Combination therapy with a mutant IDH inhibitor and a BCL-2 inhibitor

Abstract

The present invention relates to combination therapy with a mutant IDH inhibitor of formula (I) and a Bcl-2 inhibitor for the treatment of cancer. TIFF2023520338000020.tif2780(I)

Description

[Technical Field]

[0001] The present invention relates to combination therapy with mutant isocitrate dehydrogenase (IDH) inhibitors and Bcl-2 inhibitors for the treatment of cancer. [Background technology]

[0002] IDH and IDH2 catalyze the conversion of isocitrate to α-ketoglutarate (α-KG), which is converted to nicotinamide adenine dinucleotide phosphate (NADP + ) to NADPH (Megias-Vericat J, et al., Blood Lymph. Cancer: Targets and Therapy 2019; 9: 19-32).

[0003] Novel mutations of IDH1, such as those at amino acid residue R132, contribute to tumorigenesis in several types of cancer, including solid tumors and hematological malignancies (Badur MG, et al., Cell Reports 2018; 25: 1680). IDH1 mutations may cause high levels of 2-hydroxyglutarate (2-HG), inhibiting cell differentiation, and inhibitors of mutant IDH1 may reduce 2-HG levels and promote cell differentiation (Molenaar RJ, et al., Oncogene 2018; 37: 1949-1960). Mutations also occur in IDH2, e.g., at amino acid residues R172, R140, and R172 (Yang H, et al., Clin. Cancer. Res. 2012; 18: 5562-5571; Mondesir J, et al., J. Blood Med. 2016; 7: 171-180).

[0004] For example, acute myeloid leukemia (AML) is characterized by a diverse range of mutated genes and a polyclonal genomic architecture, including preleukemic and leukemic clones that dynamically evolve over time and under the selective pressure of treatment (Bloomfield CD, et al., Blood Revs. 2018; 32: 416-425). Induction chemotherapy with cytarabine and anthracycline (“7+3”) has been the standard of care for newly diagnosed AML patients for over 40 years.

[0005] Recently, five additional medications have been approved by the U.S. Food and Drug Administration to treat AML: midostaurin, enasidenib, CPX-351, gemtuzumab ozogamicin (Bloomfield CD, et al., Blood Revs. 2018; 32: 416-425), and ivosidenib (Megias-Vericat J, et al., Blood Lymph. Cancer: Targets and Therapy 2019; 9: 19-32). Approximately 60% to 70% of adults with AML can expect to achieve complete remission (CR) after appropriate induction therapy, with more than 25% of adults with AML (approximately 45% of patients achieving CR). teeth Patients can expect to live for more than three years and may be cured.

[0006] However, IDH1 resistance mutations are observed in 7-14% of AML patients, and the associated high 2-HG levels may contribute to an epigenetic hypermethylation phenotype and block differentiation, potentially contributing to leukemogenesis (Megias-Vericat J, et al., Blood Lymph. Cancer: Targets and Therapy 2019;9:19-3). Additionally, Flt3 kinase mutations are observed in approximately one-third of AML patients (Lee HJ, et al., Oncotarget 2018;9:924-936). 。 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there remains a need for alternative mutant IDH-associated cancer treatments.

[0008] Certain mutant IDH1 and IDH2 inhibitors are disclosed in WO2018 / 111707A1, such as the compound defined herein as "Compound A," which is a covalent inhibitor of mutant IDH1 that modifies a single cysteine ​​(Cys269) within the allosteric binding pocket, rapidly inactivating the enzyme and selectively inhibiting 2-HG production without affecting α-KG levels (WO2018 / 111707A1). [Means for solving the problem]

[0009] The present invention provides a method for treating cancer, comprising administering to a human cancer subject having an IDH mutation a therapeutically effective amount of (a) a first formula I: [ka] I [In formula: R 1 is -CH2CH(CH3)2, -CH2CH3, -CH2CH2OCH3, or -CH2-cyclopropyl; R 2 is -CH3 or -CH2CH3; and X is N or CH. or a pharmaceutically acceptable salt thereof; and (b) a second compound that is a Bcl-2 inhibitor, or a pharmaceutically acceptable salt thereof. The method includes administering DETAILED DESCRIPTION OF THE INVENTION

[0010] In one embodiment, the IDH mutation is an IDH1 mutation or an IDH2 mutation. In another embodiment, the IDH mutation is an IDH1 mutation. In another embodiment, the IDH1 mutation is an IDH1 R132 mutation. In another embodiment, the IDH1 mutation is R132H, R132C, R132G, R132L, or R132S. In another embodiment, the IDH1 R132 mutation is R132H. In another embodiment, the IDH1 mutation is R132C. In another embodiment, the IDH1 mutation is R132G. In another embodiment, the IDH1 mutation is R132L. In another embodiment, the IDH1 mutation is R132S.

