Combination of menin-LL1 inhibitor, DNA intercalator and pyrimidine analogue for treatment of hematopoietic dysfunction

Abstract

The present invention relates to a combination comprising: a therapeutically effective amount of a menin-mixed lineage leukemia 1 (menin-MLL) inhibitor; and a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue; and to methods of treating a subject diagnosed with cancer using such combinations.

Description

Technical Field

[0001] The present invention relates to a combination comprising: a therapeutically effective amount of a menin-mixed lineage leukemia 1 (menin-MLL) inhibitor; a therapeutically effective amount of a DNA intercalator and a pyrimidine analog; and a method relating to treating a subject diagnosed with cancer using such a combination. Background Technology

[0002] Cancer is a leading cause of death worldwide. Of the ten million cancer deaths recorded by GLOBOCAN in 2020, 7.1% were attributed to hematopoietic dysfunction. Therefore, there is an urgent need for new treatments for hematopoietic dysfunctions, including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and acute lymphoblastic leukemia (ALL), as detailed below.

[0003] AML is a common hematologic malignancy, with an incidence rate ranging from 3 in 100,000 in young adults to over 20 in 100,000 in older adults. Overall survival (OS) is 40% to 50% for patients <60 years of age, but only 5% for those >60 years. Most newly diagnosed AML patients are over 60 years of age. In this patient population, standard induction chemotherapy is often not an option due to increased treatment-related mortality from age and comorbidities. The standard of care for AML patients unsuitable for combination chemotherapy is treatment with a hypomethylating agent (azacitidine or decitabine) or low-dose cytarabine. Relapsed / refractory AML with FMS-like tyrosine kinase 3 (FLT3) mutations is treated with FLT3 kinase inhibitors (e.g., gilteritinib, midostaurin). Despite these first-line treatments, median OS is only about 10 months. In all types of AML, disease relapse is common despite an initial treatment response and is the most frequent cause of death. Standard chemotherapy and allogeneic stem cell transplantation (when used) typically cannot eradicate all proliferating tumor cells and are a selection criterion for chemotherapy-resistant proliferating subclones of leukemia. Patients refractory to salvage therapy are treated palliatively due to very limited current treatment options. These patients have a median survival of 2 months. Furthermore, patients with newly diagnosed intermediate- or higher-risk MDS and those who relapse after standard care have a poor prognosis and are at high risk of progressing to AML. Therefore, there is an urgent need for new forms of treatment for patients with relapsed / refractory (R / R) AML and MDS, newly diagnosed AML patients unsuitable for induction chemotherapy based on age and comorbidities, and newly diagnosed intermediate / high / very high-risk MDS patients.

[0004] Allergic rheumatoid arthritis (ALL) is a hematologic malignancy that spreads through impaired differentiation, proliferation, and the accumulation of lymphoprogenitor cells in the bone marrow and / or extramedullary sites. ALL represents 12% of all leukemia cases and is the most common type of childhood acute leukemia, with an estimated worldwide incidence of 1 to 4.75 per 100,000 people. ALL represents approximately 20% of adult leukemia. Despite the high complete remission (CR) rate (80% to 90%) of current therapies, most adult patients with ALL relapse. The 5-year overall survival rate for adult and elderly patients is approximately 30% to 40%. Therefore, there is an urgent need for new forms of treatment for cancer, particularly relapsed / refractory ALL, especially in adults and, particularly, elderly patients.

[0005] Compounds A, A1, A2 and A3 described herein are disclosed in PCT / CN2020 / 137266 (which was published on June 24, 2021 as WO 2021 / 121327), which is incorporated herein by reference in its entirety, and also discloses the corresponding synthesis schemes and analytical characterization.

[0006] ( R The compound )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide benzenesulfonate is disclosed in WO 2022 / 262796, which is incorporated herein by reference in its entirety, and also discloses the corresponding synthesis scheme and analytical characterization. Attached Figure Description

[0007] Figure 1 Here is the X-ray powder diffraction (XRPD) pattern of compound A4: R Crystalline form A of 2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate.

[0008] Figure 2A The plot shows the contour lines of maxR, illustrating the effect of the combination of compound A4 and idarubicin on the in vitro proliferation of MOLM-13 cells.

[0009] Figure 2B The plot shows the contour lines of maxR, illustrating the effect of the combination of compound A4 and idarubicin on the in vitro proliferation of OCI-AML3 cells.

[0010] Figure 2CThe plot shows the contour lines of maxR, illustrating the effect of compound A3 in combination with cytarabine and idarubicin on the in vitro proliferation of MOLM-13 cells.

[0011] Figure 2D The plot shows the contour lines of maxR, illustrating the effect of compound A3 in combination with cytarabine and idarubicin on the in vitro proliferation of OCI-AML3 cells.

[0012] Figure 2E The plot shows the contour lines of maxR, illustrating the effect of compound A3 in combination with cytarabine and daunorubicin on the in vitro proliferation of MOLM-13 cells.

[0013] Figure 2F The plot shows the contour lines of maxR, illustrating the effect of compound A3 in combination with cytarabine and daunorubicin on the in vitro proliferation of OCI-AML3 cells. Detailed Implementation

[0014] This invention relates to a combination (triple combination) of the following: a therapeutically effective amount of a menin-MLL inhibitor as described herein; and a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analogue.

[0015] Those skilled in the art will understand that all combinations described herein are triple combinations of menin-MLL inhibitors, DNA intercalators, and pyrimidine analogs as described herein.

[0016] As discussed in this article, the menin-MLL inhibitor involves ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide and pharmaceutically acceptable salts and solvates thereof, or subgroups thereof.

[0017] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or a pharmaceutically acceptable salt or solvate thereof; and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs.

[0018] Specifically, in one embodiment, compound A or a pharmaceutically acceptable salt or solvate thereof is any of the subgroups described herein.

[0019] Embodiments of the present invention involve combinations of the following: compound A1: Compound A1 And therapeutically effective amounts of DNA intercalators and pyrimidine analogs.

[0020] Embodiments of the present invention involve combinations of the following: compound A2: Compound A2 And therapeutically effective amounts of DNA intercalators and pyrimidine analogs.

[0021] Embodiments of the present invention involve combinations of the following: compound A3: Compound A3 And therapeutically effective amounts of DNA intercalators and pyrimidine analogs.

