Treatment of acute myeloid leukemia or lymphoma

Abstract

The present invention provides a method of treating a subject with acute myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma, comprising administering to the subject compound (I): TIFF2025500977000009.tif3068 or a pharma- ceutically acceptable salt thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 292,481, filed December 22, 2021. The entire contents of the aforementioned application are incorporated herein by reference.

[0002]

[0002] Disclosed herein is a method for treating subjects with acute myeloid leukemia (AML), as well as different subtypes of acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma, or diffuse large B-cell lymphoma (DLBCL) with 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)thieno[2,3-b]pyridin-6(7H)-one (Compound (I)) or a pharma- ceutically acceptable salt thereof. [Background technology]

[0003]

[0003] Hematopoietic progenitor kinase 1 (HPK1) is a Ste20 serine / threonine kinase restricted by hematopoietic cells. HPK1 kinase activity can be induced by activation signals generated by a variety of different cell surface receptors found on hematopoietic cells after binding to ligands. Agents that inhibit HPK1 have the potential to treat cancer. A number of potent HPK1 inhibitors are disclosed in U.S. Patent Nos. 10,501,474 and 11,059,832, the teachings of which are incorporated herein by reference in their entirety. The structure of one inhibitor disclosed in these patents is shown below as compound (I).

[0004] [ka]

[0005]

[0004] Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow. This type of cancer usually gets worse quickly if not treated. It is the most common type of acute leukemia in adults.

[0006] The development and persistence of leukemia may be due in part to alterations in (anti)apoptotic gene expression. Genome-wide transcriptome analysis revealed that 89 apoptosis-related genes were upregulated in CD34 and CD4+ cells in AML patients. + Cells and normal bone marrow (NBM) CD34 + Among these, transforming growth factor-β-activated kinase 1 (TAK1) was found to be differentially expressed between AML CD34 cells and AML CD34 cells. + TAK1 was strongly upregulated in AML cells. Genetic downregulation or pharmacological inhibition of TAK1 activity strongly impaired survival of primary AML cells and cobblestone formation in stromal cocultures. TAK1 inhibition was primarily due to blockade of the nuclear factor kB (NF-kB) pathway, whereby TAK1 inhibition reduced phosphorylated IkBa levels and p65 activity. Overexpression of a constitutively active mutant of NF-kB partially rescued TAK1-deficient cells from apoptosis. Importantly, NBM CD34 + The cells were AML CD34 + TAK1 was less sensitive to TAK1 inhibition compared to AML CD34 cells. Knockdown of TAK1 also significantly prevented leukemia development in vivo and improved overall survival in a humanized xenograft mouse model. + It is known that TAK1 is often overexpressed in cells, and inhibition of TAK1 effectively targets leukemia stem / progenitor cells in an NF-kB-dependent manner. However, since overexpression of NF-kB only partially rescues the phenotype, it cannot be excluded that other pathways besides NF-kB are affected. See Bosman et al., Blood 2014, 124(20):3130-3140.

[0007]

[0006] Internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (FLT3) gene have been described in approximately 20% of patients with adult AML; a lower incidence (5%-16.5%) has been reported in childhood leukemia. See Thiede et al., Blood 2002, 99(12):4326-4335. In AML patients, FLT3-ITD mutations were found to be associated with increased white blood cell counts and to be more frequent in patients lacking other cytogenetic abnormalities. More recently, point mutations at codon 835 of the FLT3 gene have been described in approximately 7% of patients with AML. These mutations are located in the activation loop of the second tyrosine kinase domain (TKD) of FLT3 and constitutively activate the protein.

[0008]

[0007] In addition to juxtamembrane domain mutations, tyrosine kinase domain mutations (FLT3-TKD mutations) have been described in AML. FLT3-TKD mutations are small mutations in the activation loop of FLT3, mostly representing point mutations at codon D835 or deletions at codon I836. These mutations induce constitutive tyrosine phosphorylation that activates the receptor tyrosine kinase and are hypothesized to be gain-of-function mutations. See Bacher et al., Blood 2008, 111(5):2527-2537.

[0009]

[0008] The mixed leukemia 1 (MLL1) gene (now renamed lysine [K]-specific methyltransferase 2A or KMT2A) located on chromosome 11q23 is disrupted in a unique group of acute leukemias. The majority of leukemias result from MLL1 fusions with one of about six common partner genes, but more than 80 different partner genes in these fusions have been described. Approximately 10% of all leukemias have MLL1 translocations. See Winters et al., Front. Pediatr. 2017, 5(4):1-21. t(9;11)(p21-22;q23) identifies the second largest group of translocations involving 11q23 as acquired abnormalities in hematopoietic malignancies. t(9;11) is most often associated with acute myeloid leukemia (AML), usually FAB type with substantial monocytic components, such as M4 and M5. At the molecular level, the typical rearrangement is between MLL (also known as ALL1, Htrx, HRX) at 11q23 and genes known as AF9, MLLT3 or LTG9 at 9p21-22. See Swansbury et al., Leukemia 1998, 12:792-800. Summary of the Invention [Problem to be solved by the invention]

[0010]

