mg, about 50mg to about 200mg, about 75mg to about 200mg, about 100mg to about 200mg, about 125mg to about 200mg, about 150mg to about 200mg, about 175mg to about 200mg, about 180mg to about 200mg, about 1mg to about 175mg, about 2mg to about 175mg, about 5mg to about 175mg, about 10mg to about 175mg, about 25mg to about 175mg, about 50mg to about 175mg, about 75mg to about 175mg, about 100mg to about 200mg, about 125mg to about 175mg, about 150mg to about 175mg, about 1mg to about 150mg, about 2mg to about 150mg, about 5mg to about 150mg, about 10mg to about 150mg, about 150mg to about 150mg, about 100mg to about 100mg, about 100mg to about 150mg, about 100mg to about 100mg, about 100mg to about 175mg, about 100mg, From about 1mg to about 125mg, from about 2mg to about 125mg, from about 5mg to about 125mg, from about 10mg to about 125mg, from about 25mg to about 125mg, from about 50mg to about 125mg, from about 75mg to about 125mg, from about 100mg to about 125mg, from about 1mg to about 100mg, from about 2mg to about 100mg, from about 5mg to about 100mg, from about 10mg to about 100mg, from about 25mg to about 100mg, from about 50mg to about 100mg, or from about 75mg to about 100mg of a compound of formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, for oral administration, each dosage unit comprises about 1mg, about 2mg, about 5mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 75mg, about 100mg, about 125mg, about 150mg, about 175mg, about 180mg, about 200mg, about 225mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, about 600mg, about 650mg, about 700mg, about 750mg, about 800mg, about 850mg, about 900mg, about 950mg, or about 1000mg of a compound of formula I, or a pharmaceutically acceptable salt thereof.

The above-mentioned doses for oral administration may be administered once daily (QD) or twice daily (BID). In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally in the following unit doses: about 1mg QD, about 2mg QD, about 5mg QD, about 10mg QD, about 15mg QD, about 20mg QD, about 25mg QD, about 30mg QD, about 35mg QD, about 40mg QD, about 45mg QD, about 50mg QD, about 60mg QD, about 65mg QD, about 70mg QD, about 75mg QD, about 80mg QD, about 90mg QD, about 100mg QD, about 125mg QD, about 150mg QD, about 175mg QD, about 180mg QD, about 200mg QD, about 225mg QD, about 250mg QD, about 300mg QD, about 350mg QD, about 400mg QD, about 450mg QD, about 500mg QD, about 550mg QD, about 600mg QD, about 650mg QD, about 700mg QD, about 750mg QD, about 800mg QD, about 850mg QD, about 900mg QD, about 950mg QD or about 1000mg QD. In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered orally in the following unit doses: about 1mg BID, about 2mg BID, about 5mg BID, about 10mg BID, about 15mg BID, about 20mg BID, about 25mg BID, about 30mg BID, about 35mg BID, about 40mg BID, about 45mg BID, about 50mg BID, about 75mg BID, about 80mg BID, about 90mg BID, about 100mg BID, about 125mg BID, about 150mg BID, about 175mg BID, about 200mg BID, about 225mg BID, about 250mg BID, about 300mg BID, about 350mg BID, about 400mg BID, about 450mg BID, about 500mg BID, about 550mg BID, about 600mg BID, about 650mg BID, about 700mg BID, about 750mg BID, about 800mg BID, about 850mg BID, about 900mg BID, about 950mg BID or about 1000mg BID.

In some embodiments, for parenteral administration, each dosage unit contains from 0.1mg to 1g, from 0.1mg to 700mg, or from 0.1mg to 100mg of a compound of formula I, or a pharmaceutically acceptable salt thereof.

However, for any dosage unit described herein, it will be understood that the amount of the compound actually administered will generally be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age and weight and response of the individual subject, the severity of the subject's symptoms, and the like.

To prepare solid compositions, such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition comprising a homogeneous mixture of a compound of formula I, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, this means that the active ingredient is dispersed throughout the composition uniformly and the composition may thus conveniently be subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills described herein may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action or resistance to the acidic conditions found in the stomach. For example, the tablet or pill may comprise an inner dosage component and an outer dosage component, the latter being in the form of an envelope over the former. The two components may be separated by an enteric coating layer, preventing disintegration in the stomach and allowing the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials may be used for such enteric coatings or coatings, such materials including polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

Compositions for inhalation or insufflation may include solutions and suspensions, and powders in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof. The liquid or solid composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof may comprise suitable pharmaceutically acceptable excipients as described herein. Preferably, the composition is administered by the oral or intranasal respiratory route for local or systemic effect. The composition in preferably a pharmaceutically acceptable solvent may be atomized by the use of an inert gas. The nebulized solution can be inhaled directly from the nebulizing device or the nebulizing device can be connected to a mask support or an intermittent positive pressure ventilator. The solution, suspension or powder composition may be administered from a device which delivers the formulation in a suitable manner, preferably orally or intranasally.

Dosing regimens

In the methods provided herein, a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered in a therapeutically effective amount to achieve the intended purpose. Determination of a therapeutically effective amount is within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. In some embodiments (methods of treating cancer), a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, can (I) reduce the number of cancer cells; (ii) reducing the size of the tumor; (iii) inhibit, delay, slow to some extent, and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; (v) inhibiting tumor growth; (vi) delay of appearance and/or recurrence of tumors; and/or (vii) alleviate one or more symptoms associated with cancer to some extent. In various embodiments, the amount is sufficient to ameliorate, alleviate, reduce and/or delay one or more symptoms of cancer.

The therapeutically effective amount may vary depending on the subject and the disease or condition being treated, the weight and age of the subject, the severity of the disease or condition, and the manner of administration, which can be readily determined by one of ordinary skill in the art.

In the methods provided herein, the dosage regimen of a compound of formula I, or a pharmaceutically acceptable salt thereof, may vary depending on, for example, the indication, the route of administration, and the severity of the condition. Depending on the route of administration, suitable dosages may be calculated based on body weight, body surface area, or organ size. The final dosing regimen is determined by the attending physician in view of good medical practice, considering various factors that alter the action of the drug, such as the specific activity of the compound, the identity and severity of the disease, the responsiveness of the subject, the age, condition, body weight, sex and diet of the subject, and the severity of any infection. Additional factors that may be considered include time and frequency of administration, drug combination, severity of the response, and tolerance/response to therapy. Further refinement of dosages suitable for treatment involving any of the formulations mentioned herein is routinely made by the skilled practitioner without undue experimentation, especially in light of the dosage information and tests disclosed, as well as the pharmacokinetic data observed in human clinical trials. Suitable dosages may be determined by using established tests for determining the concentration of the drug in a bodily fluid or other sample, as well as dose response data.

The formulation and route of administration selected may be tailored to the individual subject, the nature of the condition to be treated in the subject, and the judgment of the attending physician generally. For example, the therapeutic index of a compound of formula I, or a pharmaceutically acceptable salt thereof, can be enhanced by: improving or derivatizing the compound for target delivery to a cancer cell that expresses a marker thereby identifying the cancer cell. For example, the compounds may be linked to antibodies that recognize markers selective or specific for cancer cells, such that the compounds come into proximity of the cells to exert their effect locally, as described previously. See, e.g., Pieteersz et al, Immunol. Rev.,129:57 (1992); trail et al Science,261:212 (1993); and curr. opin. oncol.,4:1142(1992) by Rowlinson-Busza et al.

A therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, can be provided in a single dose or in multiple doses to achieve the desired therapeutic endpoint. As used herein, "dose" refers to the total amount of active ingredient (e.g., a compound of formula I or a pharmaceutically acceptable salt thereof) taken by a subject (e.g., a human) each time. The dose administered (e.g., the dose for oral administration described above) may be administered once daily (QD), twice daily (BID), three times daily, four times daily, or more than four times daily. In some embodiments, the dose of the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered once daily. In some embodiments, the dose of the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered twice daily.

In some embodiments, exemplary dosages of a compound of formula I, or a pharmaceutically acceptable salt thereof, for a human subject may be from about 1mg to about 5000mg, from about 1mg to about 4000mg, from about 1mg to about 3000mg, from about 1mg to about 2000mg, from about 2mg to about 2000mg, from about 5mg to about 2000mg, from about 10mg to about 2000mg, from about 1mg to about 1000mg, from about 2mg to about 1000mg, from about 5mg to about 1000mg, from about 10mg to about 1000mg, from about 25mg to about 1000mg, from about 50mg to about 1000mg, from about 75mg to about 1000mg, from about 100mg to about 1000mg, from about 125mg to about 1000mg, from about 150mg to about 1000mg, from about 175mg to about 1000mg, from about 200mg to about 1000mg, from about 225mg to about 1000mg, from about 250mg to about 1000mg, from about 300mg to about 1000mg, from about 350mg to about 1000mg, from about 400mg to about 1000mg, from about 450mg to about 1000mg, from about 1000mg to about 1000mg, About 650mg to about 1000mg, about 700mg to about 1000mg, about 750mg to about 1000mg, about 800mg to about 1000mg, about 850mg to about 1000mg, about 900mg to about 1000mg, about 950mg to about 1000mg, about 1mg to about 750mg, about 2mg to about 750mg, about 5mg to about 750mg, about 10mg to about 750mg, about 25mg to about 750mg, about 50mg to about 750mg, about 75mg to about 750mg, about 100mg to about 750mg, about 125mg to about 750mg, about 150mg to about 750mg, about 175mg to about 750mg, about 200mg to about 750mg, about 225mg to about 750mg, about 250mg to about 750mg, about 300mg to about 750mg, about 350mg to about 750mg, about 400mg to about 750mg, about 450mg to about 750mg, about 500mg to about 750mg, about 550mg to about 600mg, about 650mg to about 750mg, about 500mg, about 750mg to about 750mg, about 500mg to about 750mg, about 400mg to about 750mg, about 450mg to about 750mg, About 10mg to about 500mg, about 25mg to about 500mg, about 50mg to about 500mg, about 75mg to about 500mg, about 100mg to about 500mg, about 125mg to about 500mg, about 150mg to about 500mg, about 175mg to about 500mg, about 200mg to about 500mg, about 225mg to about 500mg, about 250mg to about 500mg, about 300mg to about 500mg, about 350mg to about 500mg, about 400mg to about 500mg, about 450mg to about 500mg, about 1mg to about 400mg, about 2mg to about 400mg, about 5mg to about 400mg, about 10mg to about 400mg, about 25mg to about 400mg, about 50mg to about 400mg, about 75mg to about 400mg, about 100mg to about 400mg, about 125mg to about 400mg, about 150mg to about 400mg, about 175mg to about 400mg, about 200mg to about 400mg, about 225mg to about 400mg, about 400mg to about 400mg, about 1mg to about 300mg, about 400mg to about 400mg, about 300mg to about 400mg, about 400mg, About 5mg to about 300mg, about 10mg to about 300mg, about 25mg to about 300mg, about 50mg to about 300mg, about 75mg to about 300mg, about 100mg to about 300mg, about 125mg to about 300mg, about 150mg to about 300mg, about 175mg to about 300mg, about 200mg to about 300mg, about 225mg to about 300mg, about 250mg to about 300mg, about 1mg to about 250mg, about 2mg to about 250mg, about 5mg to about 250mg, about 10mg to about 250mg, about 25mg to about 250mg, about 50mg to about 250mg, about 75mg to about 250mg, about 100mg to about 250mg, about 125mg to about 250mg, about 150mg to about 250mg, about 200mg to about 250mg, about 225mg to about 250mg, about 1mg to about 225mg, about 2mg to about 225mg, about 5mg to about 225mg, about 10mg to about 225mg, about 225mg to about 225mg, about 225mg, About 125mg to about 225mg, about 150mg to about 225mg, about 175mg to about 225mg, about 200mg to about 225mg, about 1mg to about 200mg, about 2mg to about 200mg, about 5mg to about 200mg, about 10mg to about 200mg, about 25mg to about 200mg, about 50mg to about 200mg, about 75mg to about 200mg, about 100mg to about 200mg, about 125mg to about 200mg, about 150mg to about 200mg, about 175mg to about 200mg, about 180mg to about 200mg, about 1mg to about 175mg, about 2mg to about 175mg, about 5mg to about 175mg, about 10mg to about 175mg, about 25mg to about 175mg, about 50mg to about 200mg, about 75mg to about 175mg, about 100mg to about 175mg, about 125mg to about 175mg, about 150mg to about 175mg, about 1mg to about 150mg, about 2mg to about 150mg, about 150mg to about 150mg, about 50mg to about 150mg, about 150mg to about 150mg, about 25mg to about 200mg, about 1mg to about 175mg, About 100mg to about 150mg, about 125mg to about 150mg, about 1mg to about 125mg, about 2mg to about 125mg, about 5mg to about 125mg, about 10mg to about 125mg, about 25mg to about 125mg, about 50mg to about 125mg, about 75mg to about 125mg, about 100mg to about 125mg, about 1mg to about 100mg, about 2mg to about 100mg, about 5mg to about 100mg, about 10mg to about 100mg, about 25mg to about 100mg, about 50mg to about 100mg, about 60mg to about 100mg, or about 75mg to about 100 mg.