[0022] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its solvates; and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs. Those skilled in the art will understand that, in this (and similar) embodiment, "or its solvates" refers to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide benzyl sulfonate.

[0023] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its hydrates; and therapeutically effective amounts of DNA intercalators and pyrimidine analogs.

[0024] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof; and therapeutically effective amounts of a DNA intercalating agent and a pyrimidine analogue.

[0025] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate; and therapeutically effective amounts of the DNA intercalating agent and the pyrimidine analogue.

[0026] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrates; and therapeutically effective amounts of the DNA intercalator and pyrimidine analogue.

[0027] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate; and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs.

[0028] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate in crystalline form A (compound A4); and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs.

[0029] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A; and therapeutically effective amounts of DNA intercalating agent and pyrimidine analogue.

[0030] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A; and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs.

[0031] According to the implementation plan, the DNA intercalating agent includes, but is not limited to, anthracyclines (e.g., daunorubicin, doxorubicin, idarubicin).

[0032] According to the implementation plan, the DNA intercalating agent is anthracycline.

[0033] According to the implementation plan, the DNA intercalating agent is daunorubicin.

[0034] According to the implementation plan, the DNA intercalating agent is doxorubicin.

[0035] According to the implementation plan, the DNA intercalating agent is Idabicin.

[0036] According to the implementation plan, pyrimidine analogues include, but are not limited to, cytarabine (ARA-C).

[0037] According to the implementation plan, the pyrimidine analogue is cytarabine.

[0038] According to the implementation plan, the DNA intercalating agent is daunorubicin, and the pyrimidine analogue is cytarabine.

[0039] According to the implementation plan, the DNA intercalating agent is doxorubicin, and the pyrimidine analogue is cytarabine.

[0040] According to the implementation plan, the DNA intercalating agent is idarubicin, and the pyrimidine analogue is cytarabine.

[0041] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or its pharmaceutically acceptable salts or solvates; and therapeutically effective amounts of doxorubicin and cytarabine.

[0042] Embodiments of the present invention involve combinations of the following: compound A1: Compound A1 And therapeutically effective doses of doxorubicin and cytarabine.

[0043] Embodiments of the present invention involve combinations of the following: compound A2: Compound A2 And therapeutically effective doses of doxorubicin and cytarabine.

[0044] Embodiments of the present invention involve combinations of the following: compound A3: Compound A3 And therapeutically effective doses of doxorubicin and cytarabine.

[0045] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its solvates; and therapeutically effective amounts of doxorubicin and cytarabine.

[0046] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its hydrates; and therapeutically effective amounts of doxorubicin and cytarabine.

[0047] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof; and therapeutically effective amounts of doxorubicin and cytarabine.

[0048] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate; and therapeutically effective amounts of doxorubicin and cytarabine.

[0049] The embodiments of the present invention involve combinations of the following: R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrates; and therapeutically effective amounts of doxorubicin and cytarabine.

[0050] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate; and therapeutically effective amounts of doxorubicin and cytarabine.

[0051] The embodiments of the present invention involve combinations of the following: R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate; and therapeutically effective amounts of doxorubicin and cytarabine.

[0052] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A; and therapeutically effective amounts of doxorubicin and cytarabine.

[0053] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A; and therapeutically effective amounts of doxorubicin and cytarabine.

[0054] The embodiments of the present invention involve combinations of the following: R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or a pharmaceutically acceptable salt or solvate thereof; and therapeutically effective amounts of idarubicin and cytarabine.

[0055] Embodiments of the present invention involve combinations of the following: compound A1: Compound A1 And therapeutically effective doses of idarubicin and cytarabine.

[0056] Embodiments of the present invention involve combinations of the following: compound A2: Compound A2 And therapeutically effective doses of idarubicin and cytarabine.

[0057] Embodiments of the present invention involve combinations of the following: compound A3: Compound A3 And therapeutically effective doses of idarubicin and cytarabine.

[0058] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its solvates; and therapeutically effective amounts of idarubicin and cytarabine.

[0059] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its hydrates; and therapeutically effective amounts of idarubicin and cytarabine.

[0060] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof; and therapeutically effective amounts of idarubicin and cytarabine.

[0061] The embodiments of the present invention involve combinations of the following: R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate; and therapeutically effective amounts of idarubicin and cytarabine.

[0062] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrates; and therapeutically effective amounts of idarubicin and cytarabine.

[0063] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate; and therapeutically effective amounts of idarubicin and cytarabine.

[0064] The embodiments of the present invention involve combinations of the following: R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate; and therapeutically effective amounts of idarubicin and cytarabine.

[0065] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A; and therapeutically effective amounts of idarubicin and cytarabine.

[0066] The embodiments of the present invention involve combinations of the following: R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A; and therapeutically effective amounts of idarubicin and cytarabine.

[0067] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or its pharmaceutically acceptable salts or solvates; and therapeutically effective amounts of daunorubicin and cytarabine.

[0068] Embodiments of the present invention involve combinations of the following: compound A1: Compound A1 In addition to therapeutically effective doses of daunorubicin and cytarabine.

[0069] Embodiments of the present invention involve combinations of the following: compound A2: Compound A2 In addition to therapeutically effective doses of daunorubicin and cytarabine.

[0070] Embodiments of the present invention involve combinations of the following: compound A3: Compound A3 In addition to therapeutically effective doses of daunorubicin and cytarabine.

[0071] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its solvates; and therapeutically effective amounts of daunorubicin and cytarabine.

[0072] Embodiments of the present invention involve combinations of the following: compound A4-a: Compound A4-a Or its hydrates; and therapeutically effective amounts of daunorubicin and cytarabine.

[0073] The embodiments of the present invention involve combinations of the following:R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof; and therapeutically effective amounts of daunorubicin and cytarabine.

[0074] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate; and therapeutically effective amounts of daunorubicin and cytarabine.

[0075] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrates; and therapeutically effective amounts of daunorubicin and cytarabine.

[0076] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate; and therapeutically effective amounts of daunorubicin and cytarabine.

[0077] The embodiments of the present invention involve combinations of the following: R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate; and therapeutically effective amounts of daunorubicin and cytarabine.

[0078] The embodiments of the present invention involve combinations of the following: R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) in crystalline form A of 0.5 to 2.0 equivalents of hydrate; and therapeutically effective amounts of daunorubicin and cytarabine.