[0009] The various subtypes of AML mentioned above, as well as ALL, NHL, Burkitt's lymphoma, or DLBCL, are difficult to treat. The 5-year overall survival rate for the majority of AML cases ranges from 5-15% in the elderly and 30% in young adults. See Kuenzi et al. Scientific Reports 2019, 9:606. This lack of improvement in patient survival is mainly due to the limited effectiveness of currently available therapies in AML. Therefore, there is a need to develop new drugs to treat these diseases. [Means for solving the problem]

[0011]

[0010] The present disclosure is based on the unexpected discovery that compound (I), identified above, actively inhibits transforming growth factor beta-activated kinase 1 (TAK1) (see Example 1). Furthermore, compound (I) selectively inhibits the proliferation of FLT3-ITD-expressing AML cells and 32D cell transfectants (see Example 5).

[0012] In one aspect, the disclosure provides a method of treating a subject with acute myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma, comprising administering to the subject a compound (I):

[0013] [ka]

[0014] or a pharma- ceutical acceptable salt thereof, wherein the acute myeloid leukemia is FLT3 mutated acute myeloid leukemia Acute myeloid leukemia with MLL-AF9 translocation. Overexpressing wild-type FLT3 Overexpression of transforming growth factor-β-activated kinase 1 (TAK1) TAK1 is mutated, or Acute myeloid leukemia with increased TAK1 signaling A method is provided.

[0015]

[0012] In another aspect, the present disclosure provides the use of compound (I) or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for treating a subject with different subtypes of acute myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma, as described above.

[0016]

[0013] In another aspect, the present disclosure provides compound (I) or a pharma- ceutically acceptable salt thereof for use in treating a subject with different subtypes of acute myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma, as described above. [Brief description of the drawings]

[0017] [Figure 1]

[0014] Figure 1A is a graph showing the inhibitory effect of compound (I) tartrate on FLT3. Figure 1B is a graph showing the inhibitory effect of compound (I) tartrate on FLT3-ITD. Figure 1C is a graph showing the inhibitory effect of compound (I) tartrate on FLT3(D835Y). Figure 1D is a graph showing the inhibitory effect of compound (I) tartrate on TAK1 / MAP3K7. [Diagram 2]

[0015] Figure 2A is a graph showing the inhibitory effect of Compound (I) tartrate on SLP-76 serine 376 phosphorylation in anti-CD3 antibody stimulated Jurkat E6.1 cells. Figure 2B is a graph showing the inhibitory effect of Compound (I) tartrate on phospho-FLT3 in MV-4-11 cells. [Diagram 3]

[0016] Figure 3A is a graph showing the change in tumor volume in female CD-1 nude mice with human AML under treatment with Compound (I) tartrate or vincristine. Figure 3B is a graph showing the change in body weight of experimental animals. PO: per-os (oral administration); IP: intraperitoneal administration; QD: quaque die (once a day); QW: quaque (once a week); TGI: tumor growth inhibition. [Figure 4]

[0017] Figure 4A shows that compound (I) tartrate treatment increases the expression of the mature 160 kDa form of FLT3-ITD in MV-4-11 cells (heterozygous "LOH" FLT3-deficient, FLT3-ITD positive cells) in a dose-dependent manner. Figure 4B shows the increase in cell surface expression of FLT3-ITD in MV-4-11 cells treated with 100 nM compound (I) tartrate or DMSO control for 4 hours at 37°C and 5% CO2. Graph showing the increase in cell surface expression of FLT3-ITD in MV-4-11 cells stained with PE-FLT3 (Beckman Coulter, Cat. IM2234U) or isotype control antibody. [Diagram 5]

[0018] FIG. 5A is a graph showing that Compound (I) tartrate treatment (48 and 72 hours at 37° C. and 5% CO2, stained with APC-CD45, PECy7-CD38, APC / Fire 750-CD33, PerCP-CD14, PE-FLT3 (Beckman Coulter, Cat. IM2234U), PE / Dazzle 594-CD117 (BioLegend, Cat. No. 313226) or isotype control antibodies) increases cell surface expression of FLT3-ITD in a dose-dependent manner in viable CD14-AML blasts. FIG. 5B is a graph showing that the same Compound (I) tartrate treatment increases cell surface expression of c-KIT (CD117) in viable CD14-AML blasts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018]

[0019] As used herein, “Compound (I)” refers to a compound having the chemical name 4-amino-5-(6-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)thieno[2,3-b]pyridin-6(7H)-one, which has the following structure:

[0019] [ka]

[0020] has.

[0020] Compound (I) has been developed as an HPK1 inhibitor and is disclosed in WO2016 / 205942. The preparation of compound (I) is described in Example A1 of WO2016 / 205942, the entire teachings of which are incorporated herein by reference.

[0021] The methods disclosed herein can be used to treat certain subtypes of AML that are difficult to cure in view of certain mutations. AML is the most common type of acute leukemia. AML develops when the bone marrow begins to make blast cells, cells that are not yet fully mature. These blast cells normally grow into white blood cells. However, in AML, these cells do not grow and are unable to ward off infection.