In some embodiments, exemplary doses of a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in a human subject may be about 1mg, about 2mg, about 5mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 60mg, about 65mg, about 70mg, about 75mg, about 100mg, about 125mg, about 150mg, about 175mg, about 180mg, about 190mg, about 200mg, about 225mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, about 600mg, about 650mg, about 700mg, about 750mg, about 800mg, about 850mg, about 900mg, about 950mg, about 1000mg, about 1200mg, about 1400mg, about 1600mg, about 1800mg, about 2000mg, about 2200mg, about 2600mg, about 2800mg, about 3200mg, about 3000mg, about 1000mg, about 3000mg, about 200mg, about 1400mg, about 200mg, about 1400, About 4200mg, about 4400mg, about 4600mg, about 4800mg, or about 5000 mg.

In other embodiments, provided methods include continuing to treat a subject (e.g., a human) by: the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered at a dose to achieve a clinical effect, or the dose is gradually reduced at a level to maintain the effect. In some embodiments, provided methods comprise administering to the subject (e.g., a human) an initial daily dose of 50mg to about 300mg of a compound of formula I, or in further embodiments, an initial daily dose of 100mg to 1000mg of a compound of formula I, or a pharmaceutically acceptable salt thereof, and administering subsequent daily doses of a compound of formula I, or a pharmaceutically acceptable salt thereof, for at least 6 days, wherein each subsequent daily dose is increased by 25mg to 300mg, or 50mg to about 400 mg. Thus, it is also understood that the dosage of a compound of formula I, or a pharmaceutically acceptable salt thereof, may be gradually increased until a clinical effect is achieved. The dosage may be increased using increments of about 10mg, about 25mg, about 50mg, about 100mg, or about 125mg, or about 150mg, or about 200mg, or about 250mg, or about 300 mg. The dose may be increased daily, every other day, twice, three, four, five or six times per week, or once per week.

The frequency of administration will depend on the pharmacokinetic parameters of the compound being administered, the route of administration and the particular disease being treated. The dose and frequency of administration may also depend on pharmacokinetic and pharmacodynamic data as well as toxicity and efficacy data. For example, pharmacokinetic and pharmacodynamic information about a compound of formula I, or a pharmaceutically acceptable salt thereof, can be collected by preclinical in vitro and in vivo studies, and then validated in humans during clinical trials. Thus, for a compound of formula I, or a pharmaceutically acceptable salt thereof, to be used in the methods provided herein, a therapeutically effective dose can be initially estimated from biochemical and/or cell-based assays. The dose can then be formulated in animal models to achieve the desired circulating concentration range that modulates Syk expression or activity. As human studies progress, further information will emerge regarding appropriate dosage levels and treatment durations for various diseases and conditions.

Toxicity and efficacy of a compound of formula I, or a pharmaceutically-acceptable salt thereof, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(50% population lethal dose) and ED₅₀(50% of the population therapeutically effective dose). The dose ratio between toxic and therapeutic effects is the "therapeutic index", which is usually expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit a large therapeutic index (i.e., a toxic dose significantly higher than the effective dose) are preferred. The data obtained from such cell culture tests and additional animal studies can be used in a range of dosages formulated for human use. The dosage of such compounds is preferably such that the dose includes an ED having little or no toxicity₅₀In the circulating concentration range of (c).

In the methods provided herein, a compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof is administered in a therapeutically effective amount to achieve the intended purpose. As used herein, when referring to a vinca alkaloid or a pharmaceutically acceptable salt thereof, a "therapeutically effective amount" is an amount sufficient to inhibit tubulin growth or formation, or inhibit or reduce microtubule formation, or interfere with spindle formation, thereby treating a subject (e.g., a human) with an indication or ameliorating or alleviating an existing symptom of an indication. For example, a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, can be an amount that: which is sufficient to reduce the symptoms of a disease or disorder responsive to inhibition of tubulin activity and/or formation.

A therapeutically effective amount of a vinca alkaloid or a pharmaceutically acceptable salt thereof according to the present invention may be provided in a single dose or in multiple doses to achieve a desired therapeutic endpoint. As used herein, "dose" refers to the total amount of active ingredient (e.g., vincristine or vinblastine or a pharmaceutically acceptable salt thereof) used by a subject (e.g., a human) each time.

Examples of the vinca alkaloids of the invention or pharmaceutically acceptable salts thereof for human subjects, depending on the identity of the vinca alkaloidThe sexual dose may be about 0.01mg-M²To about 3.0mg-M²Or about 0.01mg-M²To about 2.5mg-M²Or about 0.01mg-M²To about 2.0mg-M²Or about 0.01mg-M²To about 1.9mg-M²Or about 0.01mg-M²To about 1.8mg-M²Or about 0.01mg-M²To about 1.7mg-M²Or about 0.01mg-M²To about 1.6mg-M²Or about 0.01mg-M²To about 1.5mg-M²Or about 0.01mg-M²To about 1.4mg-M²Or about 0.01mg-M²To about 1.3mg-M ²Or about 0.01mg-M²To about 1.2mg-M²Or about 0.01mg-M²To about 1.1mg-M²Or about 0.01mg-M²To about 1.0mg-M²Or about 0.01mg-M²To about 0.9mg-M²Or about 0.01mg-M²To about 0.8mg-M²Or about 0.01mg-M²To about 0.7mg-M²Or about 0.01mg-M²To about 0.6mg-M²Or about 0.01mg-M²To about 0.5mg-M²Or about 0.01mg-M²To about 0.45mg-M²Or about 0.01mg-M²To about 0.4mg-M²Or about 0.01mg-M²To about 0.35mg-M²Or about 0.01mg-M²To about 0.33mg-M²Or about 0.01mg-M²To about 0.3mg-M²Or about 0.01mg-M²To about 0.25mg-M²Or about 0.01mg-M²To about 0.2mg-M²Or about 0.01mg-M²To about 0.15mg-M²Or about 0.01mg-M²To about 0.01mg-M²Or about 0.1mg-M²To about 1.8mg-M²Or about 0.15mg-M²To about 1.7mg-M²Or about 0.2mg-M²To about 1.6mg-M²Or about 0.25mg-M²To about 1.5mg-M²Or about 0.3mg-M²To about 1.4mg-M²Or about 0.33mg-M²To about 1.3mg-M²Or about 0.35mg-M²To about 1.2mg-M²Or about 0.4mg-M²To about 1.1mg-M²Or about 0.45mg-M²To about 1.0mg-M²Or about 0.5mg-M²To about 0.9mg-M²Or about 0.6mg-M²To about 0.8mg-M². In one embodiment, the dose of the vinca alkaloid or a pharmaceutically acceptable salt thereof of the present invention administered to the subject in the methods of the present invention is about 1.5mg-M². In one embodiment, the dose of the vinca alkaloid or a pharmaceutically acceptable salt thereof of the present invention administered to the subject in the methods of the present invention is about 1.0mg-M². In one embodiment, the dose of the vinca alkaloid or a pharmaceutically acceptable salt thereof of the present invention administered to the subject in the methods of the present invention is about 0.5mg-M²。

In other embodiments, provided methods include continuing to treat a subject (e.g., a human) by: the vinca alkaloid, or a pharmaceutically acceptable salt thereof, of the present invention is administered at a dose that achieves a clinical effect, or the dose is gradually reduced at a level that maintains the effect. The frequency of administration will depend on the pharmacokinetic parameters of the compound being administered, the route of administration and the particular disease being treated. The dose and frequency of administration may also depend on pharmacokinetic and pharmacodynamic data as well as toxicity and efficacy data.

Said vinca alkaloid of the present invention or a pharmaceutically acceptable salt thereof is administered IV. In one embodiment, the vinca alkaloid is vincristine sulfate and the amount in the vial is 1mg/1 ml. In some embodiments, the vinca alkaloid is vincristine sulfate, and the vial is 2ml containing 1mg or 2mg vincristine sulfate. In another embodiment, the "vincristine sulfate" of the united states pharmacopeia is a white to off-white powder. It is soluble in methanol, readily soluble in water, but only slightly soluble in 95% ethanol. Vincristine sulfate from the united states pharmacopoeia has a uv spectrum with a maximum at 221nm (e +47,100) in 98% ethanol.

The "vincristine sulfate injection" of the united states pharmacopeia is a sterile, preservative-free, single-use solution for intravenous injection in 2mL (1mg and 2mg) vials. Each mL of the solution contains 1mg of vincristine sulfate in United states Pharmacopeia, 100mg of mannitol and water for injection in United states Pharmacopeia. Q.s. sulfuric acid or sodium hydroxide was added to control pH. Vincristine sulfate injection, USP at a pH of 4.0 to 5.0.

Administration of a compound of formula I or a pharmaceutically acceptable salt thereof may be carried out under fed conditions. The term eating condition or variant thereof refers to the ingestion or ingestion of food or any suitable form of calories in solid or liquid form prior to or simultaneously with the administration of the compound or pharmaceutical composition thereof. For example, a compound of formula I or a pharmaceutically acceptable salt thereof can be administered to a subject (e.g., a human) within minutes or hours of ingestion of calories (e.g., a meal). In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, can be administered to a subject (e.g., a human) within 5-10 minutes, about 30 minutes, or about 60 minutes of caloric intake.

Article and kit

Compositions (including, for example, formulations and unit doses) comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, can be prepared and placed in an appropriate container and labeled for treatment of the indicated condition. Thus, also provided are articles of manufacture, such as a container comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, in unit dosage form, and a label containing instructions for use of the compound. In some embodiments, the article of manufacture is a container comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, in unit dosage form, and at least one pharmaceutically acceptable vehicle. The product may be a bottle, vial, ampoule, disposable applicator (applicator), or the like containing the pharmaceutical composition provided herein. The container may be made of a variety of materials, such as glass or plastic, and in one aspect also contains a label on or associated with the container that indicates instructions for use in treating cancer or an inflammatory condition. It will be appreciated that the active ingredient may be packaged in any material that is capable of increasing chemical and physical stability, for example, an aluminium foil pouch. In some embodiments, the disease or condition shown on the label may include, for example, treatment of cancer.

In another embodiment, the article of manufacture is a container comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, in unit dosage form, and at least one pharmaceutically acceptable vehicle, and a vial comprising a vinca alkaloid or a pharmaceutically acceptable salt.

Any of the pharmaceutical compositions provided herein can be used in a preparation as if each composition were specifically and individually enumerated for use in a preparation.