[0079] The embodiments of the present invention involve combinations of the following: R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A; and therapeutically effective amounts of daunorubicin and cytarabine.

[0080] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the combination as described in any other embodiment, as an active ingredient.

[0081] Embodiments of the present invention relate to the use of such combinations for treating subjects diagnosed with hematopoietic dysfunction (including, but not limited to, blood cancers).

[0082] Embodiments of the present invention relate to novel methods for treating subjects diagnosed with hematopoietic dysfunction using such combination therapy. Embodiments of the novel method include administering to the subject a therapeutically effective amount of a menin-MLL inhibitor as described herein; and a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analogue.

[0083] In one implementation, the subject was newly diagnosed and deemed suitable for chemotherapy.

[0084] In some embodiments, the present invention relates to methods for treating a subject who has been diagnosed with hematopoietic dysfunction, the methods comprising administering to the subject a therapeutically effective amount of a menin-MLL inhibitor; and a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analogue.

[0085] Another embodiment of the invention relates to a method for treating a subject who has been diagnosed with cancer (e.g., where the cancer is a hematopoietic disorder, such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL)), wherein the method includes administering to the subject: Therapeutic effective dose ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or its pharmaceutically acceptable salts or solvates; and Therapeutic amounts of DNA intercalating agents and pyrimidine analogs.

[0086] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or a pharmaceutically acceptable salt or solvate thereof, which, in combination with DNA intercalators and pyrimidine analogs, is used to treat cancer.

[0087] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or a pharmaceutically acceptable salt or solvate thereof, which, in combination with therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs, is used to treat cancer.

[0088] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat hematopoietic dysfunction.

[0089] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof, which, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue, is used to treat hematopoietic dysfunction.

[0090] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof, in combination with a DNA intercalator and a pyrimidine analogue for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0091] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0092] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvation thereof, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat acute myeloid leukemia (AML).

[0093] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue for the treatment of acute myeloid leukemia (AML).

[0094] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvation thereof, in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0095] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof, which, in combination with a therapeutically effective amount of daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0096] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof, in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0097] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof, in combination with a therapeutically effective amount of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0098] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof, in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0099] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof, in combination with a therapeutically effective amount of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0100] In one embodiment, the present invention relates to compound A4-a: Compound A4-a Or its solvates, which, in combination with DNA intercalators and pyrimidine analogs, are used to treat cancer.

[0101] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a: Compound A4-a Or its solvates, which, in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs, are used to treat cancer.

[0102] In one embodiment, the present invention relates to compound A4-a or a solvate thereof, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat hematopoietic dysfunction.

[0103] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or a solvate thereof, which, in combination with a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analogue, is used to treat hematopoietic dysfunction.

[0104] In one embodiment, the present invention relates to compound A4-a or a solvate thereof, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0105] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or a solvate thereof, which, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue, is used to treat myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0106] In one embodiment, the present invention relates to compound A4-a or a solvate thereof, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat acute myeloid leukemia (AML).

[0107] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or a solvate thereof, which, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue, is used to treat acute myeloid leukemia (AML).

[0108] In one embodiment, the present invention relates to compound A4-a or a solvate thereof, which, in combination with daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0109] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or a solvate thereof, which, in combination with a therapeutically effective amount of daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0110] In one embodiment, the present invention relates to compound A4-a or a solvate thereof, which, in combination with doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0111] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or a solvate thereof, which, in combination with a therapeutically effective amount of doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0112] In one embodiment, the present invention relates to compound A4-a or a solvate thereof, which, in combination with idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0113] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or a solvate thereof, which, in combination with a therapeutically effective amount of idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0114] In one embodiment, the present invention relates to compound A4-a: Compound A4-a Or its hydrate, which, in combination with DNA intercalators and pyrimidine analogs, is used to treat cancer.

[0115] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a: Compound A4-a Or its hydrate, which, in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs, is used to treat cancer.

[0116] In one embodiment, the present invention relates to compound A4-a or its hydrate, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat hematopoietic dysfunction.

[0117] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate, which, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue, is used to treat hematopoietic dysfunction.

[0118] In one embodiment, the present invention relates to compound A4-a or its hydrate, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0119] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate, which, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue, is used to treat myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0120] In one embodiment, the present invention relates to compound A4-a or its hydrate, which, in combination with a DNA intercalating agent and a pyrimidine analogue, is used to treat acute myeloid leukemia (AML).

[0121] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate, which, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue, is used to treat acute myeloid leukemia (AML).

[0122] In one embodiment, the present invention relates to compound A4-a or its hydrate, which, in combination with daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0123] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate, which, in combination with therapeutically effective amounts of daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0124] In one embodiment, the present invention relates to compound A4-a or its hydrate, which, in combination with doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0125] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate, which, in combination with therapeutically effective amounts of doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0126] In one embodiment, the present invention relates to compound A4-a or its hydrate, which, in combination with idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0127] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate, which, in combination with therapeutically effective amounts of idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0128] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, which, in combination with DNA intercalating agents and pyrimidine analogs, are used to treat cancer.

[0129] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analogue for the treatment of cancer.

[0130] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, which, in combination with DNA intercalating agents and pyrimidine analogs, are used to treat hematopoietic dysfunction.

[0131] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analogue for the treatment of hematopoietic dysfunction.

[0132] In one embodiment, the present invention relates to ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with a DNA intercalating agent and a pyrimidine analogue for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL).

[0133] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with a therapeutically effective amount of a DNA intercalator and a pyrimidine analogue for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL).

[0134] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with DNA intercalating agents and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0135] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analogue for the treatment of acute myeloid leukemia (AML).

[0136] In one embodiment, the present invention relates to ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, which, in combination with daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0137] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with therapeutically effective amounts of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0138] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, which, in combination with doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0139] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with therapeutically effective amounts of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0140] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, which, in combination with idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0141] In one embodiment, the present invention relates to a therapeutically effective amount of ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or a solvation thereof, in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0142] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, which, in combination with DNA intercalating agents and pyrimidine analogs, are used to treat cancer.

[0143] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with therapeutically effective amounts of a DNA intercalating agent and a pyrimidine analogue for the treatment of cancer.

[0144] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, which, in combination with DNA intercalating agents and pyrimidine analogs, is used to treat hematopoietic dysfunction.