[0011] In another embodiment, the IDH mutation is an IDH2 mutation. In another embodiment, the IDH2 mutation is an IDH2 R140 mutation or an IDH2 R172 mutation. In another embodiment, the IDH2 mutation is an R140 mutation. In another embodiment, the R140 mutation is R140Q, R140L, or R140W. In another embodiment, the IDH2 mutation is an R172 mutation. In another embodiment, the R172 mutation is R172K, R172M, R172G, R172S, or R172W.

[0012] In one embodiment of the method of the present invention, in the first compound of formula I or a pharmaceutically acceptable salt thereof, X is N.

[0013] In another embodiment, in the first compound of formula I or a pharmaceutically acceptable salt thereof, X is N and R 1 is -CH2-cyclopropyl, and R 2 is -CH2CH3. In another embodiment, in the first compound of formula I, X is N and R 1 is -CH2-cyclopropyl, and R 2 is -CH2CH3.

[0014] In another embodiment, the first compound is: 7-[[(1S)-1-[4-[(1R)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; or 1-Ethyl-7-[[(1S)-1-[4-[1-(4-prop-2-enoylpiperazin-1-yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][1,3]oxazin-2-one or a pharmaceutically acceptable salt thereof.

[0015] In another embodiment, the first compound is 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one.

[0016] In another embodiment, the first compound is: [ka] Compound A or a pharmaceutically acceptable salt thereof. In another embodiment, the first compound is Compound A.

[0017] In another embodiment, the subject is identified as having an IDH mutation. In another embodiment, the subject is identified as having an IDH mutation in tissue. In another embodiment, the subject is identified as having an IDH1 mutation. In another embodiment, the subject is identified as having an IDH2 mutation.

[0018] In another embodiment, the subject is identified as having an R132 IDH1 mutation. In another embodiment, the subject is identified as having an R132 IDH1 mutation in tissue. In another embodiment, the subject is identified as having an IDH2 mutation, such as an IDH2 R172, R140, or R172 mutation. In another embodiment, the subject is identified as having an IDH2 R172, R140, or R172 mutation in tissue.

[0019] In another embodiment, the cancer is a hematological malignancy and the subject has been identified as having an IDH mutation (e.g., an IDH1 R132 mutation, or an IDH2 R172, R140, or R172 mutation) in their blood, bone marrow, lymph nodes, or lymph. In another embodiment, the subject has been identified as having an IDH mutation (e.g., an IDH1 R132 mutation, or an IDH2 R172, R140, or R172 mutation) in their blood cells, bone marrow cells, lymph node cells, or lymph cells.

[0020] In another embodiment, the cancer is a solid tumor cancer and the subject is identified as having an IDH mutation (e.g., an IDH1 R132 mutation, or an IDH2 R172, R140, or R172 mutation) in the solid tumor tissue. In another embodiment, the subject is identified as having an IDH mutation (e.g., an IDH1 R132 mutation, or an IDH2 R172, R140, or R172 mutation) in the solid tumor tissue cells.

[0021] In another embodiment, the first compound, or a pharmaceutically acceptable salt thereof, is administered before the second compound, or a pharmaceutically acceptable salt thereof.

[0022] In another embodiment, the first compound, or a pharmaceutically acceptable salt thereof, is administered after the second compound, or a pharmaceutically acceptable salt thereof.

[0023] In another embodiment, a first compound, or a pharmaceutically acceptable salt thereof, is formulated with a second compound, or a pharmaceutically acceptable salt thereof.

[0024] In another embodiment of the method of the present invention, the cancer is a solid tumor cancer. In another embodiment, the solid tumor cancer is cholangiocarcinoma, head and neck cancer, chondrosarcoma, hepatocellular carcinoma, melanoma, pancreatic cancer, astrocytoma, oligodendroglioma, glioma, glioblastoma, bladder cancer, colorectal cancer, lung cancer, or undifferentiated sinonasal carcinoma. In another embodiment, the lung cancer is non-small cell lung cancer. In another embodiment, the solid tumor cancer is cholangiocarcinoma.

[0025] In another embodiment, cancer teeth In another embodiment, the hematological malignancy is acute myeloid leukemia, myelodysplastic syndrome myeloproliferative neoplasm, angioimmunoblastic T-cell lymphoma, T-cell acute lymphoblastic leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, or chronic myelogenous leukemia. In another embodiment, the hematological malignancy is acute myeloid leukemia.

[0026] The present invention provides a compound of formula I: [ka] I [In formula: R 1 is -CH2CH(CH3)2, -CH2CH3, -CH2CH2OCH3, or -CH2-cyclopropyl; R 2 is -CH3 or -CH2CH3; and X is N or CH. or a pharmaceutically acceptable salt thereof.

[0027] In one embodiment, the IDH mutation is an IDH1 mutation or an IDH2 mutation. In another embodiment, the IDH mutation is an IDH1 mutation. In another embodiment, the IDH1 mutation is an IDH1 R132 mutation. In another embodiment, the IDH1 mutation is R132H, R132C, R132G, R132L, or R132S. In another embodiment, the IDH1 R132 mutation is R132H. In another embodiment, the IDH1 mutation is R132C. In another embodiment, the IDH1 mutation is R132G. In another embodiment, the IDH1 mutation is R132L. In another embodiment, the IDH1 mutation is R132S.