[0022] In AML, the bone marrow can also make abnormal red blood cells and platelets. The number of these abnormal cells increases rapidly, and the abnormal (leukemic) cells begin to crowd out normal white blood cells, red blood cells, and platelets that the body needs.

[0023] One of the main features that distinguishes AML from the other major forms of leukemia is that it has eight different subtypes based on the type of cell from which the leukemia arises and its level of maturity. The types of acute myeloid leukemia include: Myeloblastic (M0) - in special analysis Myeloblastic (M1) - no maturation Myeloblastic (M2) - with maturation Myeloid (M3) Myelomonocytic (M4) Monocytic (M5) Erythroleukemia (M6) · Megakaryocytic (M7)

[0024] It was unexpected that Compound (I) was found to be effective against FLT3-mutated acute myeloid leukemia. Thus, in one aspect, the present disclosure provides a method of treating a subject with acute myeloid leukemia, where the acute myeloid leukemia is FLT3-mutated acute myeloid leukemia. In some embodiments, the FLT3 mutation is an FLT3-internal tandem duplication (ITD) mutation and / or an FLT3-tyrosine kinase domain (TKD) mutation. Several methods have been developed to identify FLT3 mutations. These include polymerase chain reaction (PCR)-based approaches, next-generation sequencing (NGS) approaches, and multiplex targeted NGS (i.e., gene panel) approaches. See Daver et al., Leukemia 2019, 33:299-312. In some embodiments, the acute myeloid leukemia has a D835 mutation, for example, D835Y. In some embodiments, the acute myeloid leukemia is FLT3 mutated acute myeloid leukemia and the AML is selected from AML M1, AML M2, AML M3, AML M4, AML M5, AML M6, and AML M7, e.g., AML M5.

[0024]

[0025] The present disclosure provides a method for treating a subject with acute myeloid leukemia, wherein the acute myeloid leukemia is acute myeloid leukemia with MLL-AF9 translocation.In some embodiments, AML is selected from AML M1, AML M2, AML M3, AML M4, AML M5, AML M6 and AML M7, for example, AML M4 or AML M5.

[0025]

[0026] The present disclosure also provides a method for treating a subject with acute myeloid leukemia, wherein the acute myeloid leukemia overexpresses TAK1, or TAK1 is mutated, or TAK1 signaling is increased. In some embodiments, the AML is selected from AML M1, AML M2, AML M3, AML M4, AML M5, AML M6, and AML M7, for example, AML M4.

[0026]

[0027] It is known that TAK1 is important for the survival of FLT3-mutated AML cells (Shanmugam Clin Cancer Res 2012, vol. 18(2), pp. 360-369), NHL / DLBCL cells (Palakurthi AACR Annual Meeting 2008; Ansell Blood Cancer Journal 2014, 4, e183; Wu Cell Biochem Funct 2019, vol. 37, pp. 153-160) and MLL-AF9 leukemia cells (Carretta PLoS ONE 2017, pp. 1-18).

[0027]

[0028] Thus, the present disclosure also provides a method of treating a subject with acute myeloid leukemia, where the acute myeloid leukemia is FLT3 mutated acute myeloid leukemia (e.g., has an FLT3-ITD mutation or a TKD mutation), or has an MLL-AF9 translocation and overexpresses TAK1, or is TAK1 mutated, or has increased TAK1 signaling.

[0028]

[0029] In some embodiments, the AML being treated is relapsed or refractory.

[0030] In some embodiments, the treated acute lymphoblastic leukemia is relapsed or refractory. In some embodiments, the treated ALL is mixed karyotype acute lymphoblastic leukemia.

[0029]

[0031] In some embodiments, the acute lymphocytic leukemia being treated is T-cell acute lymphocytic leukemia. In some embodiments, the acute lymphocytic leukemia being treated is B-cell acute lymphocytic leukemia.

[0030]

[0032] In some embodiments, the non-Hodgkin's lymphoma being treated is relapsed or refractory. In some embodiments, the non-Hodgkin's lymphoma being treated is a mixed karyotype non-Hodgkin's lymphoma.

[0031]

[0033] In some embodiments, the Burkitt lymphoma being treated is relapsed or refractory, hi some embodiments, the Burkitt lymphoma being treated is complex karyotype Burkitt lymphoma.

[0032]

[0034] In some embodiments, the diffuse large B-cell lymphoma being treated is relapsed or refractory. In some embodiments, the diffuse large B-cell lymphoma being treated is a mixed karyotype diffuse large B-cell lymphoma.

[0033]

[0035] In some embodiments, the diffuse large B cell lymphoma being treated is germinal center B cell-like. In some embodiments, the diffuse large B cell lymphoma being treated is activated B cell-like.

[0034]

[0036] In some embodiments, the present teachings provide a method of treating a subject with acute myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma, comprising administering to the subject an effective amount of Compound (I) in combination with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-cancer drug.

[0035]

[0037] An "anti-cancer drug" is a compound that, when administered in an effective amount to a subject with cancer, can partially or substantially accomplish one or more of the following: halting the growth of the cancer, reducing the extent of the cancer (e.g., reducing the size of a tumor), inhibiting the rate of growth of the cancer, and ameliorating or improving clinical symptoms or indicators associated with the cancer (e.g., tissue or serum components), or increasing the life span of the subject.