[0145] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with therapeutically effective amounts of a DNA intercalating agent and a pyrimidine analogue for the treatment of hematopoietic dysfunction.

[0146] In one embodiment, the present invention relates to ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with DNA intercalating agents and pyrimidine analogs for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL).

[0147] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with therapeutically effective amounts of a DNA intercalator and a pyrimidine analogue for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL).

[0148] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with DNA intercalating agents and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0149] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with therapeutically effective amounts of a DNA intercalating agent and a pyrimidine analogue for the treatment of acute myeloid leukemia (AML).

[0150] In one embodiment, the present invention relates to ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, which, in combination with daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0151] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with therapeutically effective amounts of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0152] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, which, in combination with doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0153] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with therapeutically effective amounts of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0154] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, which, in combination with idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0155] In one embodiment, the present invention relates to a therapeutically effective amount of ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate, in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0156] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which is used in combination with DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0157] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which is used in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0158] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which, in combination with DNA intercalating agents and pyrimidine analogs, is used to treat hematopoietic dysfunction.

[0159] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs, is used to treat hematopoietic dysfunction.

[0160] In one embodiment, the present invention relates to (R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, in combination with DNA intercalators and pyrimidine analogs for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0161] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalents of hydrate, in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0162] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which, in combination with DNA intercalating agents and pyrimidine analogs, is used to treat acute myeloid leukemia (AML).

[0163] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, in combination with therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0164] In one embodiment, the present invention relates to ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which is used in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0165] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0166] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which, in combination with doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0167] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0168] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, which, in combination with idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0169] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 0.5 to 2.0 equivalent hydrate, in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0170] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which is used in combination with DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0171] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which is used in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0172] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which is used in combination with DNA intercalating agents and pyrimidine analogs for the treatment of hematopoietic dysfunction.

[0173] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs, is used to treat hematopoietic dysfunction.

[0174] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which, in combination with DNA intercalators and pyrimidine analogs, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0175] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0176] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which is used in combination with DNA intercalators and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0177] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs, is used to treat acute myeloid leukemia (AML).

[0178] In one embodiment, the present invention relates to ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which is used in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0179] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of daunorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0180] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which is used in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0181] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of doxorubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0182] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which is used in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0183] In one embodiment, the present invention relates to a therapeutically effective amount of (R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate, which, in combination with therapeutically effective amounts of idarubicin and cytarabine, is used to treat acute myeloid leukemia (AML).

[0184] In one embodiment, the present invention relates to ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0185] In one embodiment, the present invention relates to a therapeutically effective amount of ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0186] In one embodiment, the present invention relates to ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with DNA intercalators and pyrimidine analogs for the treatment of hematopoietic dysfunction.

[0187] In one embodiment, the present invention relates to a therapeutically effective amount of ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of hematopoietic dysfunction.

[0188] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate crystalline form A (compound A4), which, in combination with DNA intercalators and pyrimidine analogs, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL).

[0189] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate crystalline form A (compound A4), which, in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0190] In one embodiment, the present invention relates to ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with DNA intercalators and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0191] In one embodiment, the present invention relates to a therapeutically effective amount of ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with therapeutically effective amounts of a DNA intercalator and a pyrimidine analogue for the treatment of acute myeloid leukemia (AML).

[0192] In one embodiment, the present invention relates to (R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0193] In one embodiment, the present invention relates to a therapeutically effective amount of ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with therapeutically effective amounts of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0194] In one embodiment, the present invention relates to ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0195] In one embodiment, the present invention relates to a therapeutically effective amount of ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with therapeutically effective amounts of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0196] In one embodiment, the present invention relates to ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0197] In one embodiment, the present invention relates to a therapeutically effective amount of ( R The crystalline form A (compound A4) of 2-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate is used in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0198] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0199] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0200] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with DNA intercalating agents and pyrimidine analogs for the treatment of hematopoietic dysfunction.

[0201] In one embodiment, the present invention relates to a therapeutically effective amount of ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which, in combination with therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs, is used to treat hematopoietic dysfunction.

[0202] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which, in combination with DNA intercalators and pyrimidine analogs, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0203] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which, in combination with therapeutically effective amounts of a DNA intercalator and a pyrimidine analogue, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0204] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with DNA intercalating agents and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0205] In one embodiment, the present invention relates to a therapeutically effective amount of ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which, in combination with therapeutically effective amounts of a DNA intercalator and a pyrimidine analogue, is used to treat acute myeloid leukemia (AML).

[0206] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0207] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0208] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0209] In one embodiment, the present invention relates to a therapeutically effective amount of ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0210] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0211] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbenzenesulfonate in 0.5 to 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0212] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0213] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of cancer.

[0214] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with DNA intercalating agents and pyrimidine analogs for the treatment of hematopoietic dysfunction.

[0215] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs for the treatment of hematopoietic dysfunction.

[0216] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which, in combination with DNA intercalators and pyrimidine analogs, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0217] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which, in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs, is used to treat myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0218] In one embodiment, the present invention relates to ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with DNA intercalating agents and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0219] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of DNA intercalators and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0220] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0221] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0222] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0223] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0224] In one embodiment, the present invention relates to ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0225] In one embodiment, the present invention relates to a therapeutically effective amount of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) 2.0 equivalent hydrate crystalline form A, which is used in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0226] Specifically, in any of these embodiments, the DNA intercalating agent is daunorubicin, and the pyrimidine analogue is cytarabine.

[0227] Specifically, in any of these embodiments, the DNA intercalating agent is doxorubicin, and the pyrimidine analogue is cytarabine.

[0228] Specifically, in any of these embodiments, the DNA intercalating agent is idarubicin, and the pyrimidine analogue is cytarabine.

[0229] A method for treating a subject diagnosed with hematopoietic dysfunction, the method comprising administering to the subject a combination of the following: a therapeutically effective dose ( R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or a pharmaceutically acceptable salt or solvate thereof; and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs.

[0230] Pharmaceutically acceptable salts include acid addition salts and base addition salts. Such salts can be formed by conventional methods, such as by reacting a free acid or free base with one or more equivalents of a suitable base or acid, optionally in a solvent or in a medium in which the salt is insoluble, followed by removal of the solvent or medium using standard techniques (e.g., vacuum, by freeze-drying, or by filtration). Salts can also be prepared by exchanging the counter ion of the compound of the invention in salt form with another counter ion, for example using a suitable ion exchange resin.