[0028] In another embodiment, the IDH mutation is an IDH2 mutation. In another embodiment, the IDH2 mutation is an IDH2 R140 mutation or an IDH2 R172 mutation. In another embodiment, the IDH2 mutation is an R140 mutation. In another embodiment, the R140 mutation is R140Q, R140L, or R140W. In another embodiment, the IDH2 mutation is an R172 mutation. In another embodiment, the R172 mutation is R172K, R172M, R172G, R172S, or R172W.

[0029] In one embodiment, the subject is identified as having an IDH mutation (eg, an IDH1 R132 mutation, or an IDH2 R172, R140, or R172 mutation).

[0030] In one embodiment, the compound for use is a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein X is N. In another embodiment, the compound for use is a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein R 1 is -CH2-cyclopropyl, or a pharmaceutically acceptable salt thereof. In another embodiment, the compound for use is 2 is -CH2CH3 or a pharmaceutically acceptable salt thereof. In another embodiment, the compound for use is a compound of formula I, wherein X is N and R 1 is -CH2-cyclopropyl, and R 2is -CH2CH3 or a pharmaceutically acceptable salt thereof. In another embodiment, the compound is a compound of formula I, wherein X is N and R 1 is -CH2-cyclopropyl, and R 2 is a compound of formula I where

[0031] In another embodiment, the compound is: 7-[[(1S)-1-[4-[(1R)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; or 1-Ethyl-7-[[(1S)-1-[4-[1-(4-prop-2-enoylpiperazin-1-yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][1,3]oxazin-2-one or a pharmaceutically acceptable salt thereof.

[0032] In another embodiment, the compound is 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one or a pharmaceutically acceptable salt thereof.

[0033] In another embodiment, the compound is: [ka] (Compound A), or a pharmaceutically acceptable salt thereof.

[0034] In another embodiment, the compound is Compound A.

[0035] Provided herein are novel methods for using a compound of Formula I in combination with a Bcl-2 inhibitor to treat cancer. Accordingly, some embodiments of the present invention provide a compound of Formula I for use in simultaneous, separate, or sequential combination with a Bcl-2 inhibitor in the treatment of cancer. Furthermore, some embodiments of the present invention provide a compound of Formula I for use in simultaneous, separate, or sequential combination with a Bcl-2 inhibitor in the treatment of solid tumor cancer. Additionally, some embodiments of the present invention provide a compound of Formula I for use in simultaneous, separate, or sequential combination with a Bcl-2 inhibitor in the treatment of hematological malignancies.

[0036] The present invention also provides a pharmaceutical composition comprising a compound of Formula I for use in treating cancer in a human subject having an IDH mutation in blood cells, bone marrow cells, lymph nodes, or lymph fluid.

[0037] In one embodiment, the IDH mutation is an IDH1 mutation or an IDH2 mutation. In another embodiment, the IDH mutation is an IDH1 mutation. In another embodiment, the IDH1 mutation is an IDH1 R132 mutation. In another embodiment, the IDH1 mutation is R132H, R132C, R132G, R132L, or R132S. In another embodiment, the IDH1 R132 mutation is R132H. In another embodiment, the IDH1 mutation is R132C. In another embodiment, the IDH1 mutation is R132G. In another embodiment, the IDH1 mutation is R132L. In another embodiment, the IDH1 mutation is R132S.

[0038] In another embodiment, the IDH mutation is an IDH2 mutation. In another embodiment, the IDH2 mutation is an IDH2 R140 mutation or an IDH2 R172 mutation. In another embodiment, the IDH2 mutation is an R140 mutation. In another embodiment, the R140 mutation is R140Q, R140L, or R140W. In another embodiment, the IDH2 mutation is an R172 mutation. In another embodiment, the R172 mutation is R172K, R172M, R172G, R172S, or R172W.

[0039] The invention also provides use of a compound of Formula I in the manufacture of a medicament for the treatment of cancer in a human subject identified as having an IDH mutation (e.g., an IDH1 R132 mutation, or an IDH2 R172, R140, or R172 mutation) in blood, bone marrow, lymph nodes, lymph, blood cells, bone marrow cells, lymph node cells, or lymph cells.

[0040] In one embodiment of the method of the present invention, the cancer is a frontline cancer. In another embodiment, the frontline cancer is a solid tumor cancer. In another embodiment, the frontline cancer is a hematological malignancy. In another embodiment, the frontline hematological malignancy is frontline AML.

[0041] In another embodiment of the method of the present invention, the cancer is a recurrent cancer. In another embodiment, the recurrent cancer is a solid tumor cancer. In another embodiment, the recurrent cancer is a hematological malignancy. In another embodiment, the recurrent hematological malignancy is recurrent AML.