Also provided are kits comprising a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof. For example, a kit can comprise a compound of formula I, or a pharmaceutically acceptable salt thereof, in unit dosage form and a package insert comprising instructions for use of the composition in the treatment of a medical condition. In some embodiments, the kit comprises a compound of formula I, or a pharmaceutically acceptable salt thereof, in unit dosage form, and at least one pharmaceutically acceptable vehicle. In some embodiments, the kit comprises a compound of formula I, or a pharmaceutically acceptable salt thereof, in unit dosage form, and at least one pharmaceutically acceptable vehicle, and a vial comprising a solution of vinca alkaloid, or a pharmaceutically acceptable salt thereof. The instructions for use in the kit can be used to treat cancer, including, for example, hematological malignancies. In some embodiments, the instructions are directed to the use of the pharmaceutical composition for treating cancer, such as leukemia or lymphoma, including relapsed and refractory leukemia or lymphoma. In some embodiments, the instructions for use in the kit can be used to treat a hematologic cancer selected from small lymphocytic lymphoma, non-hodgkin's lymphoma, indolent non-hodgkin's lymphoma, refractory non-hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, marginal zone lymphoma, immunoblastic large cell lymphoma, lymphoblastic lymphoma, splenic marginal zone B cell lymphoma (+/-villous lymphocytes), nodal marginal zone lymphoma (+/-monocytic B cells), extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type, cutaneous T cell lymphoma, extranodal T cell lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, mycosis fungoides, B cell lymphoma, diffuse large B cell lymphoma, chronic lymphocytic leukemia, Mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, small non-dividing cell lymphoma, burkitt's lymphoma, multiple myeloma, plasmacytoma, acute lymphocytic leukemia, acute T-cell lymphoblastic leukemia, acute B-cell lymphocytic leukemia, B-cell prolymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, juvenile myelomonocytic leukemia, minimal residual disease, hairy cell leukemia, primary myelofibrosis, secondary myelofibrosis, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorders, and waldenstrom's macroglobulinemia. In one embodiment, the instructions for use in the kit can be used to treat chronic lymphocytic leukemia or non-hodgkin's lymphoma. In one embodiment, the NHL is diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, and marginal zone lymphoma. In one embodiment, the hematologic malignancy is indolent non-hodgkin's lymphoma. In some embodiments, the disease or condition shown on the label may include, for example, cancer treatment.

In some cases, the instructions relate to the use of the pharmaceutical composition for the treatment of a solid tumor, wherein the solid tumor is a cancer selected from the group consisting of: pancreatic cancer, urinary organ cancer, bladder cancer, colorectal cancer, colon cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, thyroid cancer, gallbladder cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), ovarian cancer, cervical cancer, gastric cancer, endometrial cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain tumors (e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma), bone cancer, soft tissue sarcoma, retinoblastoma, neuroblastoma, peritoneal leakage, malignant pleural effusion, mesothelioma, wilms 'tumor, trophoblastic tumors, vascular pericellular tumors, kaposi's sarcoma, mucus cancer, round cell carcinoma, squamous cell carcinoma, esophageal squamous cell carcinoma, oral cancer, adrenal cortical cancer, ACTH producing tumors.

In some cases, the instructions relate to the use of the pharmaceutical composition for the treatment of an allergic disease and/or an autoimmune disease and/or an inflammatory disease and/or an acute inflammatory response. In some embodiments, the instructions relate to the use of the pharmaceutical composition for treating an autoimmune disease. In some embodiments, the instructions relate to the use of the pharmaceutical composition for treating an autoimmune disease selected from the group consisting of: systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, Sjogren's syndrome, psoriasis, autoimmune hemolytic anemia, asthma, ulcerative colitis, Crohn's disease, irritable bowel disease, and chronic obstructive pulmonary disease. In some embodiments, the autoimmune disease is selected from asthma, rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, and systemic lupus erythematosus.

Any of the pharmaceutical compositions provided herein can be used in a kit as if each composition were specifically and individually listed for use in a kit.

Synthesis of

The compounds of the present invention can be prepared using the methods disclosed herein and conventional variations apparent in light of the present disclosure as well as methods well known in the art. Conventional well-known synthetic methods may also be used in addition to those taught in the present application. The synthesis of a typical compound of formula I, or a pharmaceutically acceptable salt thereof, may be effected as described in the examples below. Reagents may be purchased commercially, for example, from Sigma Aldrich or other chemical suppliers, if commercially available.

General Synthesis

Typical embodiments of the compounds of the present invention may be synthesized using the general reaction schemes described below. It is evident in view of the description of the present application that this general synthetic route can be varied by replacing the starting materials by other materials having a similar structure, thus obtaining different products accordingly. The following synthetic description gives a number of examples of how the starting materials may be varied to give the corresponding products. In the case of the desired products defined by a given substituent group, the necessary starting materials can generally be determined by inspection. The starting materials are typically obtained from commercial sources or synthesized using published methods. In order that compounds of embodiments of the present invention may be synthesized, examining the structure of the compound that needs to be synthesized will provide for the determination of the individual substituents. The determination of the end product usually makes the determination of the necessary starting materials apparent by means of simple inspection procedures (in view of the examples given in this application).

Parameters of the Synthesis reaction

The compounds of the present invention may be prepared from readily available starting materials using, for example, the following general procedures and procedures. It is understood that where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reagents, solvents, pressures, etc.) are given, other process conditions may also be used unless otherwise indicated. The optimized reaction conditions may vary with the particular reagents or solvents used, but such conditions may be determined by one skilled in the art through routine optimization steps.

Furthermore, it will be apparent to those skilled in the art that conventional protecting groups may be necessary to prevent certain functional groups from undergoing unwanted reactions. Suitable protecting groups for various functional groups and conditions for protecting and deprotecting specific functional groups are well known in the art. For example, various Protecting Groups are described in T.W.Greene and G.M.Wuts (1999) Protecting Groups in Organic Synthesis,3rd Edition, Wiley, New York, which is incorporated herein by reference.

Furthermore, the compounds of the present invention may contain chiral centers. Accordingly, such compounds may be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or stereoisomerically enriched mixtures, if desired. All such stereoisomers (and enriched mixtures) are within the scope of the invention unless otherwise specified. Pure stereoisomers (or enriched mixtures) may be prepared, for example, using optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated, for example, using chiral column chromatography, chiral resolving agents, and the like.

The starting materials for the following reactions are generally known compounds or can be prepared by known methods or obvious variations thereof. For example, many starting materials are commercially available from suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA). Other starting materials can be prepared by procedures described in standard reference texts or obvious variations thereof, such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15(John Wiley, and Sons,1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and supplements (Elsevier Science Publishers,1989) Organic reactions, Volumes1-40(John Wiley, and Sons,1991), March's Advanced Organic Chemistry, (John Wiley, and Sons,5th Edition 2001), and Larock's comprehensive Organic Transformations (VCH publications, Inc., 1989).

The terms "solvent", "inert organic solvent" or "inert solvent" refer to a solvent (including, for example, benzene, toluene, acetonitrile, tetrahydrofuran ("THF"), dimethylformamide ("DMF"), chloroform, dichloromethane (methylene chloride or dichloromethane)), diethyl ether, methanol, pyridine, and the like) that is inert under the reaction conditions described therewith. Unless specified to the contrary, the solvent used in the reaction of the present invention is an inert organic solvent, and the reaction is carried out in an inert gas (preferably nitrogen).

The term "q.s." means adding an amount sufficient to achieve the function, for example, adjusting the solution to the desired volume (i.e. 100%).

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be understood to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

List of abbreviations and acronyms

Meaning of abbreviations

DEG C

anal of

ATP adenosine-5' -triphosphate

ATX II Mediterranean anemone toxin

AcOH acetic acid

ACN acetonitrile

CAN ammonium ceric nitrate

CDI 1,1' -carbonyldiimidazole

CHO Chinese hamster ovary

con. concentration

d doublet peak

DABCO 1, 4-diazabicyclo [2.2.2] octane

DAST (diethylamino) sulfur trifluoride

dd doublet of doublets

DCE 1, 2-dichloroethane

DCM dichloromethane

DEAD azodicarboxylic acid diethyl ester

DIPEA N, N-diisopropylethylamine

Meaning of abbreviations

DMAP 4-dimethylaminopyridine

DME 1, 2-dimethoxyethane

DMF dimethyl formamide

DMSO dimethyl sulfoxide

dppf 1,1' -bis (diphenylphosphino) ferrocene

EA ethanol

ECF extracellular fluid

EDTA ethylene diamine tetraacetic acid

EGTA ethylene glycol tetraacetic acid

equiv/eq equivalent

ESI electrospray ionization

Ac acetate salt/ester

Et Ethyl group

EtOAc ethyl acetate

g

HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid)

HATU 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyl

Uronium hexafluorophosphate

hERG human Ether-a-go-go related gene

HMDS hexamethyldisilazane (azide)

HPLC high performance liquid chromatography

h hours

Hz

IC₅₀Half maximal inhibitory concentration

IMR-32 human neuroblastoma cell line

J coupling constant

Kg kilogram

kHz kilohertz

LAH lithium ammonium hydride

LCMS/LC-MS liquid chromatography-mass spectrometry

M molarity

m multiplet

m/z mass to charge ratio

M + mass peak

M + H hydrogenation mass peak

mCPBA 3-chloroperoxybenzoic acid

Me methyl group

MeOH methanol

Meaning of abbreviations

mg of

MHz

min/m min

mL/mL mL

mM millimolar concentration

mmol millimole

nanomole of nmol

mOsmol milliosmol

MRM magnetic resonance microscopy

MS Mass Spectrometry

ms

mV millivolt

mw microwave

N equivalent

mol mole of

NMP N-methylpyrrolidone

NMR nuclear magnetic resonance

pA Pian's medicine

Ph phenyl

parts per million ppm

preparation of prep

q.s. an amount sufficient to achieve said function

Rf Retention factors

RP inverse

RT/RT Room temperature

s second

s single peak

SEM 2- (trimethylsilyl) ethoxymethyl group

t triplet peak

TB tensile Block (sonic Block)

TEA Triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TMS trimethylsilyl group

TTX tetrodotoxin

UDB use dependent blocking

WT wild type

Meaning of abbreviations

Chemical shift

Microgram of μ g

μ L/. mu.l microliter

Micromolar concentration of μ M

Micron diameter of

Micromole of mu mol

Examples

Preparation of common intermediate

Intermediate 1.01 preparation of tert-butyl (6-bromoimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamate IV and tert-butyl 4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl (6- (tributylstannyl) imidazo [1,2-a ] pyrazin-8-yl) carbamate V

1- (4-nitrophenyl) -4- (oxetan-3-yl) piperazine I: in a 500mL round bottom flask, 1- (oxetan-3-yl) piperazine (3.02g,21.26 mmol), potassium carbonate (5.87g,42.52 mmol), 1-fluoro-4-nitrobenzene (3.00g,21.26 mmol) were mixed in acetonitrile (33mL) and stirred under nitrogen at 100 ℃ overnight. The mixture was diluted with water (100mL) and extracted with DCM (100mL x3), dried over anhydrous sodium carbonate, filtered and the filtrate concentrated. The residue was dissolved in a minimum amount of DCM using an sonicator and precipitated with hexane. The precipitate was filtered, washed with hexanes and dried to give the title compound I as an orange solid (4.70g,17.85 mmol, 84%).

4- (4- (oxetan-3-yl) piperazin-1-yl) aniline II: in the hydrogenation vessel, 1- (4-nitrophenyl) -4- (oxetan-3-yl) piperazine I (4.70g,17.85 mmol) was dissolved as much as possible in MeOH (26mL) and DCM (5 mL). Pd/C (10%) (2.85g,2.68 mmol) was added and the reaction mixture was stored under nitrogen. The reaction mixture was shaken on a Parr hydrogenator at 45 PSI. After 15 minutes, the reaction mixture was fully recharged to 45PSI and shaken for an additional 1 hour. The material was filtered through celite, washed with 25% MeOH/DCM and concentrated to give the title compound II as a light brown solid (4.16g,17.85 mmol, 98%).

6-bromo-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine III: to 4- (4- (oxetan-3-yl) piperazin-1-yl) aniline II (2.00g,8.57 mmol) were added hunig's base (3.29mL) and 6, 8-dibromoimidazo [1,2-a ] pyrazine (2.37g,8.57 mmol) in DMF (43 mL). The reaction mixture was stirred in a pressure tube at 85 ℃ overnight. The material was quenched with saturated sodium bicarbonate, extracted with DCM (120mL x3), the organic layers were combined and washed with water (120mL x3), dried over anhydrous sodium carbonate and concentrated. The crude material was purified using a 120g isco column and eluted using a step gradient of 0-60% (10% MeOH/DCM). The desired fractions were combined and concentrated to give the title compound III as a pale yellow solid (3.00g,6.99 mmol, 82%).