[0036]

[0038] Anti-cancer drugs suitable for use in the methods described herein include anti-cancer drugs approved for the treatment of cancer. In one embodiment, anti-cancer drugs include, but are not limited to, targeted antibodies, angiogenesis inhibitors, alkylating agents, antimetabolites, vinca alkaloids, taxanes, podophyllotoxins, topoisomerase inhibitors, anti-tumor hormone drugs and other anti-tumor drugs.

[0037]

[0039] In one embodiment, anti-cancer drugs that can be used in the methods described herein include, but are not limited to, paclitaxel, docetaxel, 5-fluorouracil, trastuzumab, lapatinib, bevacizumab, letrozole, goserelin, tamoxifen, cetuximab, panitumumab, gemcitabine, capecitabine, irinotecan, oxaliplatin, carboplatin, cisplatin, doxorubicin, epirubicin, cyclophosphamide, methotrexate, vinblastine, vincristine, melphalan, cytarabine, etoposide, daunorubicin, bleomycin, mitomycin, and adriamycin, and combinations thereof.

[0038]

[0040] In one embodiment, the anticancer drug is venetoclax. In one embodiment, the anticancer drug is 5-azacytidine. In one embodiment, the anticancer drug is decitabine.

[0039]

[0041] In the methods disclosed herein, Compound (I) and the additional therapeutic agent are administered simultaneously or sequentially.

[0042] The present teachings include pharma- ceutically acceptable salts of compound (I). Compound (I) has a basic amine group, and therefore can form pharma- ceutically acceptable salts with pharma- ceutically acceptable acids. Suitable pharma- ceutically acceptable acid addition salts of compound (I) include salts of inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid) and salts of organic acids (e.g., acetic acid, benzenesulfonic acid, benzoic acid, ethanesulfonic acid, methanesulfonic acid, succinic acid, and trifluoroacetic acid).

[0040]

[0043] In some embodiments, the present disclosure provides Compound (I) as a tartrate salt. In certain embodiments, the molar ratio between Compound (I) and tartaric acid is 1:1.

[0044] The present disclosure also includes crystalline forms of Compound (I) or corresponding pharma- ceutically acceptable salts.For example, crystalline forms and their preparation methods are disclosed in International Application No. PCT / CA2021 / 050645, the entire teachings of which are incorporated herein by reference.

[0041]

[0045] The term "effective amount" refers to an amount that, when administered to a subject, produces beneficial or desired results, including clinical outcomes, such as an amount that inhibits, suppresses, or reduces cancer in a subject (e.g., as determined by clinical symptoms or amount of cancer cells) compared to a control. In particular, "treating a subject with cancer" includes partially or substantially achieving one or more of the following: stopping the growth or spread of cancer, reducing the extent of cancer (e.g., reducing the size of a tumor or reducing the number of affected areas), inhibiting the rate of cancer growth, and improving or enhancing clinical symptoms or indicators (e.g., tissue or serum components) associated with cancer.

[0042]

[0046] In general, the effective amount of the compound of the present invention varies depending on various factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject or host to be treated, but can nevertheless be routinely determined by those skilled in the art. The effective amount of the compound of the present invention can be easily determined by those skilled in the art by conventional methods known in the art.

[0043]

[0047] In some embodiments, an effective amount of Compound (I) or a pharma- ceutically acceptable salt thereof ranges from about 0.01 to about 1000 mg / kg body weight, alternatively from about 0.05 to about 500 mg / kg body weight, alternatively from about 0.1 to about 200 mg / kg body weight. One of skill in the art will appreciate that certain factors may affect the dosage required to effectively treat a subject suffering from cancer, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present.

[0044]

[0048] In some embodiments, the methods disclosed herein comprise administering to a subject in need thereof an amount of Compound (I) between 1 mg and 200 mg, or a pharma- ceutically acceptable salt thereof, equivalent to 1 mg and 200 mg of Compound (I) once daily.

[0045]

[0049] As used herein, the terms "treat", "treating" or "treatment", when used in relation to a disorder or condition, include effects that result in the improvement of the disorder or condition, such as the different subtypes of acute myeloid leukemia discussed above, acute lymphocytic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma or diffuse large B-cell lymphoma. Improvement or reduction in severity of any symptoms of the disorder or condition can be readily assessed according to standard methods and techniques known in the art.

[0046]

[0050] As used herein, the term "treatment resistant" refers to a cancer that does not respond to treatment. The cancer may be resistant at the start of treatment or may become resistant during treatment.

[0047]

[0051] Compound (I) and / or pharma- ceutically acceptable salts thereof as described herein are useful as materials for preparing pharmaceutical compositions incorporating active pharmaceutical ingredients (API) and one or more pharma- ceutically acceptable excipients, and are suitable for administration to human subjects.