[0042] In another embodiment of the method of the present invention, the cancer is a refractory cancer. In another embodiment, the refractory cancer is a solid tumor cancer. In another embodiment, the refractory cancer is a hematological malignancy. In another embodiment, the refractory hematological malignancy is refractory AML.

[0043] In another embodiment of the method of the present invention, the cancer is an advanced cancer. In another embodiment, the advanced cancer is an advanced solid tumor cancer. In another embodiment, the advanced cancer is an advanced hematological malignancy. In another embodiment, the advanced hematological malignancy is advanced AML.

[0044] In another embodiment, the AML is acute promyelocytic leukemia.

[0045] In one embodiment of the method of the invention, the Bcl-2 inhibitor is venetoclax, obatoclax, navitoclax, or a pharmaceutically acceptable salt of any one of them. In another embodiment, the Bcl-2 inhibitor is venetoclax or a pharmaceutically acceptable salt thereof. In another embodiment, the Bcl-2 inhibitor is venetoclax.

[0046] The present invention provides (a) a first formula I: [ka] I [In formula: R 1 is -CH2CH(CH3)2, -CH2CH3, -CH2CH2OCH3, or -CH2-cyclopropyl; R 2 is -CH3 or -CH2CH3; and X is N or CH. or a pharmaceutically acceptable salt thereof; and (b) a second compound that is a Bcl-2 inhibitor, or a pharmaceutically acceptable salt thereof. 1. A method for identifying a human subject for treatment with Identifying a human subject with cancer; and Determining whether a human subject has an IDH mutation in blood, bone marrow, lymph nodes, lymph fluid, blood cells, bone marrow cells, lymph node cells, lymph fluid cells, or solid tissue. a method comprising: Also provided is a method in which the subject has cancer.

[0047] In one embodiment, the IDH mutation is an IDH1 mutation or an IDH2 mutation. In another embodiment, the IDH mutation is an IDH1 mutation. In another embodiment, the IDH1 mutation is an IDH1 R132 mutation. In another embodiment, the IDH1 mutation is R132H, R132C, R132G, R132L, or R132S. In another embodiment, the IDH1 R132 mutation is R132H. In another embodiment, the IDH1 mutation is R132C. In another embodiment, the IDH1 mutation is R132G. In another embodiment, the IDH1 mutation is R132L. In another embodiment, the IDH1 mutation is R132S.

[0048] In another embodiment, the IDH mutation is an IDH2 mutation. In another embodiment, the IDH2 mutation is an IDH2 R140 mutation or an IDH2 R172 mutation. In another embodiment, the IDH2 mutation is an R140 mutation. In another embodiment, the R140 mutation is R140Q, R140L, or R140W. In another embodiment, the IDH2 mutation is an R172 mutation. In another embodiment, the R172 mutation is R172K, R172M, R172G, R172S, or R172W.

[0049] In one embodiment of the method of the present invention for identifying a human cancer subject for treatment, in the first compound of formula I, X is N or a pharmaceutically acceptable salt thereof. In another embodiment, in the first compound of formula I, X is N and R 1 is -CH2-cyclopropyl, and R 2 is —CH2CH3, or a pharmaceutically acceptable salt thereof. In another embodiment, in the first compound, X is N and R 1 is -CH2-cyclopropyl, and R 2 is -CH2CH3.

[0050] In another embodiment of the method of identifying a human cancer subject for treatment, the first compound is: 7-[[(1S)-1-[4-[(1R)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; or 1-Ethyl-7-[[(1S)-1-[4-[1-(4-prop-2-enoylpiperazin-1-yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][1,3]oxazin-2-one or a pharmaceutically acceptable salt thereof.

[0051] In another embodiment, the first compound is 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one.

[0052] In another embodiment, the first compound is: [ka] (Compound A) or a pharmaceutically acceptable salt thereof. In another embodiment, the first compound is Compound A.

[0053] Methods for assaying mutant IDH1 and IDH2 enzyme activity are known to those skilled in the art and are described, for example, in WO2018 / 111707A1.

[0054] As used above, and throughout the description of the present invention, the following terms, unless otherwise specified, shall be understood to have the following meanings:

[0055] The term "blood tissue" refers to blood, bone marrow, spleen, lymph nodes, or lymph fluid.

[0056] The term "solid tumor tissue" refers to tissue that is not blood tissue. Non-limiting examples of solid tissue are bile duct tissue, pancreatic tissue, head tissue, neck tissue, liver tissue, skin tissue, astrocyte tissue, oligodendrocyte tissue, glial tissue, brain tissue, bladder tissue, colorectal tissue, and lung tissue.

[0057] As used herein, a "Bcl-2 inhibitor" refers to a compound that binds to Bcl-2 and causes one or more of the following: cytotoxicity in cancer cells, downregulation of Bcl-2 expression in cancer cells, mitochondrial dysfunction in cancer cells, and apoptosis in cancer cells. Methods for determining the effect are known to those skilled in the art and are described, for example, in Wen M, et al., Front. Pharmacol. 2019; 10: 391.