(6-Bromoimidazo [1, 2-a)]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester IV: reacting 6-bromo-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a]Pyrazin-8-amine III (1000mg,2.33mmol), di-tert-butyl dicarbonate (1016.72mg,4.66mmol) and N, N-dimethylPyridin-4-amine (21.34mg,0.17mmol) was stirred in DCM (1.01ml) and refluxed at 65 ℃ for 3 hours. The reaction mixture was diluted with 100mL DCM and H₂O wash (x3), dry, filter and concentrate. The crude material was dissolved in minimal DCM, loaded onto a pre-loaded silica loader and eluted from the 40g column over 20 column volumes using 0-30% MeOH/DCM. The desired fractions were combined and concentrated to give the title compound IV (1.2g, 97%). This compound was used in example 2.

Tert-butyl 4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl (6- (tributylstannyl) imidazo [1,2-a ] pyrazin-8-yl) carbamate V: in a 350mL p-tube, tert-butyl 6-bromoimidazo [1,2-a ] pyrazin-8-yl (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamate IV (8150mg,15.39mmol), 1,1,1,2,2, 2-hexabutyldititanane (11.67mL,23.09mmol), tetrakis (triphenylphosphine) palladium (889.43mg,0.77mmol) and tetrabutylammonium iodide (5686.03mg,15.39mmol) were mixed in dioxane (62mL) and heated to 110 ℃ overnight. No starting material remained according to LCMS. The reaction mixture was adsorbed onto celite and eluted from a 160g alumina column over 50-60 column volumes using a 0-10-20-30-100% (50% EtOAc/Hex-Hex) gradient (10-15 column volumes were held at 50%) to give the title compound V (5.83g, 51%). This compound was used in examples 1 and 2.

Intermediate 1.02 preparation of tert-butyl (6-bromo-5-methylimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamate X

6-methylpyrazin-2-amine VI: to a solution of anhydrous zinc (II) chloride (26.3g,193mmol) in THF (150mL) was added dropwise a 3M solution of methyl magnesium bromide in ether (129mL) at 0 deg.C over 1 hour. Then [1, 3-bis (diphenylphosphino) propane ] is added]Nickel (II) chloride (2.08g,3.85mmol) and the mixture was warmed to room temperature. To the above mixture was added 6-chloro-2-aminopyrazine (5.00g,38.6mmol) in anhydrous THF (25mL)And the reaction mixture was stirred under nitrogen atmosphere at reflux for 6 hours. The mixture was then cooled to room temperature, then cooled to 0 ℃ and carefully quenched with saturated aqueous ammonium chloride (50 mL). The organic layer was separated and dried over sodium sulfate. The drying agent was filtered and the filtrate was concentrated under reduced pressure to give crude 6-methylpyrazin-2-amine VI (2.60g, 62%) as a pale yellow solid, which was used in the subsequent step without purification:¹H NMR(400MHz,CDCl₃)：7.63(s,1H),7.53(s,1H),4.96(bs,2H),2.16(s,3H)。

3, 5-dibromo-6-methylpyrazin-2-amine VII: to a solution of 6-methylpyrazin-2-amine VI (2.00g,18.3mmol) in THF (40mL) at 10 ℃ over 15 min N-bromosuccinimide (6.70g,37.6mmol) was added portionwise and the mixture was warmed to room temperature with stirring. After 2 hours, the reaction mixture was concentrated under reduced pressure and the resulting residue was purified by column chromatography (silica gel, gradient, hexanes to EtOAc) to give 3, 5-dibromo-6-methylpyrazin-2-amine VII as an orange solid (3.10g, 64%):¹H NMR(400MHz,CDCl₃)：4.93(bs,2H),2.38(s,3H)。

6, 8-dibromo-5-methylimidazo [1,2-a ]]Pyrazine VIII: a mixture of 2-bromo-1, 1-diethoxyethane (3.21mL,20.7mmol) and 48% aqueous hydrobromic acid (1.0mL) was stirred at reflux for 2 hours. The reaction mixture was then cooled to room temperature and treated with sodium bicarbonate until gas evolution ceased. The mixture was filtered and the filtrate was diluted with ethanol (15 mL). To this mixture was added 3, 5-dibromo-6-methylpyrazin-2-amine VII (3.00g,11.2mmol), and the reaction mixture was stirred under reflux for 16 hours. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure to a volume of about 10 mL. The suspension was filtered and the filter cake was washed with cold ethanol (5 mL). The filter cake was then taken up in water (50mL) and the pH adjusted to-8 with potassium carbonate. The resulting suspension was filtered and the filter cake was dried under vacuum to constant weight to give 6, 8-dibromo-5-methylimidazo [1,2-a ] as a pale brown solid]Pyrazine VIII (1.97g, 60%):¹H NMR(400MHz,CDCl₃)：7.90(s,1H),7.72(s,1H),2.74(s,3H)。

6-bromo-5-methyl-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine IX: compound IX was prepared from 6, 8-dibromo-5-methylimidazo [1,2-a ] pyrazine VIII using the procedure as described for the preparation of 6-bromo-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine III in intermediate example 1.01.

(6-bromo-5-methylimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester X: compound X was prepared from 6-bromo-5-methyl-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine IX using the procedure as described for the preparation of tert-butyl (6-bromoimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamate IV in intermediate example 1.01. This compound was used in example 4.

Synthesis of examples 1 to 7

Example 16 preparation of (6-amino-5-methylpyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine (1)

2-bis (tert-butoxycarbonyl) amino-6-bromo-3-chloropyrazine XI: 6-bromo-3-chloropyrazin-2-amine (2000mg,9.59mmol), triethylamine (3.99ml,28.78mmol), di-tert-butyl dicarbonate (4188.12mg,19.19mmol) and N, N-dimethylpyridin-4-amine (87.91mg,0.72mmol) were successively dissolved in DCM (48 ml). The reaction mixture was stirred at room temperature overnight. The crude material was washed with water, dried, filtered and concentrated. The crude material was dissolved in minimal DCM and loaded onto a 25g pre-packed silica gel loader and eluted from a 40g column using 0-30% MeOH/DCM. The title compound XI (3900mg, 99%) was isolated and identified by LCMS and NMR. The product was observed by NMR to be a mixture of mono and bis boc-protected materials, mainly bis boc-protected materials.

tert-Butoxycarbonyl (6- (8- ((tert-butyloxycarbonyl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) amino) imidazo [1, 2-a)]Pyrazin-6-yl) -3-chloropyrazin-2-yl) carbamic acid tert-butyl ester XII: 4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl (6- (tributylstannyl) imidazo [1, 2-a) in 1, 4-dioxane (11.27ml)]Pyrazin-8-yl) carbamic acid tert-butyl ester V (1000mg,1.4mmol), 2-bis (tert-butoxycarbonyl) amino-6-bromo-3-chloropyrazine XI (552mg,1.35mmol) and PdCl₂(PPh₃)₂(142.77mg,0.20mmol) was irradiated in a microwave at 140 ℃ for 20 minutes. The reaction mixture was adsorbed onto celite and eluted from a 40g gold Isco column over 20 column volumes using 0-10-100% (30% MeOH/DCM). Fractions 34-39 were collected and concentrated. The title compound XII was identified and isolated according to NMR (590mg, 55%).

(6- (6-amino-5-methylpyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XIII: in a microwave vial, tert-butoxycarbonyl (6- (8- ((tert-butoxycarbonyl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) amino) imidazo [1, 2-a)]Pyrazin-6-yl) -3-chloropyrazin-2-yl) carbamic acid tert-butyl ester XII (300mg,0.44mmol), methylboronic acid (794.39mg,13.27mmol), tetrakis (triphenylphosphine) palladium (51.12mg,0.04mmol) and 2M Na₂CO₃(0.44ml) were mixed in DME (1.77ml) and irradiated in a microwave at 150 ℃ for 20 minutes. The reaction was worked up using 25% MeOH/DCM and water. The organic layers were combined, dried, filtered and concentrated. The crude material was loaded onto silica gel and eluted from a 40g Gold column over 45 column volumes using 0-5-15-25-50% (30% MeOH/DCM). Concentrating the desired fraction to obtain (6- (6-amino-5-methylpyrazin-2-yl) imidazo [1, 2-a) as a secondary product]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XIII and the desired final compound 1 as an inseparable mixture (208 mg total) were used in the TFA reaction.

6- (6-amino-5-methylpyrazin-2-yl)) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a]Pyrazin-8-amine (1): to 6- (6-amino-5-methylpyrazin-2-yl) imidazo [1,2-a]Pyrazin-8-yl (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XIII (48mg,0.09mmol) and 6- (6-amino-5-methylpyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a]A solution of pyrazin-8-amine (1,160mg,0.35mmol) in DCM (2.5ml) was added TFA (0.16ml,2.15 mmol). The reaction mixture was stirred at room temperature overnight. The reaction was incomplete according to LCMS. Additional TFA (0.16ml,2.15mmol) was added to the reaction mixture and stirred for 1 hour. The reaction was incomplete by LCMS, additional TFA (0.32ml,4.3mmol) was added to the reaction mixture and stirred for an additional 2 hours. The reaction mixture was then cooled to 0 ℃ and quenched with saturated NaHCO3, then extracted with DCM (5ml x3), and the combined organic layers were washed with water (5ml x 2), brine (5ml x1), dried (Na2SO4), and concentrated to give the crude product. The crude material was adsorbed onto silica gel and eluted from a 24g Gold Isco column using 0-15-25-40-100% (30% MeOH/DCM). The desired fractions were combined and concentrated to give the desired compound (1,67.5mg, 34%). LCMS-ESI⁺(m/z)：[M+H]⁺：458.22。¹H NMR(300MHz,d₆-DMSO): 9.48(s,1H),8.54(s,1H),8.41(s,1H),8.11(s,1H),7.95(d,2H),7.6(s,1H),6.98(d,2H),6.2(s,2H),4.58-4.45(dt,4H),3.3(m,1H),3.14(t,4H),2.50-2.4(dt,4H),2.33(s, 1H). Alternatively, compound XII can be used directly in this step and similarly deprotected to provide a 5-chloropyrazine substituted analog.

Example 2.6- (6-Aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine (2) preparation

2-bis (tert-butoxycarbonyl) amino-6-bromopyrazine XIV: to 6-bromopyrazin-2-amine (5g,28.7mmol) and bisTo a mixture of di-tert-butyl carbonate (25.09g,114.94mmol) was added DCM (10ml), followed by DMAP (0.351g,29 mmol). The reaction was heated at 55 ℃ for 1 hour, cooled to room temperature, the reaction mixture partitioned between water and DCM, purified on silica gel and concentrated to give 10.75g (87% yield) of 2-bis (tert-butoxycarbonyl) amino-6-bromopyrazine XIV. LCMS-ESI⁺(m/z)：[M+H]⁺：374.14.¹H NMR(DMSO)：8.84(d,2H),1.39(s,18H)。

(6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XVI-route a: 4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl (6- (tributylstannyl) imidazo [1, 2-a)]Tert-butyl pyrazin-8-yl) carbamate V (215mg,0.291mmol) was combined with 2-bis (tert-butoxycarbonyl) amino-6-bromopyrazine XIV (217.58mg,0.581mmol), bis (triphenylphosphine) palladium (II) dichloride (30.61mg,0.044mmol) and 1, 4-dioxane (5 ml). The reaction mixture was stirred in a microwave reactor at 120 ℃ for 30 minutes. The reaction mixture was quenched with saturated KF, extracted with EtOAc, purified on silica gel, eluting with EtOAc. The desired fractions were combined and concentrated to give 100mg (46% yield) of (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XVI. LCMS-ESI⁺(m/z)：[M+H]⁺：744.4。¹H NMR(300MHz d₆-DMSO)：9.37(s,1H),9.18(s,1H),8.77(s,1H),8.33(d,1H),7.87(d,1H),7.28-7.25(d,2H),6.92-6.89(d,2H),4.55-4.41(m,4H),3.4(m,1H),3.14-3.11(m,4H),2,37-2.34(m,4H),1.37(s,18H),1.3(s,9H)。

(6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XVI-route chemical B: step 1: to a dry 250mL round bottom flask was added 2-bis (tert-butoxycarbonyl) amino-6-bromopyrazine XIV (1.0g,1.0 equiv., 2.67mmol), KOAc (790mg,8.02mmol,3.0 equiv.), 4,4,4',4',5,5,5',5' -octamethyl-2, 2' -bis (1,3, 2-dioxaborolane) (750mg,2.94mmol,1.1 equiv.), pd (dba) (171mg,0.187mmol,0.07 equiv.), and X-phos (128mg,0.267mmol,0.1 equiv.), followed by addition of 1, 4-dioxane (25mL) and sonication of the solution for 5 minutes followed by purging with nitrogen for 5 minutes. The flask with contents was then placed under a nitrogen atmosphere and heated at 110 ℃ for 90 minutes. Once complete conversion to pinacol boronate was found to be achieved by LCMS, the reaction mixture was removed from heating and cooled to room temperature. Once cooled, the reaction contents were filtered through celite and the filter cake was washed with 3x 20mL EtOAc. The resulting solution was then concentrated to a dark red-orange slurry to give N, N-bis (tert-butoxycarbonyl) 6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazin-2-amine XV, which was used directly in the subsequent step.