[0231] Pharmaceutically acceptable salts as described above or below are intended to include ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide and its solvates can form non-toxic acid and base salts with therapeutic activity.

[0232] Suitable acids include, for example, inorganic acids such as hydrohalic acids (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid (i.e., oxalic acid), malonic acid, succinic acid (i.e., succinic acid), maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexane, salicylic acid, para-aminosalicylic acid, pyric acid, etc. Conversely, the salt form can be converted into a free base form by treatment with a suitable base.

[0233] Containing acidic protons ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide and its solvates can also be converted into their non-toxic metal or amine salt forms by treatment with appropriate organic and inorganic bases.

[0234] Suitable salt forms of alkalis include, for example, ammonium salts, alkali metal and alkaline earth metal salts (e.g., lithium, sodium, potassium, cesium, magnesium, calcium salts, etc.), salts with organic bases such as primary, secondary and tertiary aliphatic and aromatic amines, such as methylamine, ethylamine, propylamine, isopropylamine, four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinine ring, pyridine, quinoline and isoquinoline; benzathine, N-methyl-D-glucosamine, hebamin salts, and salts with amino acids (such as, for example, arginine, lysine, etc.). Conversely, the salt form can be converted to the free acid form by acid treatment.

[0235] Within the scope of this invention, all isotopes and mixtures of isotopes of any particular atom or element as specified herein are contemplated, whether naturally occurring or synthetically produced, and whether in natural abundance or isotopically enriched forms. Exemplary isotopes that may be incorporated into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 O、 17 O、 18 O、 32 P, 33 P, 35 S, 18 F, 36 Cl、 122 I, 123 I, 125 I, 131 I, 75 Br、 76 Br、 77 Br and 82 Br. Preferably, the isotope is selected from... 2 H, 3 H, 11 C 13 C and 18 Group F. Preferably, the isotopes are selected from... 2 H, 3 H, 11 C and 18 Group F. More preferably, the isotope is... 2 H, 3 H or 13 C. More preferably, the isotope is 2 H or 13 C. More preferably, the isotope is 2 H. Specifically, deuterated compounds and rich in 13Compounds of type C are intended to be included within the scope of this invention. Specifically, deuterated compounds are intended to be included within the scope of this invention.

[0236] Certain isotope-labeled compounds (e.g., with) 3 H and 14 Those labeled with C can be used, for example, for substrate tissue distribution determination. Tritium ( 3 H) and carbon-14 ( 14 C) Isotopes are useful due to their ease of preparation and detectability. Furthermore, heavier isotopes such as deuterium (i.e., 2 H) substitution can provide certain therapeutic advantages due to greater metabolic stability (e.g., prolonged in vivo half-life or reduced required dose) and is therefore preferred in some cases. Positron-emitting isotopes such as 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies. PET imaging in cancer can help locate and identify tumors, stage the disease, and determine appropriate treatments. Human cancer cells overexpress many receptors or proteins that are potential disease-specific molecular targets. Radiolabeled tracers that bind to such receptors or proteins on tumor cells with high affinity and specificity have great potential for diagnostic imaging and targeted radionuclide therapy (Charron, Carlie L. et al., Tetrahedron Lett. 2016, 57(37), 4119-4127). In addition, target-specific PET radiotracers can be used as biomarkers to examine and evaluate pathology by, for example, measuring target expression and treatment response (Austin R. et al., Cancer Letters (2016), doi: 10.1016 / j.canlet.2016.05.008).

[0237] As used herein, the term "hematopoietic dysfunction" refers to any disorder related to the production of cellular components in the blood and plasma, including but not limited to blood cancers.

[0238] According to one embodiment, the present invention provides a combination as described herein.

[0239] According to one embodiment, the present invention provides combinations as described herein for use as pharmaceutical agents.

[0240] According to one embodiment, the present invention provides a combination as described herein for preparing a pharmaceutical agent.

[0241] According to one embodiment, the present invention provides a combination as described herein for preparing a medicament for treating or preventing any of the disease conditions mentioned herein.

[0242] According to one embodiment, the present invention provides combinations as described herein for the prevention or treatment (especially for the treatment) of diseases as described herein.

[0243] According to one embodiment, the present invention provides combinations as described herein for the prevention or treatment (especially the treatment) of hematopoietic dysfunction (including but not limited to blood cancers, including but not limited to lymphoma, myeloma and leukemia).

[0244] According to one embodiment, the present invention provides a combination as described herein for the prevention or treatment (especially the treatment) of hematopoietic dysfunction.

[0245] According to one implementation plan, hematopoietic dysfunction is selected from, but not limited to, lymphoma, myeloma, spinal dysplasia, and leukemia.

[0246] According to one implementation plan, hematopoietic dysfunction is spinal dysplasia, including but not limited to myelodysplastic syndrome (MDS).

[0247] According to one implementation plan, hematopoietic dysfunction is leukemia.

[0248] According to one implementation plan, hematopoietic dysfunction is leukemia, selected from acute leukemia and chronic leukemia. According to another implementation plan, the leukemia is acute leukemia. According to yet another implementation plan, the leukemia is chronic leukemia.

[0249] According to one implementation plan, the hematopoietic dysfunction is myeloid leukemia, myeloid leukemia, lymphoblastic leukemia, or lymphocytic leukemia. According to one implementation plan, the hematopoietic dysfunction is leukemia selected from, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), acute myeloid leukemia (AML), chronic idiopathic myelofibrosis (MF), chronic myeloid leukemia (CML), T-cell prolymphoblastic leukemia (T-PLL), B-cell prolymphoblastic leukemia (B-PLL), chronic neutrophilic leukemia (CNL), hairy cell leukemia (HCL), T-cell large granular lymphoblastic leukemia (T-LGL), and aggressive NK cell leukemia. According to one implementation plan, AML is acute megakaryoblastic leukemia (AMKL).

[0250] According to one implementation, leukemia is MDS, CLL, SLL, ALL, or AML. According to one implementation, leukemia is CLL, SLL, or AML. According to one implementation, leukemia is CLL or SLL. In some implementations, CLL or SLL is a CD20-expressing cancer. According to one implementation, leukemia is ALL or AML. According to one implementation, leukemia is ALL. According to one implementation, leukemia is AML. According to one implementation, the hematopoietic dysfunction is Waldenström macroglobulinemia.