[0058] The term "mutant IDH inhibitor" refers to a compound that inhibits the enzymatic activity and / or production of 2-HG by a mutant IDH enzyme (e.g., a mutant IDH1 enzyme or a mutant IDH2 enzyme). Methods for assaying mutant IDH1 and mutant IDH2 enzyme activity are known to those skilled in the art and are described, for example, in WO2018 / 111707A1. In the term "mutant IDH inhibitor," the word "mutation" refers to the IDH gene, not the inhibitor.

[0059] The term "solid tumor cancer" means that the cancer originates in tissue that is not the blood or bone marrow.

[0060] The term "hematologic malignancy" relates to cancers that originate in the blood, bone marrow, lymph nodes, or lymphatic fluid.

[0061] The term "frontline cancer" means that for the cancer being treated, no human cancer subjects have been previously treated.

[0062] The term "refractory cancer" refers to cancer that has been treated but in which the human cancer subject has not responded to the treatment.

[0063] The term "recurrent cancer" means that a human cancer subject has responded to treatment for a period of time, but the cancer has returned.

[0064] The term "advanced cancer" refers to cancer that has spread to lymph nodes or other tissues other than the site of origin of the cancer, for example, AML that has spread to tissues other than the blood or bone marrow.

[0065] The term "cancer subject" means a subject diagnosed with cancer.

[0066] The term "solid tumor subject" means a subject diagnosed with a solid tumor cancer. In one embodiment, the solid tumor cancer is cholangiocarcinoma.

[0067] The term "hematological malignancy subject" refers to a subject diagnosed with a hematological malignancy. In one embodiment, the hematological malignancy subject is an AML subject. The term "AML subject" refers to a subject diagnosed with AML. Methods for diagnosing AML are known to those skilled in the art and are described, for example, in Dohner H, et al., Blood 2017; 129: 424-447. 。

[0068] The terms "acute myeloid leukemia," "acute myelogenous leukemia," and "acute nonlymphocytic leukemia" are synonymous.

[0069] 「 "Response to hematologic malignancy (e.g., AML) treatment" includes improved overall survival, partial response to treatment, prolonged stable disease, or improved long-term survival characterized by complete remission (defined by less than 5% myeloblasts in the bone marrow or the absence of circulating blasts) or hematologic recovery (evidenced by no red blood cell transfusions, absolute peripheral blood neutrophil count greater than 1,000 cells / μL, platelet count greater than 100,000 / μL, and the absence of extramedullary disease) (Bloomfield CD, et al., Blood Revs. 2018;32:416-425). 。

[0070] The term "IDH1 R132 mutation" is synonymous with "R132 IDH1 mutation" and refers to an IDH1 mutation at amino acid residue 132 of a subject's IDH1 gene, eg, as determined in the subject's nucleic acid (eg, DNA).

[0071] The term "identified as having an IDH mutation" means that nucleic acid (e.g., DNA) from tissues or cells of a human subject has been analyzed to determine whether the human subject has an IDH mutation (e.g., an IDH1 R132 mutation, an IDH2 R140 mutation, or an IDH2 R172 mutation). In one embodiment, the blood, blood cells, bone marrow, bone marrow cells, lymph nodes, lymph node cells, lymph, or lymph cells of the human subject have been analyzed for IDH mutations. In another embodiment, the solid tissue of the human subject has been analyzed for IDH mutations. In another embodiment, the solid tissue of the human subject has been analyzed for IDH mutations.

[0072] In the methods of the present invention, the party who identifies the human subject as having an IDH mutation (e.g., an IDH1 R132 mutation, an IDH2R140 mutation, or an IDH2 R172 mutation) can be different from the party who administers the first and second compounds. In one embodiment, the party who identifies the human subject as having an IDH mutation (e.g., an IDH1 R132 mutation, an IDH2R140 mutation, or an IDH2 R172 mutation) is different from the party who administers the first and second compounds.

[0073] Analytical methods for identifying genetic mutations are known to those skilled in the art (Clark, O., et al., Clin. Cancer. Res. 2016; 22: 1837-42), including, but not limited to, chromosome analysis (Guller JL, et al., J. Mol. Diagn. 2010; 12: 3-16), fluorescence in situ hybridization (Yeung DT, et al., Pathology 2011; 43: 566-579), Sanger sequencing (Lutha, R et al., Haematologica 2014; 99: 465-473), metabolic profiling (Miyata S, et al., Scientific Reports 2019; 9: 9787), polymerase chain reaction (Ziai, JM and AJ Siddon, Am. J. Clin. Pathol. 2015; 144: 539-554), and next-generation sequencing (e.g., whole transcriptome sequencing) (Lutha, R et al., Haematologica 2014; 99: 465-473; and Wang HY, et al., J. Exp. Clin. Cancer Res. 2016; 35: 86).