Step 2: freshly formed N, N-bis (tert-butoxycarbonyl) 6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazin-2-amine XV (2.67mmol, based on 100% conversion, 2.0 equivalents, based on bromide) is dissolved in 20ml of 1, 2-dimethoxyethane and (6-bromoimidazo [1,2-a ] is added to this solution]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester IV (707mg,1.34mmol,1.0 equiv.), Na₂CO₃(283mg,2.67mmol,2.0 equiv.), Pd (PPh)₃)₄(155mg,0.134mmol,0.1 equiv.) and water (10mL), and the solution was degassed using nitrogen for 5 minutes. The reaction was then placed in N₂Heated under an atmosphere at 110 ℃ for 90 minutes. LCMS showed complete consumption of the bromide starting material and the reaction mixture was removed from heating and cooled to room temperature. The reaction mixture was diluted with 100mL of water and 100mL of 20% MeOH/DCM, and the organic layer was recovered and saturated with 1X NaHCO₃1X saturated brine, then Na₂SO₄And (5) drying. The solution was then filtered and concentrated to an orange-red solid. The sample was then slurried in warm MeOH, sonicated, then filtered, washed with 2x 20mL of cold MeOH, and the cream-colored solid was then dried under high vacuum overnight to give 905mg of (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1, 2-a) imidazo [1]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XVI, 90% yield over 2 steps (95% in each step).

6- (6-Aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a]Pyrazin-8-amine (2): to (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1, 2-a)]A solution of pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XVI (200mg,0.269mmol) in DCM (2ml) was added TFA (0.5ml,6.578 mmol). The reaction mixture was stirred at rt for 16 h, saturated sodium bicarbonate was added, extracted with EtOAC and purified on silica gel eluting with 5% MeOH/EtOAC, 20% MeOH/EtOAC. The desired fractions were combined and concentrated to yield 100mg (83% yield) of the title compound 2. LCMS-ESI⁺(m/z)：[M+H]⁺：444.2。¹H NMR(300MHz d₆-DMSO)：9.5(s,1H),8.588(s,1H),8.47(s,1H),8.12(d,1H),7.95-7.92(d,2H),7.88(s,1H),7.62(s,1H),6.99-6.96(d,2H),6.46(s,2H),4.57-4.53(m,2H),4.48-4.44(m,2H),3.43(m,1H),3.15-3.12(m,4H),2.41-2.38(m,4H)。

EXAMPLE 3 preparation of (R) - (4- (4- ((6- (6-aminopyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) phenyl) morpholin-2-yl) methanol (3)

(R) - (4- (4- ((6-bromoimidazo [1,2-a ] pyrazin-8-yl) amino) phenyl) morpholin-2-yl) methanol XVII: in a 250mL round bottom flask equipped with a condenser was added 6, 8-dibromoimidazo [1,2-a ] pyrazine (2000mg,7.22mmol) and 30mL isopropanol, followed by N, N-diisopropylethylamine (2.52mL,14.44mmol) and (R) - (4- (4-aminophenyl) morpholin-2-yl) methanol (1504.12mg,7.22 mmol). The reaction mixture was heated to reflux (95 ℃ oil bath) overnight. The reaction mixture was cooled and the precipitate was collected by filtration and washed with isopropanol then hexane to give the desired compound XVII, 2.92g, 95% yield.

(R) - (6-Bromoimidazo [1, 2-a)]Pyrazin-8-yl) (4- (2)- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) carbamic acid tert-butyl ester XVIII: a250 mL round bottom flask was charged with (R) - (4- (4- ((6-bromoimidazo [1, 2-a))]Pyrazin-8-yl) amino) phenyl) morpholin-2-yl) methanol XVII (2.80g,6.9mmol) and DCM are added, followed by triethylamine (2.9mL,2.1g,20.8mmol), DMAP (63g,0.52mmol) and di-tert-butyl dicarbonate (3.8g,17.3 mmol). The reaction mixture was stirred overnight, then diluted with DCM and water, separated, washed with brine, over Na₂SO₄Dried, filtered and concentrated under reduced pressure. The crude material was purified by chromatography: ISCO 40g silica gel with 25g silica gel loader, eluting with 0-100% EtOAc/hexanes, provided Compound XVIII (1.66g, 40%).

(R) - (4- (2- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) (6- (tributylstannyl) imidazo [1,2-a ] pyrazin-8-yl) carbamic acid tert-butyl ester XIX: in a similar manner to intermediate 1.01, tert-butyl (R) - (6-bromoimidazo [1,2-a ] pyrazin-8-yl) (4- (2- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) carbamate XVIII was reacted to give tert-butyl (R) - (4- (2- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) (6- (tributylstannyl) imidazo [1,2-a ] pyrazin-8-yl) carbamate XIX.

(R) - (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) (4- (2- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) carbamic acid tert-butyl ester XX: following a similar procedure to chemistry a as described in example 2, tert-butyl (R) - (4- (2- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) (6- (tributylstannyl) imidazo [1,2-a ] pyrazin-8-yl) carbamate XIX was reacted with 2-bis (tert-butoxycarbonyl) amino-6-bromopyrazine XIV to give the desired compound tert-butyl (R) - (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) (4- (2- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) carbamate XX.

(R) - (4- (4- ((6- (6-amino-5-methylpyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) amino) phenyl) morpholin-2-yl) methanol (3): (R) - (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazine-2) -carboxylic acid in DCMYl) imidazo [1,2-a]Pyrazin-8-yl) (4- (2- (((tert-butoxycarbonyl) oxy) methyl) morpholino) phenyl) carbamic acid tert-butyl ester XX (460mg,0.56mmol) was added to a round bottom flask and TFA (1.29ml,16.85mmol) was added. The reaction was partially complete after stirring for-5 hours. An additional 10eq TFA was added and stirred overnight, then concentrated under reduced pressure. Add 10% MeOH/DCM (. about.100 mL) and saturated aqueous sodium bicarbonate and stir 15 min, separate, and extract with. about.100 mL 10% MeOH/DCM. The organic layers were combined, washed with brine, and Na₂SO₄Dry, filter, and concentrate under reduced pressure and dry under vacuum. The resulting solid was triturated with DCM, the solid collected by filtration and dried under vacuum to give a brown solid, 125mg, identified by NMR and LC-MS as compound 3. LCMS-ESI⁺(m/z)：[M+H]⁺：419.2。¹H NMR(300MHz d₆-DMSO): 9.57(s,1H),8.59(s,1H),8.47(s,1H),8.13(d, J ═ 1.2Hz,1H), 8.06-7.90 (m,2H),7.87(s,1H),7.62(d, J ═ 1.1Hz,1H), 7.05-6.93 (m,2H),6.49(s,2H),4.78(t, J ═ 5.8Hz,1H), 3.98-3.87 (m,1H), 3.71-3.36 (m,7H),2.63(td, J ═ 11.7,3.4Hz,1H),2.37(dd, J ═ 12.1,10.5Hz, 1H). The corresponding (S) isomer or racemic mixture of compounds is similarly prepared in the first step using a racemic mixture of (S) - (4- (4-aminophenyl) morpholin-2-yl) methanol or (4- (4-aminophenyl) morpholin-2-yl) methanol, respectively.

Example 4.6- (6-Aminopyrazin-2-yl) -5-methyl-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine (4) preparation

(6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) -5-methylimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XXI: following the procedure of chemistry B as described in example 2, tert-butyl (6-bromo-5-methylimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamate X is reacted with XV to give the desired compound XXI.

6- (6-Aminopyrazin-2-yl) -5-methyl-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a]Pyrazin-8-amine (4): the compound (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) -5-methylimidazo [1, 2-a) was synthesized by a similar method to that described in example 2]Deprotection of t-butyl pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) carbamate XXI affords the desired compound 4. LCMS-ESI + (m/z): [ M + H ]]⁺：458.32。¹H NMR(300MHz,d₆-DMSO)：9.28(s,1H),8.28(s,1H),8.04(s,1H),7.89(d,2H),7.83(s,1H),7.7(s,1H),6.91(d,2H),6.46(s,2H),4.6-4.4(dt,4H),3.43(m,1H),3.1(t,4H),2.49(s,3H),2.4(t,4H)。

Example 5 preparation of 2- (5- ((6- (6-aminopyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol (5)

2- (2- (2-fluoro-5-nitrophenoxy) ethoxy) tetrahydro-2H-pyran XXII: a mixture of 2-fluoro-5-nitrophenol (4g,25mmol), 2- (2-bromoethoxy) tetrahydro-2H-pyran (4.4mL,28mmol) and potassium carbonate (4.2g, 30mmol) in DMF (50mL) was stirred at 50 ℃ for 16H. The reaction mixture was cooled to room temperature, washed with EtOAc and H₂And (4) diluting with oxygen. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts are washed with H₂O (5 times to remove DMF) and brine, and dried over sodium sulfate. The resulting residue was purified by column chromatography ISCO Rf (40g column) eluting with a gradient of 100% hexane-1: 1 hexane: EtOAc to give 2- (2- (2-fluoro-5-nitrophenoxy) ethoxy) tetrahydro-2H-pyran XXII (1.5g, 21%).

1- (4-Nitro-2- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy)Yl) phenyl) -4- (oxetan-3-yl) piperazine XXIII: a mixture of 2- (2- (2-fluoro-5-nitrophenoxy) ethoxy) tetrahydro-2H-pyran XXII (1550mg,5.43mmol), 1- (oxetan-3-yl) piperazine (772mg,5.43mmol), and potassium carbonate (1126.41mg,8.15mmol) in NMP (6mL) was stirred at 100 ℃ for 8 hours. The aqueous layer was separated and extracted with EtOAc. The combined organic extracts are washed with H₂O (5 times to remove NMP) and brine and dried over sodium sulfate. The resulting residue was purified by column chromatography ISCORf (24g column) with 100% DCM-60: 35:5DCM: Et₂Purification by MeOH gradient elution afforded 1- (4-nitro-2- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) -4- (oxetan-3-yl) piperazine XXIII (2.1g, 94%).

4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) aniline XXIV: to a suspension of 1- (4-nitro-2- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) -4- (oxetan-3-yl) piperazine XXIII (2100mg,5.1mmol) in ethanol (50mL) was added 10% Pd/C (50% wet, 390mg dry weight) in a 500-mL Parr hydrogenation bottle. The bottle was evacuated, filled with hydrogen to a pressure of 50psi and shaken on a Parr hydrogenation apparatus at room temperature for 2 hours. The reaction mixture was filtered and washed with ethanol. The filtrate was concentrated in vacuo to give 4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) aniline XXIV (1850mg, 95%).

6-bromo-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) imidazo [1,2-a]Pyrazine-8-amine XXV: to 4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) aniline XXIV (619mg,2.17mmol) and 6, 8-dibromoimidazo [1,2-a ]]To a solution of pyrazine (601mg,2.2mmol) in IPA (15mL) was added N, N-diisopropylethylamine (0.95mL,5.43 mmol). The mixture was stirred at 110 ℃ for 16 hours. Then DCM (10mL) and saturated NaHCO were added₃Aqueous solution (15mL), aqueous layer separated and extracted with DCM (2 × 10mL), combined organic extracts washed with brine (10mL) and dried over sodium sulfate, the resulting residue passed through column chromatography ISCO Rf (24g column) with 100% DCM-60:35:5DCM:Et₂MeOH gradient elution purification to give 6-bromo-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) imidazo [1,2-a]Pyrazin-8-amine XXV (1.2g, quantitative).