[0048]

[0052] In some embodiments, the present disclosure provides a pharmaceutical composition comprising Compound (I) and / or a pharma- ceutically acceptable salt thereof and at least one additional pharma- ceutically acceptable excipient. As used herein, the term "pharma- ceutically acceptable excipient" refers to a pharma- ceutically acceptable material, composition, and / or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each excipient must be "pharma- ceutically acceptable" in the sense that it is compatible with the subject composition and its components and is not harmful to the patient. The use of any conventional pharma- ceutical acceptable excipient is contemplated within the scope of the present disclosure, except that it is incompatible with Compound (I) and / or a pharma- ceutical acceptable salt thereof, for example, by causing any undesirable biological effect or by otherwise interacting in a deleterious manner with any other component of the pharma- ceutical acceptable composition.

[0049]

[0053] Some non-limiting examples of materials that may serve as pharma- ceutically acceptable excipients include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and the like. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffers; and (21) other non-toxic compatible substances used in pharmaceutical formulations. Remington: The Science and Practice of Pharmacy, 21st ed., 2005, edited by D.B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York, the contents of each of which are incorporated herein by reference, also disclose additional non-limiting examples of pharma- ceutically acceptable excipients, as well as known techniques for preparing and using the same.

[0050]

[0054] Compound (I) or corresponding pharma-ceutically acceptable salt used in the disclosed method can be administered to patients in various forms depending on the selected route of administration, as understood by those skilled in the art.The compounds of the present teachings can be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and pharmaceutical compositions formulated accordingly.Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration.Parenteral administration can be by continuous infusion over a selected period of time.

[0051]

[0055] Compound (I) or corresponding pharmaceutically acceptable salt used in the disclosed method can be suitably formulated into a pharmaceutical composition for administration to a subject. The pharmaceutical composition of the present teachings may optionally include one or more pharmaceutically acceptable carriers and / or diluents therefor, such as lactose, starch, cellulose and dextrose. Other excipients, such as flavoring agents; sweeteners; and preservatives, such as methyl, ethyl, propyl and butyl paraben, may also be included. A more complete list of suitable excipients can be found in Handbook of Pharmaceutical Excipients (5th Edition, Pharmaceutical Press (2005)). Those skilled in the art should know how to prepare suitable formulations for various types of administration routes. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003-20th Edition) and the United States Pharmacopeia: National Formulary, 1999 (USP24 NF19). The carriers, diluents and / or excipients are "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0052]

[0056] Typically, for oral therapeutic administration, Compound (I) or a corresponding pharma- ceutically acceptable salt used in the methods of the present disclosure will be incorporated into an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0053]

[0057] In general, for parenteral administration, the solution of compound (I) or corresponding pharma-ceutically acceptable salt used in the disclosed method can be prepared in water, generally mixed with a surfactant, such as hydroxypropylcellulose, appropriately. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, DMSO and their mixtures, with or without alcohol, and in oil. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0054]

[0058] Typically, for use in injections, sterile aqueous solutions or dispersions, and sterile powders of Compound (I) or the corresponding pharma- ceutically acceptable salts used in the disclosed methods, for extemporaneous dissolution preparation of sterile injections or dispersions, are suitable.

[0055]

[0059] For nasal administration, the compound (I) or corresponding pharma- ceutically acceptable salt used in the disclosed method can be formulated as aerosols, drops, gels and powders. Aerosol formulations usually contain a solution or fine suspension of active substance in a physiologically acceptable aqueous or non-aqueous solvent, usually presented in a sterile form in a sealed container in single or multiple doses, which can take the form of cartridges or refills for use with a nebulizer device. Alternatively, the sealed container can be an integrated dispensing device, such as a single-dose nasal inhaler or an aerosol dispenser fitted with a metering valve for disposal after use. When the dosage form includes an aerosol dispenser, it contains a propellant that can be a compressed gas, such as compressed air, or an organic propellant, such as a fluorochlorohydrocarbon. The aerosol dosage form can also take the form of a pump-action atomizer.

[0056]

[0060] For buccal or sublingual administration, Compound (I) or a corresponding pharma- ceutically acceptable salt used in the disclosed methods can be formulated as tablets, lozenges or pastilles with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerin.

[0057]

[0061] For rectal administration, Compound (I) or a corresponding pharma- ceutically acceptable salt used in the disclosed methods can be formulated in the form of suppositories containing a conventional suppository base, e.g., cocoa butter. EXAMPLES

[0058]

[0062] The following examples are intended to be illustrative and are not meant to limit the scope of the disclosure in any way.

[0059] Example 1 In vitro cytotoxicity (IC 50 ) Assay

[0063] Kinase IC 50 The IC value was determined by Reaction Biology Corp. (Malvern, PA, USA) in a Radiometric HotSpot™ assay using recombinantly expressed kinase enzyme. 50 The curves were tested in duplicate. Enzymes were tested at the Km[ATP] of each enzyme using the binning structure of Reaction Biology. Kinase reactions were performed using a protein or peptide substrate and [γ- 33 The reaction was initiated by the addition of [P]-adenosine triphosphate (ATP). The phosphorylated substrate was captured by spotting the reaction mix onto a filter membrane, and enzyme activity was quantified by liquid scintillation counting. The substrate and ATP concentrations were as follows:

[0060] FLT3: peptide substrate, [EAIYAAPFAKKK], 5 μM; ATP 50 μM FLT3-ITD (internal tandem duplication): peptide substrate, [EAIYAAPFAKKK], 20 μM; ATP, 30 μM FLT3(D835Y): peptide substrate, [EAIYAAPFAKKK], 5 μM; ATP 20 μM TAK1: casein, 1 mg / mL; ATP, 20 μM

[0064] These results are shown in Figures 1A to 1D. In particular, Compound (I) tartrate inhibited FLT3 via IC 50 Inhibits FLT3-ITD at IC 50 Inhibits FLT3(D835Y) at IC 50 Inhibits TAK1 at IC 50 Inhibitory activity was determined at 18 nM.