[0074] The term "in combination with" means that a compound of Formula I is or will be used in combination simultaneously, separately or sequentially with a Bcl-2 inhibitor in the treatment of cancer.

[0075] The terms "treatment," "treat," and "treating" and the like are meant to include slowing, preventing, or halting the progression of cancer. These terms also include alleviating, ameliorating, attenuating, eliminating, or alleviating one or more symptoms of a disorder or condition, even if the cancer is not actually eliminated and even if the progression of the cancer itself is not slowed, prevented, or halted.

[0076] "Therapeutically effective amount" refers to the amount of a compound or a pharmaceutically acceptable salt thereof administered to a subject that elicits a biological or medical response or desired therapeutic effect in the subject. A therapeutically effective amount can be readily determined by the attending clinician, as one skilled in the art, by observing results obtained under analogous circumstances using known techniques. In determining the effective amount for a subject, the attending clinician will consider numerous factors, including, but not limited to, the subject's size, age, and general health, the specific disease or disorder involved, the extent, involvement, or severity of the disease or disorder, the individual subject's response, the specific compound administered, the method of administration, the bioavailability characteristics of the administered formulation, the selected dosage regimen, the use of concomitant medications, and other relevant circumstances.

[0077] The compound administered in the method of the present invention can be optionally formulated as a pharmaceutical composition that can be administered by any route that makes the compound bioavailable, including oral, intravenous, or transdermal routes. In one embodiment, such a composition is formulated for oral administration. Such pharmaceutical compositions and methods for their preparation are well known in the art. (See, for example, Remington: The Science and Practice of Pharmacy (DB Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006)).

[0078] A "pharmaceutically acceptable carrier, diluent, or excipient" is a medium generally accepted in the art for delivery of a biologically active agent to a mammal, eg, a human.

[0079] Those skilled in the art will understand that the compound administered in the method of the present invention can form salt.Compound can react with any of many inorganic and organic acids to form pharmaceutically acceptable acid addition salt.Such pharmaceutically acceptable acid addition salts and the general methodology for preparing them are well known in the art.For example, see P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA / Wiley-VCH, 2008).

[0080] "Pharmaceutically acceptable salts" or "pharmaceutically acceptable salts" refer to the relatively non-toxic inorganic and organic salts or bases of the compounds of the present invention (SM Berge, et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977). [Example]

[0081] material and method 2-HG inhibition assay Cell line composition MOLM14 wild-type human leukemia cells and the MOLM14_R132 construct cell line were generated by lentiviral transfection of MOLM14 cells with IDH1 WT (doxycycline-inducible) pSLIK-IDH1-FLAG (Addgene plasmid #66802) and IDH1 R132H (doxycycline-inducible) pSLIK-IDH1-R132H-FLAG (Addgene plasmid #66803), and were kindly provided by Dr. Jean-Emmanuel Salley (Centre de Recherches en Cancerologie de Toulouse, UMR1037, Inserm, Université de Toulouse 3 Paul Sabatier, Toulouse, France; bioRxiv 749580; doi.org / 10.1101 / 749580). MOLM14-WT and MOLM14-R132 mutant cells are seeded (1 x 10) in 6-well plates in 5 mL of complete RPMI medium containing 10% FBS (Sigma) and 1x Pen / Strep (Sigma). 6 / well). 2 μg / ml doxycycline Guidance by Afterwards, the cells are incubated at 37°C for 4 days.

[0082] Cells are seeded in triplicate in 1 mL of 12-well plates (100,000 cells / well). Cells are treated with 2 μg / ml doxycycline to induce IDH1 expression, then treated with DMSO, or 0.1 or 1 μM Compound A, and incubated at 37° C. for 4 days.

[0083] 65 μL of medium is collected and centrifuged, and 60 μL of supernatant medium is collected and frozen at -80°C.

[0084] LCMS 2-HG metabolite analysis. The effect of IDH1 inhibition on the concentrations of total 2-HG and α-KG is determined by liquid chromatography-mass spectrometry (LC-MS) analysis of cell culture supernatants. A calibration curve is generated by spiking 2-HG and α-KG into cell culture medium. This method utilizes derivatization with O-benzylhydroxylamine prior to analysis by LC-MS. 10 μL of each standard or sample is placed in a deep-well 96-well plate and combined with 100 μL of an internal standard solution containing 10 μM d5-2-hydroxyglutarate and 10 μM d6-α-KG. 50 μL of 1M O-benzylhydroxylamine in pyridine buffer (8.6% pyridine, pH 5) and 50 μL of 1M N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) in pyridine buffer are added to each sample. The derivatization reaction is allowed to proceed at room temperature for 1 hour. Using a Beckman Biomek FX Liquid Handler, 300 μL of ethyl acetate was added to each sample. The plate was sealed, vortexed for 5 minutes, and then centrifuged at 4000 rpm in an Eppendorf 5810R centrifuge for 5 minutes. 220 μL of the upper layer was transferred to a new 96-well plate. The samples were dried under heated nitrogen at 50°C and reconstituted with 100 μL of methanol / water (1:1). 1 μL of the derivatized sample was injected into an LC-MS system consisting of a Shimadzu Prominence 20A HPLC system and a Thermo Quantum Ultra™ triple quadrupole mass spectrometer. Analytes were separated on a Water XBridge™ C18 column (2.1 x 50 mm, 3.5 μm) at a flow rate of 0.6 mL / min. Mobile phase A was 0.1% formic acid in water, and mobile phase B was methanol. The gradient profile was: 0 min, 5% B; 2 min, 100% B; 4.00 min, 100% B; 4.1 min, 5% B; 5.50 min, and stop. The mass spectrometer utilized a HESI-II probe operated in positive ion selective reaction monitoring mode. Calibration curves were generated by plotting analyte concentration vs. analyte / internal standard peak area ratio and performing a quadratic fit of the data using 1 / concentration weighting in Xcalibur™ software. Analyte concentrations of unknowns were back-calculated from the calibration curve.