[0251] According to one implementation plan, hematopoietic dysfunction is MLL rearrangement leukemia, MLL partial tandem duplication (PTD) leukemia, MLL amplification leukemia, MLL positive leukemia, or leukemia exhibiting elevated HOX / MEIS1 gene expression tags.

[0252] According to one implementation scheme, leukemia is MLL rearrangement leukemia and / or nucleophosphoprotein 1 (NPM1) mutation leukemia.

[0253] According to one implementation plan, hematopoietic dysfunction is MLL rearrangement leukemia.

[0254] According to one implementation scheme, hematopoietic dysfunction is nucleophosphorus protein 1 (NPM1) mutant leukemia (e.g., NPM1c).

[0255] According to one implementation plan, hematopoietic dysfunction is myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN).

[0256] According to one implementation plan, hematopoietic dysfunction is acute lymphoblastic leukemia (ALL).

[0257] According to one implementation plan, hematopoietic dysfunction is acute myeloid leukemia (AML).

[0258] According to one implementation plan, the hematopoietic dysfunction is small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL).

[0259] According to one implementation plan, hematopoietic dysfunction is SLL or CLL, where SLL or CLL is a cancer that expresses CD20.

[0260] According to one implementation plan, hematopoietic dysfunction is myelodysplastic syndrome (MDS).

[0261] According to one implementation plan, hematopoietic dysfunction is myeloproliferative neoplasm (MPN).

[0262] According to one implementation plan, hematopoietic dysfunction is NPM1-mutant leukemia with an FLT3 mutation.

[0263] According to one implementation plan, hematopoietic dysfunction is FLT3-dependent leukemia.

[0264] According to one implementation plan, hematopoietic dysfunction is MEF2G-dependent leukemia.

[0265] According to one implementation, hematopoietic dysfunction has one or more MLL1 (KMT2A) gene rearrangements or alterations (e.g., duplication or amplification) and / or NPM1 mutations.

[0266] According to one implementation, hematopoietic dysfunction has (i) one or more MLL1 (KMT2A) gene rearrangements or alterations (e.g., duplication or amplification) and / or NPM1 mutations plus (ii) FLT3 mutations.

[0267] According to one implementation plan, hematopoietic dysfunction is MLL rearrangement leukemia.

[0268] According to one implementation plan, hematopoietic dysfunction is acute myeloid leukemia (AML).

[0269] According to one implementation plan, hematopoietic dysfunction is small lymphocytic lymphoma (SLL).

[0270] According to one implementation plan, hematopoietic dysfunction is chronic lymphocytic leukemia (CLL).

[0271] According to one implementation plan, hematopoietic dysfunction is defined as acute leukemia, chronic leukemia, myeloid leukemia, myeloid leukemia, lymphoblastic leukemia, lymphocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), T-cell prolymphoblastic leukemia (T-PLL), large granular lymphocytic leukemia, hairy cell leukemia (HCL), MLL rearrangement leukemia, MLL-PTD leukemia, MLL amplification leukemia, MLL-positive leukemia, or leukemia showing elevated levels. HOX / MEIS1 Leukemia with gene expression tags.

[0272] According to one implementation plan, hematopoietic dysfunction is AML, particularly AML with nucleophosphorus protein (NPM1) mutations (i.e., NPM1). mut AML), and more specifically, the NPM1 mutation AML.

[0273] According to one implementation plan, hematopoietic dysfunction is MLL rearrangement leukemia, particularly MLL rearrangement AML or ALL.

[0274] According to one implementation plan, hematopoietic dysfunction includes MLLGenetic alterations, specifically, hematopoietic dysfunction, are... MLL Genetically altered AML or ALL. In some implementations, MLL Genetic alterations are repetitive. In some implementations, MLL Genetic alteration is amplification.

[0275] According to one implementation plan, hematopoietic dysfunction includes NPM1 gene mutations and / or MLL1 (also known as NPM1 gene mutations and / or MLL1 mutations). KMT2A Gene mutation.

[0276] According to one implementation scheme, MLL1 gene mutations include, but are not limited to, MLL1 gene rearrangements, duplications, or amplifications.

[0277] According to one implementation plan, hematopoietic dysfunction is mixed lineage leukemia (MLL), MLL-associated leukemia, MLL-related leukemia, MLL-positive leukemia, MLL-induced leukemia, MLL-related leukemia, acute leukemia, chronic leukemia, myelodysplastic syndrome (MDS), or myeloproliferative neoplasm (MPN).

[0278] All embodiments of the methods for treating disorders described herein are also applicable to use in treating said disorders.

[0279] All implementation schemes described herein for use in treating disorders also apply to methods for treating said disorders.

[0280] All implementation schemes described herein for use in treating disorders also apply to methods for treating said disorders.

[0281] All implementation schemes described herein for use in treating disorders are also applicable to use in treating said disorders.

[0282] All embodiments of the methods for treating disorders described herein are also applicable to use in methods for treating said disorders.

[0283] All implementation schemes described herein for use in methods of treating disorders are also applicable to methods of treating said disorders.

[0284] As used herein, the term “therapeutic effective amount” means the amount of an active compound or agent that elicits a biological or pharmaceutical response (including relief or reversal of symptoms of the disease or disorder being treated) in an tissue system, animal, or human body that is being sought by researchers, veterinarians, physicians, or other clinicians.

[0285] All abbreviations used in the general regimens and examples of the menin-MLL inhibitor described herein are defined as in Table 1. Variables are as defined in the ranges or as specifically defined in the general regimens. Example