[0061] Example 2 In vitro phosphorylation assay A.

[0065] Jurkat E6.1 cells were starved overnight in RPMI-1640 medium (Invitrogen) without fetal bovine serum (FBS). Starved cells (2 × 10 per sample in 1 mL) were cultured at 4 °C for 1 h. 6 (1000 x 1000 cells) were pretreated with Compound (I) tartrate for 3 hours and then stimulated with 5 μg / mL anti-CD3 antibody (clone OKT3) for 15 minutes. Cells were immediately lysed in cell lysis buffer plus protease and phosphatase inhibitors and then assayed for SLP-76 Ser376 phosphorylation with the PathScan Phospho-SLP-76 (Ser376) Sandwich ELISA kit according to the kit instructions (Cell Signaling Technology, Inc.). Duplicate data points for each concentration were collected (Molecular Devices M5). Raw absorbance (A = 450 nm) values ​​were background subtracted and then the individual values ​​were averaged for each Compound (I) tartrate concentration. Cell EC 50 was determined using a simple one-site dose-response curve fitting model (XLfit4, IDBS). Compound (I) tartrate had an EC 50The results are shown in Figure 2A.

[0062] B.

[0066] MV-4-11 cells were grown in RPMI-1640 medium (Invitrogen) containing 10% FBS. Cells (1 × 10 per sample in 1 mL) were 6 ) were treated with Compound (I) tartrate for 4 hours and then lysed. Lysates were tested with PathScan Phospho-FLT3(panTyr)Sandwich ELISA kit according to the kit instructions (Cell Signaling Technology, Inc). Duplicate data points for each concentration were collected (Molecular Devices M5). Raw absorbance (A=450 nm) values ​​were background subtracted and then individual values ​​were averaged for each Compound (I) tartrate concentration. Cell EC 50 was determined using a simple one-site dose-response curve fitting model (XLfit4, IDBS). Compound (I) tartrate was determined to inhibit phospho-FLT3 in MV-4-11 cells, and the EC 50 The results are shown in FIG. 2B.

[0063] Example 3 In vivo efficacy of compound (I) tartrate on xenograft models of human AML

[0067] Eight to ten week old female CD-1 nude mice (Charles River Laboratories) with established MV-4-11 xenografts were treated for 21 days (n=8). MV-4-11 cells are a human AML cell line that endogenously express an Fms-related receptor tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation. Animal body weights were monitored daily and tumor volumes were measured three times a week. Tumor volumes (mm 3 ) as follows: length x width 2 Percentage tumor growth inhibition (%TGI) was defined as 100 × [1-(TV f,処置 -TV i,処置 ) / (TV f,対照 -TV i,対照)] and TV f is the mean tumor volume at the end of the study, and TV i is the average tumor volume at the start of treatment. In cases where tumor shrinkage occurred, the tumor shrinkage rate was calculated as follows: 100 × [1-(TV f,処置 / TV i,処置 After completion of the study, mice were sacrificed by anesthetic overdose. The Institutional Animal Care and Use Committee of the University Health Network approved all animal studies. No tumors were detected in mice receiving Compound (I) tartrate at 50 mg / kg or 150 mg / kg QD orally 10 days after treatment (Figure 3A). In contrast, vincristine had a moderate effect on tumor growth (vincristine 0.5 mg / kg IP, QW×4 vs vehicle, day 10 TGI=62%, p=0.137). Compound (I) tartrate (50 mg / kg) was administered to mice with large tumors (≈900 mm 3 ) and complete tumor regression was observed after 7 days of treatment. Data are presented as mean ± SEM. p values ​​were calculated using Student's t-test.

[0064]

[0068] Compound (I) tartrate was well tolerated as measured by minimal weight loss and normal behavior (Figure 3B). These results indicate that compound (I) tartrate inhibits human AML cell proliferation and may be effective in the clinical setting, even in advanced tumors.

[0065] Example 4 Inhibition of growth of human leukemia and lymphoma cells in vitro by compound (I) tartrate.