(6-bromoimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) carbamic acid tert-butyl ester XXVI: following an analogous procedure (III conversion to IV) as described in intermediate example 1.01, 6-bromo-N- (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) imidazo [1,2-a ] pyrazin-8-amine XXV (1.2g,2.4mmol) was reacted to give tert-butyl (6-bromoimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) carbamate XXVI (639mg, 37%).

(6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) carbamic acid tert-butyl ester XXVII: following the procedure of chemistry B as described in example 2, tert-butyl (6-bromoimidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) carbamate XXVI is reacted with XV to give the desired compound (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) amino Tert-butyl formate XXVII (313mg, 59%).

2- (5- ((6- (6-aminopyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol (5): the compound (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1, 2-a) was prepared by a similar method to that described in example 2]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) carbamic acid tert-butyl ester XXVII (313mg,0.35mmol) deprotection to give 2- (5- ((5- (oxetan-3-yl) amino acid6- (6-Aminopyrazin-2-yl) imidazo [1,2-a]Pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol (5). LCMS-ESI⁺(m/z)：[M+H]⁺：504.3。¹H NMR(300MHz,d₆-DMSO)：9.52(s,1H),8.61(s,1H),8.51(s,1H),8.14(d,J＝1.1Hz,1H),7.89(s,1H),7.81(d,J＝2.3Hz,1H),7.74–7.60(m,2H),6.90(d,J＝8.6Hz,1H),6.47(s,2H),5.74(s,1H),4.86–4.76(m,1H),4.50(dt,J＝25.6,6.3Hz,4H),4.04(t,J＝5.1Hz,2H),3.73(q,J＝5.1Hz,2H),3.51–3.42(m,1H),3.02(s,4H),2.40(s,4H).

Example 6.2 preparation of- ((4- (4- ((6- (6-aminopyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) phenyl) piperazin-1-yl) methyl) propane-1, 3-diol (6)

Oxetane-3-carbaldehyde XXVIII: in a round bottom flask equipped with a stir bar, oxetan-3-ylcarbinol (2.00g,22.7mmol) was dissolved in DCM (50mL) and dess-martin periodinane (10.67g,28.38mmol) was added in one portion. The reaction mixture was stirred at room temperature overnight. The solid was filtered through celite and washed with DCM (3mL × 5). The filtrate was removed and concentrated in vacuo and the resulting crude oxetane-3-carbaldehyde XXVIII was used directly in the next step.

1- (4-nitrophenyl) -4- (oxetan-3-ylmethyl) piperazine XXIX: to a round bottom flask equipped with a stir bar was added oxetane-3-carbaldehyde XXVIII (0.977g,11.35mmol), 1- (4-nitrophenyl) piperazine (1.18g,5.68mmol) in DCM (100mL), and HOAc (1.70g,28.38mmol) in DCM (2 mL). After 5 minutes, add NaBH (OAc)₃(24.06g,113.05 mmol). The resulting mixture was stirred at room temperature for 2 hours. Most of the volatiles were removed in vacuo. DCM (200mL) was added followed by saturated NaHCO₃Aqueous solution (20mL) and the resulting mixture was stirred for 20 minutes. The organic phase was separated and washed with saturated NaHCO₃Aqueous (20mL x3), brine (20mL x1), washed over Na₂SO₄Dry, filter and remove the solvent in vacuo. The residue was passed through a silica gel column (MeOH: DCM ═ 0:100 to 5:95 to 25:75) to give the desired compound XXIX as a yellow solid, 2.10 g.

4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) aniline XXX: to a round bottom flask equipped with a stir bar was added 1- (4-nitrophenyl) -4- (oxetan-3-ylmethyl) piperazine XXIX (3.20g,11.54mmol), ethanol (60mL), and water (60 mL). After addition of iron (4.51g,80.77mmol) and ammonium chloride (4.32g,80.77mmol), the reaction mixture was heated at 80 ℃ for 1 hour, then filtered through celite and washed with DCM (5mL x 5). The resulting filtrate was extracted with DCM (20mL × 3). The combined organic extracts were washed with water (20mL x 2), brine (20mL x1), dried over Na2SO4, and concentrated in vacuo. To give the desired 4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) aniline XXX, 1.19 g.

6-bromo-N- (4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) phenyl) imidazo [1,2-a]Pyrazine-8-amine XXXI: to a sealed tube equipped with a stir bar were added 4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) aniline XXX (1.19g,4.81mmol), 6, 8-dibromoimidazo [1,2-a ] amine]Pyrazine (1.33g,4.81mmol), isopropanol (24.1mL), and diisopropylethylamine (1.37g,10.58mmol), and the reaction mixture was heated at 100 ℃ overnight. Most of the solvent was removed in vacuo and DCM (200mL) was added to the mixture. Subjecting the solution to H₂O (20mL x 2), brine (20mL x1), Na₂SO₄Dry, filter and remove the solvent in vacuo. The resulting residue was passed through a silica gel column (MeOH: DCM ═ 5:95) to give 0.692g (32.4% yield) of light red solid as the desired compound XXXI.

(6-Bromoimidazo [1, 2-a)]Pyrazin-8-yl) (4- (1- (oxetan-3-ylmethyl) piperidin-4-yl) phenyl) carbamic acid tert-butyl ester XXXII: to a round bottom flask equipped with a stir bar was added 6-bromo-N- (4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) phenyl) imidazo [1,2-a]Pyrazin-8-amine XXXI (560mg,1.27mmol), DCM (11mL), di-tert-butyl dicarbonate (414.4mg,1.90mmol), and triethylamine (640.5mg,6.33 mmol).The reaction mixture was heated at 50 ℃ overnight. DCM (200mL) was added and the resulting solution was washed with water (20mL x 2), brine (20mL x1) and Na₂SO₄Dry, filter and remove the solvent in vacuo. Column chromatography afforded the desired compound XXXII as a yellow solid; 640mg (yield 93.2%).

(6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) (4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XXXIII: to a round bottom flask equipped with a stir bar was added (6-bromoimidazo [1,2-a ]]Pyrazin-8-yl) (4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) phenyl) carbamic acid tert-butyl ester XXXII (150mg,0.276mmol), N-bis (tert-butoxycarbonyl) 6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazin-2-amine XV (255.8mg,0.607mmol) in DME (2.3mL), Pd (PPh)₃)₄(16.0mg,0.14mmol)、Na₂CO₃Aqueous solution (1.0N,0.91mL,0.91mmol), and DME (2 mL). The mixture was heated at 75 ℃ for 2h, then DCM (200mL) was added and the resulting mixture was washed with water (30mL x3), brine (30mL x1), over MgSO₄Dried, filtered and the solvent removed in vacuo. Purification through a silica gel column (MeOH: DCM ═ 5:95) afforded the desired compound XXXIII, 250 mg.

2- ((4- (4- ((6- (6-aminopyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) amino) phenyl) piperazin-1-yl) methyl) propane-1, 3-diol (6): to (6- (6- (bis (tert-butoxycarbonyl) amino) pyrazin-2-yl) imidazo [1, 2-a)]A solution of tert-butyl pyrazin-8-yl) (4- (4- (oxetan-3-ylmethyl) piperazin-1-yl) phenyl) carbamate XXXIII (250mg,0.33mmol) in DCM (30mL) was added TFA (940.3mg,8.25 mmol). The resulting mixture was stirred at room temperature overnight. TFA (752.2mg,6.60mmol) was added again and stirred at room temperature overnight. Most of the solvent was removed in vacuo, DCM (200mL) and saturated aqueous NaHCO3 (30mL) were added, and the resulting mixture was stirred for 30 min. The organic phase was separated and washed with saturated aqueous NaHCO3 (20 mL. times.4), brine (20 mL. times.1). The aqueous phase was extracted with DCM (30mL × 2). The combined organic phases were washed with brine (20 mL. times.1) and Na₂SO₄Drying, filtering, and drying the mixture,and the solvent was removed in vacuo. The crude material was purified on an ISCO column (MeOH: DCM ═ 0:100 to 5:95 to 7.5:92.5 to 25:75 to elute the desired compound). Two compounds were obtained, the first being an oxetane compound (26.8 mg); the other was the desired compound (6, 49.3mg, 31.4% yield). LCMS-ESI⁺(m/z)：[M+H]⁺：476。¹H NMR(300MHz,d₆-DMSO): 9.51(S,1H),8.60(S,1H),8.49(S,1H),8.14(d, J ═ 1.5Hz,1H),7.95(d, J ═ 9Hz,2H),7.90(S,1H),7.64(S,1H),6.99(d, J ═ 9Hz,2H),6.48(S,2H),4.51 (wide S,2H),3.43(d, J ═ 6Hz,4H),3.12 (wide m,4H),2.54 (wide m,4H),2.34(d, J ═ 7.2Hz,2H),1.83(m, 1H).

Example 7.2 preparation of 2- (5- ((6- (6-amino-5-methylpyrazin-2-yl) imidazo [1,2-a ] pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol (7)

tert-Butoxycarbonyl (6- (8- ((tert-butyloxycarbonyl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) amino) imidazo [1,2-a]Pyrazin-6-yl) -3-chloropyrazin-2-yl) carbamic acid tert-butyl ester XXXIV: to a flask equipped with a reflux condenser was added (6-bromoimidazo [1,2-a ]]Pyrazin-8-yl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) carbamic acid tert-butyl ester XXVI (prepared as described in example 5) (352mg,0.52mmol), 2- (bis-boc-amino) -3-chloro-6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyrazine (prepared by a method analogous to that used in example 2 to prepare compound XV) (500mg,1.1mmol), 1M H in sodium carbonate (1.6mL, 1M)₂O solution) Pd (PPh)₃)₄(30mg,0.03mmol) and DME (4.8 mL). The mixture was heated to reflux for 1 hour. The reaction mixture was cooled to room temperature, quenched with DCM and H₂And (4) diluting with oxygen. The aqueous layer was separated and extracted with DCM. Combining the organic extractsThe extract was washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was passed through a column chromatography ISCO Rf (4g column) with 100% DCM-100% 60/35/5DCM/Et₂Purification with a gradient of O/MeOH elution and combining the appropriate fractions and concentrating to afford the desired compound tert-butoxycarbonyl (6- (8- ((tert-butoxycarbonyl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) amino) imidazo [1,2-a]Pyrazin-6-yl) -3-chloropyrazin-2-yl) carbamic acid tert-butyl ester XXXIV (258mg, 53%).

tert-Butoxycarbonyl (6- (8- ((tert-butyloxycarbonyl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) amino) imidazo [1,2-a]Pyrazin-6-yl) -3-methylpyrazin-2-yl) carbamic acid tert-butyl ester XXXV: adding tert-butoxycarbonyl (6- (8- ((tert-butoxycarbonyl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) amino) imidazo [1, 2-a) to a microwave vial]Pyrazin-6-yl) -3-chloropyrazin-2-yl) carbamic acid tert-butyl ester XXXIV (258mg,0.28mmol), methylboronic acid (503mg,8.4mmol), sodium carbonate (0.8mL,1M H₂O solution) Pd (PPh)₃)₄(32mg,0.03mmol) and DME (2.5 mL). The mixture was heated at 150 ℃ for 20 minutes. The reaction mixture was cooled to room temperature, quenched with DCM and H₂And (4) diluting with oxygen. The aqueous layer was separated and extracted with DCM. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was passed through a column chromatography ISCO Rf (4g column) with 100% DCM-100% 75/18/7DCM/Et₂Gradient elution purification of O/MeOH afforded the desired compound tert-butoxycarbonyl (6- (8- ((tert-butoxycarbonyl) (4- (4- (oxetan-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) amino) imidazo [1,2-a]Pyrazin-6-yl) -3-methylpyrazin-2-yl) carbamic acid tert-butyl ester XXXV (165mg, 65%).