[0286] Table 1 - Abbreviations . abbreviation meaning <![CDATA[Ag(Phen)2OTf]]> Silver trifluoromethanesulfonate-bis(1,10-phenanthroline) complex 2-MeTHF 2-Methyltetrahydrofuran ACN Acetonitrile AcCl Acetyl chloride AcOH Acetic acid <![CDATA[Ac2O]]> Acetic anhydride aq. Water content Ar Argon <![CDATA[BBr3]]> Tribromoborane bn benzyl Boc tert-butoxycarbonyl <![CDATA[Boc2O]]> Ditert-butyl dicarbonate -BuLi n-Butyllithium Cbz benzyloxycarbonyl <![CDATA[CD3OD]]> <![CDATA[Methanol-d4]]> <![CDATA[CHCl3]]> Chloroform <![CDATA[Cs2CO3]]> cesium carbonate conc. Concentrated DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC Dicyclohexylcarbodiimide DCE dichloroethane DCM dichloromethane DDQ 4,5-Dichloro-3,6-dioxocyclohexyl-1,4-diene-1,2-dicarboxynitrile DEA Diethylamine DIBAL-H Diisobutylaluminum hydride DIEA or DIPEA ,-Diisopropylethylamine DMAP ,-Dimethylpyridine-4-amine DMF ,-Dimethylformamide DMP Dess-Martin periodine DMSO Dimethyl sulfoxide dppf 1,1'-Ferrocenyl-bis(diphenylphosphine) EDCI -(3-Dimethylaminopropyl)-′-ethylcarbodiimide hydrochloride EA or EtOAc Ethyl acetate EtOH ethanol eq. equivalent FA Formic acid FCC Rapid column chromatography h Hour <![CDATA[H2]]> hydrogen HATU 1-[bis(dimethylamino)methylene]-1-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate <![CDATA[H2O]]> water HCl hydrochloric acid HOBt 1-Hydroxybenzotriazole HPLC High performance liquid chromatography <![CDATA[ICH2Cl]]> Chloroiodim IPA Isopropanol IPAc Isopropyl acetate <![CDATA[K2CO3]]> Potassium carbonate KI Potassium iodide <![CDATA[K2HPO4]]> dipotassium hydrogen phosphate <![CDATA[K3PO4]]> Tripotassium phosphate <![CDATA[LiAlD4]]> Lithium aluminum deuteride LAH Lithium aluminum hydride <![CDATA[LiBH4]]> Lithium boroide LDA Lithium diisopropylaminodimethylamine LiCl Lithium chloride LG Leaving group Me methyl MeOH methanol 2-MeTHF 2-Methyltetrahydrofuran min minute mL milliliters mmol millimole mg mg <![CDATA[MgSO4]]> Magnesium sulfate MSA mesylate MsCl Methanesulfonyl chloride MS Molecular sieve MTBE Methyl tert-butyl ether <![CDATA[N2]]> Nitrogen NA Unavailable <![CDATA[NaBH3CN]]> Sodium cyanoborohydride <![CDATA[NaBH(OAc)3]]> Sodium triacetoxyborohydride <![CDATA[NaBD3CN]]> Sodium cyanoboronide <![CDATA[Na2CO3]]> Sodium carbonate NaH Sodium hydride <![CDATA[NaHCO3]]> Sodium bicarbonate NaI Sodium iodide NaOAc Sodium acetate NaOH Sodium hydroxide <![CDATA[Na2SO3]]> Sodium sulfite <![CDATA[Na2SO4]]> Sodium sulfate <![CDATA[NH4Cl]]> ammonium chloride NMM 1-4-Methylmorpholine <![CDATA[Pd2dba3]]> Tris(dibenzylacetone)dipalladium(0) <![CDATA[Pd(dppf)Cl2·DCM]]> The complex of [1,1'-bis(diphenylphosphino)ferrocene]-palladium(II) dichloride and dichloromethane <![CDATA[Pd(PPh3)4]]> Tetra(triphenylphosphine)palladium(0) PE petroleum ether PG Protecting groups Phen phenanthroline psi pounds per square inch -TsOH p-Toluenesulfonic acid <![CDATA[ p -TsOH·H2O]]> p-Toluenesulfonic acid monohydrate <![CDATA[R t ]]> Retention time Rochelle salt Potassium sodium tartrate tetrahydrate RT room temperature sat. saturation SFC Supercritical fluid chromatography TBAF Tetrabutylammonium fluoride TBDMS tert-butyldimethylsilyl TBDPS tert-butyldiphenylsilyl -BuOK Potassium tert-butoxide TEA Triethylamine Tf Trifluoromethanesulfonyl TFA Trifluoroacetic acid THF Tetrahydrofuran <![CDATA[Ti(OiPr)4]]> Titanium isopropoxide (IV) TLC Thin-layer chromatography TMEDA ,,,-Tetramethylethylenediamine TMG 1,1,3,3-Tetramethylguanidine TMSI Trimethyliodosilane Ts p-Toluenesulfonyl TsCl p-Toluenesulfonyl chloride v / v Volume / Volume vol. volume wt weight Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethyloxanthracene

[0287] As will be understood by those skilled in the art, compounds synthesized using the illustrated schemes may exist as solvates (e.g., hydrates) and / or contain residual solvents or trace amounts of impurities. Compounds or intermediates isolated as salts may be integer stoichiometric, i.e., single or disalts, or intermediate stoichiometry. When an intermediate or compound in the experimental section below is referred to as “HCl salt” without specifying the equivalent number of HCl, this means that the equivalent number of HCl has not yet been determined. The same principle will also apply to all other salt forms mentioned in the experimental section, such as “oxalate,” “formate,” “benzenesulfonate,” or “…” ".

[0288] Technicians will recognize that, even if not explicitly mentioned in the following experimental protocol, the desired fraction is typically collected and the solvent evaporated after column chromatography purification.

[0289] In the absence of a specified stereochemistry, this means that it is a mixture of stereoisomers unless otherwise indicated or is clear from the context.

[0290] Unless otherwise specified, when “RS” is used to denote a stereocenter, it means that a racemic mixture is obtained at the specified center.

[0291] Example 1 - ( R )- N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)) (amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3,4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide Synthesis-Preparation Method A of (Compound A) intermediate 1 - (5- methyl -4- Oxyhexyl ) Preparation of tert-butyl carbamate To a solution of tert-butyl 2-oxopyrrolidine-1-carboxylate (5.0 g, 27 mmol) cooled to -70 °C and TMEDA (5.0 mL, 33 mmol) in THF (60 mL), a solution of isopropyl magnesium bromide (19 mL, 55 mmol, 2.9 M in 2-methyltetrahydrofuran) was slowly added. The resulting mixture was slowly heated to room temperature and stirred for 12 hours. The mixture was poured into a saturated aqueous solution of NH4Cl (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a crude product. This crude product was further purified by FCC (PE / EtOAc = 1:0 to 100:1) to provide a yellow oily title intermediate (3.7 g, 60% yield).