[0069] Cell lines were obtained from the American Type Culture Collection (ATCC), maintained according to the supplier's instructions, and obtained from Dr. Mark Minden (Princess Margaret Cancer Centre, Toronto, Canada). Short tandem repeat (STR) profiling was used to verify the authenticity of the cell lines. Peripheral blood from healthy human donors was obtained from the Hematology Malignancy Tissue Bank of the University Health Network. Peripheral blood mononuclear cells (PBMCs) were prepared from blood by density gradient centrifugation using Ficoll-Paque PLUS according to the manufacturer's instructions (GE Healthcare Life Sciences). Growth inhibition IC 50 For the assay, various numbers of cells were seeded in 96-well plates (Thermo Fisher Scientific) according to cell proliferation rate 24 hours before compound overlay and cultured at 37°C with 5% CO2. Compound (I) tartrate was prepared as a 10 mM stock solution in 100% DMSO. Stock solutions were diluted in RPMI-1640 (Invitrogen) containing 10% FBS (fetal bovine serum) to give final concentrations ranging from 300 fM to 300 μM. Aliquots (20 μL) from each concentration were overlayed onto 180 μL of pre-seeded cells to give final concentrations ranging from 30 fM to 30 μM. After 3 days, cell proliferation in each well was assessed by Alamar Blue assay reagent (Invitrogen). Raw fluorescence (lambdaEM=590 nm) values ​​were adjusted by subtracting the average of baseline readings from untreated cells assessed 1 day after cell overlay. Relative cell proliferation was calculated by comparing with cells treated with DMSO. The concentration at which cell proliferation was inhibited by 50% (proliferation inhibition IC 50 ) were calculated using GraphPad PRISM software (GraphPad Software Inc.) The results are shown in Table 1 below.

[0066] [Table 1]

[0067] Example 5 Inhibition of proliferation of FLT3-ITD expressing cells in vitro by compound (I) tartrate

[0070] Cell lines were obtained from the American Type Culture Collection (ATCC), maintained according to the supplier's instructions, and obtained from Dr. Mark Minden. 32D cell transfectants were obtained from Dr. Mark Minden. Wild-type FLT3-expressing 32D cells were maintained in IMDM medium supplemented with 10% heat-inactivated FBS and 50 ng / mL human FLT3 ligand (PeproTech, Inc.). FLT3-ITD-expressing 32D cells were maintained in IMDM medium supplemented with 10% heat-inactivated FBS without cytokines. Growth inhibition IC 50 Assay is carried out as described in Example 4. Compound (I) tartrate selectively inhibits the proliferation of FLT3-ITD expressing AML cells and 32D cell transfectants. The proliferation inhibition of FLT3-ITD expressing 32D cell transfectants is reversed by adding mouse IL-3, indicating the selective inhibition profile of compound (I) tartrate against constitutively active FLT3 kinase. These results are shown in Table 2 below.

[0068] [Table 2]

[0069] Example 6 Compound (I) treatment increases cell surface expression of the glycosylated mature form of FLT3-ITD in AML cells.

[0070]

[0071] MV-4-11 cells (FLT3 LOH, FLT3-ITD positive cells) were treated with the indicated concentrations of Compound (I) tartrate for 4 hours at 37°C and 5% CO2. Lysates were analyzed by immunoblot analysis using antibodies against FLT3 (Cell Signaling Technology, Inc., Cat. No. 3462), PARP (Cell Signaling Technology, Inc., Cat. No. 9542) and GAPDH (Millipore, Cat. No. MAB374). Protein bands were visualized using a LI-COR Odyssey near-infrared imager, and representative data are shown. Compound (I) tartrate treatment increases the expression of the mature 160 kDa form of FLT3-ITD in MV-4-11 cells in a dose-dependent manner. Apoptotic cell death, as measured by PARP cleavage, was induced by Compound (I) tartrate treatment. See Figure 4A. Increased FLT3-ITD maturation following treatment with compound (I) tartrate was also observed in MOLM-14 cells co-expressing FLT3-ITD and wild-type FLT3, thus suggesting that this mechanism is not limited to FLT3 LOH cells (data not shown).

[0071]

[0072] MV-4-11 cells were treated with 100 nM compound (I) tartrate or DMSO control for 4 hours at 37° C. and 5% CO2, stained with PE-FLT3 (Beckman Coulter, catalog IM2234U) or isotype control antibody, and analyzed on a BD LSRFortessa II flow cytometry analyzer. Data was analyzed using FlowJo software, and representative data is shown. As demonstrated in FIG. 4B, compound (I) tartrate treatment increases cell surface expression of FLT3-ITD in MV-4-11 cells.

[0072] Example 7 Compound (I) treatment increases cell surface expression of FLT3-ITD and c-KIT (CD117) in primary human AML cells.

[0073]

[0073] Patient samples were obtained from Dr. Mark Minden (Princess Margaret Cancer Centre, Toronto, Ontario, Canada). FLT3-ITD positive AML cells were treated with the indicated concentrations of Compound (I) for 48 and 72 hours at 37°C and 5% CO2, stained with APC-CD45, PECy7-CD38, APC / Fire 750-CD33, PerCP-CD14, PE-FLT3 (Beckman Coulter, Cat. IM2234U), PE / Dazzle 594-CD117 (BioLegend, Cat. No. 313226) or isotype control antibodies and analyzed on a BD LSRFortessa II flow cytometry analyzer. Data were analyzed using FlowJo software and representative data are shown. Compound (I) treatment reduced viable CD14 - In AML blasts, cell surface expression of FLT3 and c-KIT was increased in a dose-dependent manner. See Figures 5A and 5B. Similar results were obtained in additional FLT3-ITD positive primary AML samples (data not shown).