2- (5- ((6- (6-amino-5-methylpyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol (7): to tert-butoxycarbonyl (6- (8- ((tert-butoxycarbonyl) (4- (4- (oxy)) carbonylHeterocyclobutane-3-yl) piperazin-1-yl) -3- (2- ((tetrahydro-2H-pyran-2-yl) oxy) ethoxy) phenyl) amino) imidazo [1,2-a]A solution of tert-butyl pyrazin-6-yl) -3-methylpyrazin-2-yl) carbamate XXXV (165mg,0.18mmol) in DCM (2.2mL) was added TFA (1.1mL,0.11 mmol). The mixture was stirred at room temperature for 16 hours. The reaction mixture was washed with 9:1DCM: MeOH and H₂And (4) diluting with oxygen. The aqueous layer was separated and extracted with 9:1DCM: MeOH. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using 100% 75/18/7DCM/Et₂O/MeOH-100％70/20/10DCM/Et₂Gradient elution purification of O/MeOH to afford the desired compound 2- (5- ((6- (6-amino-5-methylpyrazin-2-yl) imidazo [1, 2-a)]Pyrazin-8-yl) amino) -2- (4- (oxetan-3-yl) piperazin-1-yl) phenoxy) ethanol (7,56mg, 59%). LCMS-ESI⁺(m/z)：[M+H]⁺：518.2。¹H NMR(300MHz,d₆-DMSO): 9.49(s,1H),8.56(s,1H),8.44(s,1H),8.13(d, J ═ 1.1Hz,1H), 7.85-7.66 (m,2H),7.62(d, J ═ 1.1Hz,1H),6.90(d, J ═ 8.6Hz,1H),6.25(s,2H), 4.87-4.77 (m,1H),4.50(dt, J ═ 25.2,6.3Hz,4H),4.04(t, J ═ 5.1Hz,2H),3.74(q, J ═ 5.2Hz,2H), 3.51-3.39 (m,1H),3.10-2.95(m,4H),2.45-2.35(m,4H),2.34(s, 3H). Alternatively, compound XXXIV can be used directly in this step and similarly deprotected to provide a 5-chloropyrazine substituted analog.

Biological examples

Example 8: high throughput Syk biochemical test

Syk activity was tested using kinease (cisbio), time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, Syk catalyzes the phosphorylation of XL 665-labeled peptide substrates. Europium-conjugated phosphotyrosine-specific antibodies bind the resulting phosphorylated peptides. Formation of phosphorylated peptides was quantified by TR-FRET using europium as the donor and XL665 as the acceptor in a 2-step endpoint assay. Briefly, an Echo 550Acoustic Liquid Dispenser was usedTest compounds serially diluted in DMSO were delivered to Corning white, small volume, non-binding 384-well plates. Syk enzyme and substrate were dispensed to the test plate using Multi-Flo (Bio-Tek Instruments). The standard 5. mu.L reaction mixture was contained in reaction buffer (50mM Hepes, pH 7.0, 0.02% NaN)₃0.1% BSA,0.1mM orthovanadate, 5mM MgCl₂1mM DTT, 0.025% NP-40), 20. mu.M ATP, 1. mu.M biotin-labeled peptide, 0.015nM Syk. After incubation at room temperature for 30 minutes, 5. mu.L of stop solution and detection solution (1:200 europium cryptate-labeled anti-phosphorylated peptide antibody solution and 125nM streptavidin-XL 665Tracer, detection buffer (containing sufficient EDTA) at 50mM Hepes pH 7.0) were added. The plates were then incubated at room temperature for an additional 120 minutes, with excitation/emission/FRET emission readings at 340nm/615nm/665nm, respectively, using an Envision2103 multilabelled reader (PerkinElmer). The fluorescence intensities at the emission wavelengths of 615nm and 665nm are expressed as a ratio (665nm/615 nm). Percent inhibition was calculated as follows: % inhibition of 100x (ratio)_{Sample (I)}The proportion is 0%_{Suppression of}) V (ratio)_{100% inhibition}-ratio of_{0% inhibition}) With 0.1% DMSO (0% inhibition) as negative control and 1uMK252a (100% inhibition) as positive control. The activity of the compounds of examples 1-7 is provided in the table below, confirming that the compounds are IC₅₀Syk inhibitors below 50 nM.

Example 9: 384-well HTBS whole blood CD63 basophil assay

Syk Activity vs. basophils measured by expression of CD63 in the human whole blood basophil assay (25% blood)Decreased activation of the cells was evaluated in relation thereto. Basophil activation was measured in human whole blood using the Flow CAST kit (Buhlmann Laboratories AG, baselasse, Switzerland), following the protocol provided by the manufacturer with minor modifications. Fresh human whole blood in heparin was collected and delivered on the same day (AllCells, Emeryville, CA). Whole blood samples were incubated with DMSO (final 1%) or serial dilutions of compounds in DMSO for 60 minutes at 37 ℃. Basophils were activated with anti-FceRI mAb and stained with anti-CD 63-FITC and anti-CCR 3-PE at 37 ℃ for 20 minutes (per well: 50 μ L whole blood was mixed with 113 μ L stimulation buffer, 8.5 μ L anti-FceRI mAb, 8.5 μ L Ab stain CCR3-PE/CD 63-FITC). Cells were centrifuged at 1000x g for 18 minutes and 150. mu.L/well of supernatant was removed. Erythrocytes were lysed and cells were fixed by 2 rounds of cell lysis: the cell pellet was resuspended with 150. mu.L/well of 1 Xlysis buffer, incubated at room temperature for 10 minutes, and the cell pellet was collected by centrifugation at 1200rpm for 5 minutes. Cells were washed twice with 150 μ L/well wash buffer and resuspended in a final volume of 75 μ L/well wash buffer for immediate flow cytometric analysis or incubated overnight at 4 ℃ prior to flow cytometric analysis. Degranulation (basophil activation) was detected by surface expression of CD63 on CCR3 positive cells. The percentage of CD63 positive cells in the gated (gated) basophil population was determined and normalized to DMSO (negative control) and control compound (positive control). The activity of the compounds of examples 1-7 is provided in the table below, demonstrating that the compounds are effective in reducing basophil activation, EC₅₀Less than 200 nM.

Example 10: dynamic solubility

The kinetic solubility of the compounds in phosphate buffer at pH 7.4 was evaluated. The compound to be tested was dissolved in dimethyl sulfoxide at a concentration of 10 mM. Mu.l of stock sample was diluted with 297. mu.l of phosphate buffer pH 7.4 (Dubiaceae phosphate buffered saline (Sigma-Aldrich D8662), total molar concentration 0.149M and pH 7.43). The samples were then incubated at 37 ℃ for 24 hours under shaking, centrifuged and aliquots taken for testing against a known standard concentration of 0.1 mM. The kinetic solubilities of the compounds of examples 1-7 are provided in the table below, confirming that the kinetic solubilities of the compounds at pH 7.4 are greater than 90. mu.M.

Example 11: human hepatocyte stability test

Human blood cell stability (in L/hr/kg) as predicted clearance of hepatocytes was evaluated for the compounds. The compounds to be tested were diluted to 200. mu.M (4. mu.l of a 10mM DMSO stock was diluted to 196. mu.l of ACN: H₂O (50: 50). Propranolol was used as a positive control and buffer without hepatocytes alone was used as a 0% control. Mu.l of these materials were further diluted with 891. mu.l KHB buffer (InVitroGRO Cat. No. Z99074) to provide a 2X dosing solution. In each well of a 24-well plate, 250 μ Ι of 2X dosing solution was added to each well with 250 μ Ι of hepatocytes per well (1X 10 per well)⁶Individual viable cells/ml) or KHB for control samples, a final compound concentration of 1 μ M was achieved during incubation. The final solvent concentration was 0.01% DMSO and 0.25% ACN. The plates were placed on a shaker and incubated at 37 ℃ with 5% CO₂And (4) incubating. Samples were collected at 0, 1,3 and 6 hours. Loss of parent compound was determined against a standard curve using LC-MS method. The activity of the compounds of examples 1-7 is provided in the table below, showing a clearance of hepatocytes of about 0.12L/hr/kg or less.

Example 12: comparison with known Syk inhibitors

The tests of examples 8-11 were used to compare the compounds described herein with compounds known in the art. Data comparing the compounds of examples 1-7 with the foregoing compounds is provided in the table below. From these results it is evident that the compounds described herein are ideal as Syk inhibitors, having improved Syk and CD63 activity, improved kinetic solubility (solubility at least about 9-fold higher than known compounds) and hepatocyte clearance (clearance up to about 1/2 of known compounds) relative to known compounds. Likewise, the combination of improved Syk and CD63 inhibitory activity with improved kinetic solubility and clearance provides compounds that are expected to be effective in treating the diseases described herein with improved pharmacokinetic properties.

The following examples are included to illustrate embodiments of the invention and are not intended to limit the scope of the invention. It should be appreciated by those of skill in the art that the techniques disclosed herein represent techniques for practicing the invention. Those skilled in the art will appreciate that variations may be made in accordance with the present invention without departing from the spirit and scope thereof.

Example 13

Comparison of the Combined Effect of Compound 6- (6-Aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and vincristine of formula I with the Combined Effect of Compound 6- (6-Aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and vinblastine of formula I in the malignant DLBCL B cell line DHL-10

This example evaluates the efficacy of the compound of formula I, 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine, or a pharmaceutically acceptable salt thereof, in combination with vincristine, in inhibiting malignant diffuse large B-cell lymphoma (DLBCL) cell line DHL-10. This example also evaluates and compares the efficacy of the compound of formula I6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine, or a pharmaceutically acceptable salt thereof, in combination with vinblastine in the malignant diffuse large B-cell lymphoma (DLBCL) cell line DHL-10, to inhibit the viability of malignant B-cells.

Cell titer Glo viability assay: vincristine and vinblastine were assayed alone or in combination with 100nM of the compound of formula (I). The DLBCL cell line DHL-10 was plated twice at 10,000 cells per well and in RPMI supplemented with 10% FBS and 100U/L penicillin-streptomycin at 37 ℃, 5% CO₂Incubate for 72 hours. Cell viability was assessed using the cell titer glo (ctg) (Promega, Madison, WI) following the manufacturer's instructions. The CTG signals were recorded for single compound treatment and combined treatment. The results are shown in FIG. 1.

Figure 1 shows and summarizes the inhibitory effect of the compound 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine of figure 1 in combination with one of two vinca alkaloids, vincristine (figure 1A) and vinblastine (figure 1B), respectively, when the compounds are co-administered in DLBCL cell line DHL-10 (figure 1).

Example 14

Comparison of the Effect of the combination of the Compound of formula I6- (6-Aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and vincristine in solid tumor cell lines expressing Syk with the Effect of the combination of the Compound of formula I6- (6-Aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and vincristine in solid tumor cell lines not expressing Syk

This example evaluates the efficacy of the compound of formula I, 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine, or a pharmaceutically acceptable salt thereof, in combination with vincristine, in inhibiting cell viability in the malignant colon cell line MiaPaca expressing Syk. This example also evaluated the efficacy of the combination of the compound of formula I, 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine and vincristine, in inhibiting cell viability in the cell line HepG2, which does not express Syk.

Cell titer Glo viability assay: vincristine was assayed in the malignant colon cell line MiaPaca and in HepG2 cells alone or in combination with 4 concentrations of the compound of formula (I) (300, 100, 33 and 11 nM). Cells were plated at 5,000 cells per well, repeated twice, and in RPMI supplemented with 10% FBS and 100U/L penicillin-streptomycin, 5% CO at 37 ℃₂Incubate for 72 hours. Cell viability was assessed using the cell titer glo (ctg) (Promega, Madison, WI) following the manufacturer's instructions. The CTG signals were recorded for single compound treatment and combined treatment. The results are shown in FIG. 2.

Figure 2 summarizes the inhibitory effect of the combination of compound 6- (6-aminopyrazin-2-yl) -N- (4- (4- (oxetan-3-yl) piperazin-1-yl) phenyl) imidazo [1,2-a ] pyrazin-8-amine (compound a) of formula I and vincristine on cell viability in the malignant colon cell line MiaPaca expressing Syk (figure 2A) and in the malignant colon cell line HepG2 not expressing Syk (figure 2B).