[0085] In experiments carried out essentially as described above, the results in Table 1 are obtained. [Table 1]

[0086] The results in Table 1 demonstrate that Compound A inhibits 2-HG production by mutant IDH1 in the MOLM14_R132 cell line construct.

[0087] Cell viability assay MOLM14 wild-type and MOLM14_R132 cells are treated with 2 μg / ml doxycycline to induce expression of IDH1 WT and IDH1-R132H, and then incubated at 37° C. for 4 days. 1 mL of growth medium containing 2 μg / mL doxycycline Seed 10,000 cells / well into 4 (12-well) plates in a medium.

[0088] Cells were treated with DMSO (in triplicate) or 0, 125, 250, 500, or 1000 nM of Compound A (each in triplicate) as described below. 。 After the initial incubation described above, 0.5 mL of medium containing 1x doxycycline + 1x Compound A or DMSO is added and incubated for 3 days. After 3 days, another 0.5 mL aliquot of fresh medium containing 1x doxycycline + 1x Compound A or DMSO is added and incubated for an additional 3 days. Venetoclax (0, 25, 50, 100, or 200 nM) is then added as a single agent or in combination with 1x Compound A. R After 2 days, collect cells in flow cytometry filtration tubes. ,centre Process the cells for flow cytometry. Wash the cells with Annexin V Binding Buffer (ABB, 2 mL) [1 M Hepes buffer (10 mL), 5 M NaCl (28 mL), 1 M CaCl2 (5 mL), HO (957 mL)] and then centrifuge at 1500 rpm for 5 minutes. Stain the cells with ABB (50 μL) and then with APC Annexin V antibody (1 μL, BioLegend, Cat. No. 640941).、2 Place in darkness for 0 minutes Ku Wash the cells with ABB (2 mL) and centrifuge at 1500 rpm for 5 min. do DAPI (Invitrogen Cat. No. D3571) and counting beads (Invitrogen Cat. No. C36950) were added to Annexin V binding buffer [DAPI (5 μL, 2 g / mL) + counting beads (5 μL (520,000 beads / 50 μL)) + ABB (150 μL)] to a total volume of 160 μL / tube, and then analyzed on a Gallios flow cytometer (Beckman Coulter). do Cell collection is stopped when the counting beads reach 250 beads / sample. Kalusa software is used for analysis. The percentage of gated Annexin V-negative / DAPI-negative cells constitutes the % viable cells, while the percentage of Annexin V-positive cells constitutes the % apoptotic cells.

[0089] In experiments carried out essentially as described above, the results in Tables 2A-2C and 3 are obtained. [Table 2]

[0090] The results in Tables 2A-2C show that the α- and β-blockers were treated with either compound alone. Ta It has been demonstrated that the combination of venetoclax and Compound A results in a further reduction in leukemia cell viability compared to the level of leukemia cell viability in the cells. [Table 3]

[0091] The results in Table 3 demonstrate that the combination of venetoclax and Compound A results in increased levels of apoptosis in leukemia cells compared to the levels of apoptosis in cells treated with each compound alone.

[0092] AML-PDX model Male NSG mice (6–9 weeks old, Jackson Laboratories) were irradiated with 250 cGy, and the following day, AML-PDXs (1 × 10 6 cells / 100 μL) intravenously do Peripheral blood is collected via the retro-orbital route, processed, and hCD45+ cells are measured by flow cytometry to confirm the development of leukemia.

[0093] Once leukemia engraftment exceeds 1%, mice are randomly assigned to treatment with vehicle, Compound A (10 mg / kg, daily by gavage), venetoclax (50 mg / kg, 2 weeks by gavage followed by 1 week off (2 cycles)), or a combination of venetoclax (50 mg / kg, 2 weeks by gavage followed by 1 week off (2 cycles)) and Compound A (10 mg / kg, daily by gavage). Additional dosing beyond these two cycles may be utilized based on assessment of the level of residual disease remaining after 6 weeks of treatment.