[0074] Significance: FLT3-ITD is largely sequestered in the endoplasmic reticulum (ER), where constitutive activation of the oncogenic receptor prevents glycosylation, maturation, and translocation to the cell surface. Compound (I) inhibits the activity of FLT3-ITD, thus allowing its processing into a fully glycosylated mature form and increasing cell surface expression. These data demonstrate that compound (I) increases the cell surface expression of FLT3-ITD and c-KIT (CD117) in AML cells, thus providing a rationale for combining compound (I) with FLT3-directed and c-KIT-directed immunotherapies (e.g., monoclonal antibodies, bispecific antibodies, and CAR T cell therapy) for AML patients. This therapeutic strategy can not only overcome the limited availability of FLT3 antigen in FLT3-ITD-positive AML cells, but also prevent adaptive resistance, a frequent problem in single-agent treatment.

Claims

1. 1. A method of treating a subject with acute myeloid leukemia comprising administering to a subject a compound (I): 【Chemistry 1】 or a pharma- ceutical acceptable salt thereof, wherein the acute myeloid leukemia is FLT3 mutated acute myeloid leukemia, acute myeloid leukemia with MLL-AF9 translocation, Overexpressing wild-type FLT3, Overexpressing transforming growth factor-β-activated kinase 1 (TAK1), TAK1 is mutated, or Acute myeloid leukemia with increased TAK1 signaling. The above method.

2. 2. The method of claim 1, wherein the acute myeloid leukemia is FLT3 mutated acute myeloid leukemia.

3. The method of claim 2, wherein the FLT3 mutation is an FLT3-internal tandem duplication (ITD) mutation.

4. The method of claim 2, wherein the FLT3 mutation is a FLT3 tyrosine kinase domain (TKD) mutation.

5. The method of any one of claims 2 to 4, wherein the acute myeloid leukemia has a D835 mutation, such as D835Y.

6. 6. The method of any one of claims 2 to 5, wherein the acute myeloid leukemia is selected from AML M1, AML M2, AML M3, AML M4, AML M5, AML M6, and AML M7.

7. 7. The method of claim 6, wherein the acute myeloid leukemia is AML M5.

8. The method according to claim 1, wherein the acute myeloid leukemia is acute myeloid leukemia associated with MLL-AF9 translocation.

9. 9. The method of claim 8, wherein the acute myeloid leukemia is selected from AML M1, AML M2, AML M3, AML M4, AML M5, AML M6, and AML M7.

10. 10. The method of claim 9, wherein the acute myeloid leukemia is AML M4 or AML M5.

11. The method of any one of claims 1 to 5 and 8, wherein the acute myeloid leukemia overexpresses TAK1, or TAK1 is mutated, or TAK1 signaling is increased.

12. 12. The method of claim 11, wherein the acute myeloid leukemia is selected from AML M1, AML M2, AML M3, AML M4, AML M5, AML M6, and AML M7.

13. 13. The method of claim 12, wherein the acute myeloid leukemia is AML M4.

14. The method according to any one of claims 1 to 13, wherein the acute myeloid leukemia is relapsed or resistant to therapy.

15. 1. A method of treating a subject with acute lymphocytic leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma, comprising administering to the subject compound (I): 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof.

16. 16. The method of claim 15, wherein the subject has acute lymphoblastic leukemia.

17. 17. The method of claim 16, wherein the acute lymphoblastic leukemia is T-cell acute lymphoblastic leukemia or B-cell acute lymphoblastic leukemia.

18. 16. The method of claim 15, wherein the subject has non-Hodgkin's lymphoma.

19. 16. The method of claim 15, wherein the subject has Burkitt's lymphoma.

20. 16. The method of claim 15, wherein the subject has diffuse large B-cell lymphoma.

21. 21. The method of claim 20, wherein the diffuse large B-cell lymphoma is germinal center B-cell-like or activated B-cell-like.

22. 22. The method of any one of claims 15 to 21, wherein the acute lymphocytic leukemia, chronic myelogenous leukemia, non-Hodgkin's lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma is relapsed or refractory to treatment.

23. The method according to any one of claims 1 to 22, wherein compound (I) is a tartrate salt.

24. 24. The method according to claim 23, wherein the molar ratio between compound (I) and tartaric acid is 1:

1.

25. The method of any one of claims 1 to 24, further comprising the step of co-administering an additional therapeutic agent.

26. 26. The method of claim 25, wherein the additional therapeutic agent is an anti-cancer agent.

27. 27. The method of claim 26, wherein the anticancer drug is venetoclax.

28. 27. The method of claim 26, wherein the anticancer drug is 5-azacytidine.

29. 27. The method of claim 26, wherein the anticancer drug is decitabine.

30. The method of any one of claims 25 to 29, wherein compound (I) and the additional therapeutic agent are administered simultaneously.

31. The method of any one of claims 25 to 29, wherein compound (I) and the additional therapeutic agent are administered sequentially.

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