Syk protein assay: cell lines were grown overnight in RPMI supplemented with 10% FBS and 100U/L penicillin-streptomycin in log. Collect 1X 10 in 50mL tubes by centrifugation at 300x g for 8 min at room temperature⁷And (4) cells. The cell pellet was treated with 200uL of protease (R)oche, Palo Alto CA) and phosphatase inhibitors (Sigma, Saint Louis MO; santa Cruz Technologies, Dallas TX) in 1XRIPA buffer (Cell Signaling Technology, Danvers MA) was lysed on ice for 15 minutes. Cell lysates were transferred to 96-well V-plates and used directly or frozen at-80 ℃ for the next day. Proteins were separated using 4-12% SDS-Bis/Tris gels and blotted onto nitrocellulose. Blots were blocked in Rockland Odyssey blocking solution and used with Total Syk antibody, 4D10(Santa Cruz) and pSyk-Y_525/6(Cell signalling Technologies.) Primary antibodies were diluted at 1: 1000 and incubated for 1 hour at room temperature, blots were washed 3 times with Tris-buffered saline containing 1.0% Tween (TBS-T) for 5 minutes, then blots were incubated for 1 hour at room temperature with goat α mouse IgG (H + L), AlexaFluor 680(Life Sciences, Inc) and goat α rabbit IgG (H + L), DyLight800(Thermo Scientific) (each diluted 1:20,000 in blocking solution). The blots were then washed 3 times for 5 minutes in TBS-T and analyzed on an Odyssey gel imager (LI-COR).

Figure 3 shows the Syk expression levels in the MiaPaca and HepG2 malignant colon cell lines (figure 3).

Example 15

Determination of Syk expression in several malignant solid tumor cell lines

Syk protein assay: the cell lines were grown overnight logarithmically and collected in 50mL tubes at 1X 10 by centrifugation at 300x g for 8 min at room temperature⁷And (4) cells. The Cell pellet was lysed in 200uL of 1XRIPA buffer (Cell Signaling Technology, Danvers MA) containing protease (Roche, Palo Alto CA) and phosphatase inhibitors (Sigma, Saint Louis MO; Santa Cruz Technologies, Dallas TX) on ice for 15 minutes. Cell lysates were transferred to 96-well V-plates and used directly or frozen at-80 ℃ for the next day. Proteins were separated using 4-12% SDS-Bis/Tris gels and blotted onto nitrocellulose. Blots were blocked in Rockland Odyssey blocking solution and used with total Syk antibody, 4D10 (San)ta Cruz) the primary antibody was diluted 1: 1000 and incubated at room temperature for 1 hour, the blot was washed with Tris-buffered saline containing 1.0% Tween (TBS-T) for 5 minutes 3 times, then the blot was incubated at room temperature with goat α mouse IgG (H + L), AlexaFluor 680(Life Sciences, Inc) (each diluted 1:20,000 in blocking solution) for 1 hour, then the blot was washed in TBS-T for 5 minutes 3 times and analyzed on an oederi gel imager (LI-COR), see fig. 4.

Throughout the specification, various patents, patent applications, and other types of publications (e.g., journal articles) are referenced. The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference for all purposes.

Claims

1. A method of treating a disease or disorder selected from inflammatory diseases, allergic diseases, autoimmune diseases, and cancer in a human in need thereof, comprising administering to the human in need thereof a therapeutically effective amount of a vinca alkaloid, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a compound having the structure of formula I, or a pharmaceutically acceptable salt thereof:

wherein:

R²is H or 2-hydroxyethoxy;

R³is H or methyl; and

R⁴is H or methyl.

2. The method of claim 1, wherein the disease or disorder is a cancer selected from the group consisting of hematological malignancies and solid tumors.

3. The method of claim 2, wherein the disease or disorder is a hematological malignancy selected from lymphoma, multiple myeloma, or leukemia.

4. The method of claim 2, wherein the disease or disorder is selected from the group consisting of small lymphocytic lymphoma, non-hodgkin's lymphoma, indolent non-hodgkin's lymphoma, refractory non-hodgkin's lymphoma, mantle cell lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, marginal zone lymphoma, immunoblastic large cell lymphoma, lymphoblastic lymphoma, splenic marginal zone B cell lymphoma (+/-villous lymphocytes), nodal marginal zone lymphoma (+/-monocyte-like B cells), extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type, cutaneous T cell lymphoma, extranodal T cell lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, mycosis fungoides, B cell lymphoma, diffuse large B cell lymphoma, mediastinal large B cell lymphoma, Large B cell lymphoma in a blood vessel, primary effusion lymphoma, small non-dividing cell lymphoma, burkitt's lymphoma, multiple myeloma, plasmacytoma, acute lymphocytic leukemia, acute T cell lymphoblastic leukemia, acute B cell lymphocytic leukemia, B cell prolymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, juvenile myelomonocytic leukemia, minimal residual disease, hairy cell leukemia, primary myelofibrosis, secondary myelofibrosis, chronic myelocytic leukemia, myelodysplastic syndrome, myeloproliferative disorders, and waldenstrom's macroglobulinemia.

5. The method of claim 2, wherein the disease or disorder is a solid tumor, wherein the solid tumor is a cancer selected from the group consisting of: pancreatic cancer, urinary organ cancer, bladder cancer, colorectal cancer, colon cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular cancer, thyroid cancer, gallbladder cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), ovarian cancer, cervical cancer, gastric cancer, endometrial cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancers, brain tumors (e.g., gliomas, anaplastic oligodendrogliomas, adult glioblastoma multiforme, and adult anaplastic astrocytomas), bone cancers, soft tissue sarcomas, retinoblastoma, neuroblastoma, peritoneal leakage, malignant pleural effusion, mesothelioma, wilms 'tumor, trophoblastic tumors, vascular pericellular tumors, kaposi's sarcoma, mucous cancers, round cell carcinoma, squamous cell carcinoma, esophageal squamous cell carcinoma, oral cancer, adrenal cortex carcinoma, and ACTH-producing tumors.

6. The method of claim 2, wherein the disease or condition is selected from the group consisting of: systemic lupus erythematosus, myasthenia gravis, Goodpasture's syndrome, glomerulonephritis, hemorrhage, pulmonary hemorrhage, atherosclerosis, rheumatoid arthritis, psoriatic arthritis, monoarthritis, osteoarthritis, gouty arthritis, spondylitis, Behcet's disease, autoimmune thyroiditis, Raynaud's syndrome, acute disseminated encephalomyelitis, chronic idiopathic thrombocytopenic purpura, multiple sclerosis, Sjogren's syndrome, autoimmune hemolytic anemia, tissue transplant rejection, hyperacute rejection of transplanted organs, allograft rejection, graft-versus-host disease, diseases involving leukocyte exudation, diseases due to leukocyte dyscrasia and metastasis, syndrome associated with granulocyte transfusion, cytokine-induced toxicity, scleroderma, vasculitis, asthma, psoriasis, chronic inflammatory bowel disease, autoimmune thyroiditis, Raynaud's syndrome, acute disseminated encephalomyelitis, chronic idiopathic thrombocytopenic purpura, multiple sclerosis, Sjogren's syndrome, autoimmune hemolytic anemia, tissue transplant rejection, hyperacut, Ulcerative colitis, Crohn's disease, necrotizing enterocolitis, irritable bowel syndrome, dermatomyositis, Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes mellitus, type I diabetes, sepsis, septic shock, endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shock syndrome, multiple organ injury syndrome secondary to sepsis, trauma, hypovolemic shock, allergic conjunctivitis, vernal conjunctivitis, thyroid-related eye disease, eosinophilic granuloma, eczema, chronic bronchitis, acute respiratory distress syndrome, allergic rhinitis, pollinosis, bronchial asthma, silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, emphysema, pneumonia, bacterial pneumonia, bronchiectasis, pulmonary oxygen poisoning, reperfusion injury of the heart muscle or brain or limbs, thermal injury, chronic bronchitis, Cystic fibrosis, keloid formation or scar tissue formation, fever and myalgia due to infection, brain or spinal cord injury due to minimal trauma, diseases involving leukocyte exudation, acute hypersensitivity, delayed-type hypersensitivity, urticaria, food allergy, sunburn of the skin, pelvic inflammatory disease, urethritis, uveitis, sinusitis, pneumonia, encephalitis, meningitis, myocarditis, nephritis, osteomyelitis, myositis, hepatitis, alcoholic hepatitis, gastritis, enteritis, contact dermatitis, atopic dermatitis, gingivitis, appendicitis, pancreatitis, cholecystitis, polycythemia vera, essential thrombocythemia, and polycystic kidney disease.

7. The method of claim 2, wherein the disease or disorder is selected from the group consisting of systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, sjogren's syndrome, psoriasis, autoimmune hemolytic anemia, asthma, ulcerative colitis, crohn's disease, irritable bowel disease, chronic obstructive pulmonary disease, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, sjogren's syndrome, psoriasis, autoimmune hemolytic anemia, asthma, ulcerative colitis, crohn's disease, irritable bowel disease, and chronic obstructive pulmonary disease.

8. The method of claim 2, wherein the disease or condition is selected from the group consisting of asthma, rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease, and systemic lupus erythematosus.

9. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt of claim 1 and a therapeutically effective amount of a vinca alkaloid or a pharmaceutically acceptable salt.

10. The method according to claim 2, wherein the compound of formula I is

And said vinca alkaloid is selected from the group consisting of vincristine, vinblastine, vindesine, vinorelbine, desoxyvincaminol, vincaminol, vinbutine, vingmatine, and vindoline.

11. The method according to claim 10, wherein the vinca alkaloid is selected from the group consisting of vincristine, vinblastine, vindesine, and vinorelbine.

12. The method of claim 11, wherein the vinca alkaloid is vincristine.

13. The method according to claim 11, wherein the vinca alkaloid is vinblastine.

14. The method of claim 11, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered prior to the administration of the vinca alkaloid or a pharmaceutically acceptable salt thereof.

15. The method according to claim 11, wherein the vinca alkaloid, or a pharmaceutically acceptable salt thereof, is administered prior to the administration of the compound of formula (I), or a pharmaceutically acceptable salt thereof.

16. The method according to claim 11, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof and the vinca alkaloid or a pharmaceutically acceptable salt thereof are administered simultaneously.

17. The method of claim 11, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered at a dose of 50mg to 300mg and the vinca alkaloid or a pharmaceutically acceptable salt thereof is 0.1mg-M²To 1.5mg-M²The dosage of (a).

18. The method of claim 11, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered at a dose of 100mg to 250mg and the vinca alkaloid or a pharmaceutically acceptable salt thereof is 0.25mg-M²To 1.0mg-M²Wherein the vinca alkaloid is selected from vincristine and vinblastine.

19. The method of claim 11, wherein the human with cancer (i) is refractory to at least one chemotherapy, or (ii) relapses after treatment with chemotherapy, or a combination thereof.

20. The method of claim 11, wherein the cancer is a hematologic malignancy.

21. The method according to claim 20, wherein the cancer is leukemia.

22. The method according to claim 20, wherein the leukemia is Chronic Lymphocytic Leukemia (CLL).

23. The method according to claim 20, wherein the cancer is lymphoma.

24. The method according to claim 23, wherein the lymphoma is non-hodgkin's lymphoma (NHL).

25. The method according to claim 20, wherein the NHL is diffuse large B-cell lymphoma (DLBCL), Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Small Lymphocytic Lymphoma (SLL), lymphoplasmacytic lymphoma (LPL), and Marginal Zone Lymphoma (MZL).

26. The method according to claim 20, wherein the cancer is selected from Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML), Burkitt's Lymphoma (BL), Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), Chronic Myeloid Leukemia (CML), Multiple Myeloma (MM), non-Hodgkin's lymphoma (NHL), indolent non-hodgkin lymphoma (iNHL), refractory non-hodgkin lymphoma, Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL), Waldenstrom Macroglobulinemia (WM), T-cell lymphoma, B-cell lymphoma, hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), lymphoplasmacytic lymphoma (LPL) and Marginal Zone Lymphoma (MZL).

27. The method according to claim 20, wherein the cancer is a solid tumor and expresses spleen tyrosine kinase (Syk) activity.

28. The method according to claim 27, wherein the solid tumor cancer is selected from the group consisting of: pancreatic cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, adenocarcinoma, esophageal cancer, and hepatocellular cancer.