[0094] Peripheral blood was collected biweekly via the retro-orbital route, processed, and analyzed by flow cytometry to measure leukemia burden (% hCD33+ / hCD45+ cells) and differentiation (% hCD14+ cells among total hCD45+ cells). 65 μL of staining / washing buffer (5% heat-inactivated (HI)-FBS in DPBS) was added to each tube, mixed by pipetting up and down 3-4 times, and 100 μL of blood suspension was transferred to a 5 mL polypropylene tube. Staining was performed according to the manufacturer's instructions (anti-human CD33-APC (BD Biosciences catalog no. 551378), anti-human CD14-PE-Cy7 (BD Biosciences catalog no. 557742), and anti-human CD45-APC-Cy7 (BD Biosciences catalog no. 557833)). Samples were incubated at room temperature for 30 min, protected from light. After incubation, 1.5 mL of 1XBD Lyse / Fix buffer (37°C, (BD#558049)) is added to each sample and incubated at room temperature for 12 minutes. The tubes are then centrifuged at 1500 rpm for 5 minutes. The BD Lyse / Fix solution is aspirated, and the cell pellet is washed twice with stain / wash buffer. The fixed cells are then resuspended in 200 μL of stain / wash buffer and then transferred to a filtration flow tube. Samples are analyzed on a Gallios flow cytometer (Beckman, Texas) using standard flow cytometry principles and techniques. Population gating and percent population data analysis are performed with Flow Jo software.

[0095] In experiments carried out essentially as described above, the results in Tables 4 and 5 are obtained. [Table 4] [Table 5]

[0096] The results in Tables 4 and 5 demonstrate that while the combination of venetoclax and Compound A does not result in an increased level of AML cell differentiation, the combination does result in a reduction in leukemia burden compared to that obtained with either compound alone, and reducing leukemia burden is a relevant clinical endpoint.

Claims

1. 1. A compound of the formula: 【Chemical 1】 [In the formula: R １ Ha-CH ２ CH (CH ３ ) ２ , -CH ２ CH ３ , -CH ２ CH ２ OCH ３ , or -CH ２ -cyclopropyl; R ２ Ha-CH ３ or -CH ２ CH ３ and X is N or CH. or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition of claim 1 , wherein the IDH mutation is an IDH1 mutation.

3. The pharmaceutical composition according to claim 2 , wherein the IDH1 mutation is an IDH1 R132 mutation.

4. The pharmaceutical composition of claim 1 , wherein the IDH mutation is an IDH2 mutation.

5. The pharmaceutical composition according to claim 4, wherein the IDH2 mutation is an IDH2 R140 or IDH2 R172 mutation.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein X is N.

7. R １ Ga-CH ２ The pharmaceutical composition according to any one of claims 1 to 6, wherein the aryl group is -cyclopropyl.

8. R ２ Ga-CH ２ CH ３ The pharmaceutical composition according to any one of claims 1 to 7, wherein

9. The compound is: 7-[[(1S)-1-[4-[(1R)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; 7-[[(1S)-1-[4-[(1S)-2-cyclopropyl-1-(4-prop-2-enoylpiperazin-1-yl)ethyl]phenyl]ethyl]amino]-1-ethyl-4H-pyrimido[4,5-d][1,3]oxazin-2-one; or 1-ethyl-7-[[(1S)-1-[4-[1-(4-prop-2-enoylpiperazin-1-yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][1,3]oxazin-2-one The pharmaceutical composition according to any one of claims 1 to 5, which is a pharmaceutically acceptable salt thereof.

10. The compound is: 【Chemistry 2】 The pharmaceutical composition according to any one of claims 1 to 5, which is a pharmaceutically acceptable salt thereof.

11. The compound is: 【Chemistry 3】 The pharmaceutical composition of claim 10, wherein

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the cancer is a solid tumor cancer.

13. 13. The pharmaceutical composition of claim 12, wherein the solid tumor cancer is cholangiocarcinoma, head and neck cancer, chondrosarcoma, hepatocellular carcinoma, melanoma, pancreatic cancer, astrocytoma, oligodendroglioma, glioma, glioblastoma, bladder cancer, colorectal cancer, or lung cancer.

14. The pharmaceutical composition of claim 13, wherein the solid tumor cancer is cholangiocarcinoma.

15. The pharmaceutical composition of any one of claims 1 to 11, wherein the cancer is a hematological malignancy.

16. 16. The pharmaceutical composition of claim 15, wherein the hematological malignancy is acute myeloid leukemia, myelodysplastic syndrome myeloproliferative neoplasm, angioimmunoblastic T-cell lymphoma, T-cell acute lymphoblastic leukemia, polycythemia vera, essential thrombocythemia, or primary myelofibrosis.

17. 17. The pharmaceutical composition of claim 16, wherein the hematological malignancy is acute myeloid leukemia.

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