[0292] intermediate 13 - 6-(3,6- dichloro -1,2,4- Triazine -5- base )-2,6- diazalozyro [3.4] Octane -2- Formic acid Preparation of tert-butyl ester To a solution of 3,5,6-trichloro-1,2,4-triazine (10.0 g, 54.2 mmol) and TEA (15.2 mL, 109 mmol) in DCM (100 mL) cooled at 0 °C, tert-butyl 2,6-diazaspiro[3,4]octane-2-carboxylate (9.21 g, 43.4 mmol) was added, and the mixture was heated to room temperature and stirred for 1 hour. The mixture was diluted with water (20 mL) and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude product, which was purified by FCC on silica gel (PE / EtOAc = 1:0 to 3:1) to provide a yellow solid title intermediate (12.0 g, 58% yield).

[0293] intermediate 27 - N- Ethyl -5- fluorine -N- Isopropyl -2- Preparation of methoxybenzamide 5-Fluoro-2-methoxybenzoic acid (8.00 g, 47.0 mmol) cooled at 0 °C and N 2-Ethylpropyl-2-amine (8.19 g, 94.0 mmol) was slowly added in portions to a mixture in anhydrous DCM (150 mL) with HATU (21.5 g, 56.5 mmol) and DIEA (9.10 g, 70.4 mmol). The resulting mixture was slowly heated to room temperature and stirred for 8 hours. The organic layer was washed with water (20 mL × 3) and dried over anhydrous Na₂SO₄. After filtration, the solvent was removed under reduced pressure, and the crude product was purified by FCC (EtOAc / PE = 0% to 20%) to provide a white solid title intermediate (12.0 g, 96% yield).

[0294] intermediate 28 - N- Ethyl -5- fluorine -2- hydroxyl -N- Preparation of isopropylbenzamide Cooled at -78°C N -Ethyl-5-fluoro- N-Isopropyl-2-methoxybenzamide (Intermediate 27) (12.0 g, 50.1 mmol) was slowly added to a solution of BBr3 (14.4 mL, 152 mmol) in anhydrous DCM (100 mL), and the resulting mixture was slowly heated to room temperature and stirred for 8 hours. The mixture was then cooled again to -78 °C, and MeOH (5 mL) was added dropwise to quench the reaction. The resulting mixture was slowly heated to room temperature, and the pH was adjusted to approximately 8 (by adding a saturated aqueous solution of NaHCO3). The aqueous layer was extracted with DCM (50 mL × 3), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a crude product. This crude product was purified by FCC (EtOAc / PE = 0% to 20%) to provide the title intermediate (9.0 g, 78% yield) as a white solid.

[0295] intermediate 28 Alternative preparation A mixture of 5-fluoro-2-hydroxybenzoic acid (14.0 kg, 89.68 mol, 1.0 equivalent) in THF (168 L, 12 volumes) was adjusted to 15°C–25°C, and 1,1-carbonyldiimidazole (17.45 kg, 107.62 mol, 1.2 equivalent) was added over a 1-hour period. After addition, the mixture was stirred at 15°C–25°C for 18 hours. Subsequently, N-ethylpropyl-2-amine (14.85 kg, 170.39 mol, 1.9 equivalent) was added to the mixture over a 2-hour period at 15°C–25°C. The resulting mixture was further aged at 15°C–25°C for 18–24 hours. The pH was adjusted to 4–5 using 10% H₂SO₄ aqueous solution (140 kg, 10 volumes), and the layers were separated. The organic phase was concentrated to between 42 and 56 L, and the temperature was kept below 40 °C. Then, n-heptane (43 kg, 4.5 V / L) was added to the mixture over a 3-hour period at 15 °C–25 °C. The mixture was then cooled to 0–10 °C and stirred for another 6 hours. The resulting slurry was filtered, and the filter cake was washed with a tert-butyl methyl ether (MTBE):n-heptane mixture (25 kg of a 2:3 V / V MTBE:n-heptane mixture, 2.5 V / L). The filter cake washing was repeated twice, and the resulting solid was vacuum dried at 50 °C to provide intermediate 28 (16.5 kg, purity: 99.1%, yield: 80.4%).

[0296] intermediate 14 - 6-(3- chlorine -6-(2-( Ethyl ( Isopropyl ) carbamoyl )-4- fluorophenoxy )-1,2,4- three azine -5- base )-2,6- diazalozyro [3.4] Octane -2- Preparation of tert-butyl formate 6-(3,6-dichloro-1,2,4-triazin-5-yl)-2,6-diazaspiro[3,4]octane-2-carboxylic acid tert-butyl ester (intermediate 13) (12.0 g, 33.3 mmol), N -Ethyl-5-fluoro-2-hydroxy- N A mixture of isopropylbenzamide (intermediate 28) (7.5 g, 33.3 mmol) and DBU (6.1 g, 40.1 mmol) in THF (120 mL) was stirred at 25 °C for 8 hours. The mixture was diluted with water (30 mL) and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a crude product, which was purified by FCC (PE / EtOAc = 1:0 to 3:1) to provide a green solid title intermediate (14.0 g, 73% yield).

[0297] intermediate 2 - 6-(6-(2-( Ethyl ( Isopropyl ) carbamoyl )-4- fluorine - phenoxy )-1,2,4- Triazine -5- base )-2,6- diazalozyro [3.4] Octane -2- Preparation of tert-butyl formate Synthesis method A of intermediate 2 : Under a nitrogen atmosphere, tert-butyl 6-(3-chloro-6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (intermediate 14) (20 g, 36.4 mmol), NaBH4 (2.48 g, 65.7 mmol), and TMEDA (8.54 g, 73.5 mmol) in THF (500 mL) was added to Pd(dppf)Cl2·DCM (1.70 g, 2.08 mmol). After addition, the reaction mixture was stirred at 25 °C for 14 hours. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by FCC (EtOAc) on silica gel to provide the title intermediate (15 g, 93% purity, 74% yield) as a brown solid.

[0298] intermediate 2 Synthesis method B : Pd / C (wet, 5.0 g, 10%) was added to a solution of tert-butyl 6-(3-chloro-6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (intermediate 14) (22.0 g, 40.1 mmol) and TEA (15 mL) in MeOH (100 mL). The resulting mixture was stirred at 25 °C for 8 hours under a H2 atmosphere (30 psi). The reaction mixture was filtered through a diatomaceous earth pad, and the filtrate was concentrated under vacuum to provide the title intermediate (25.0 g, crude), which could be used directly in the next step without further purification.

[0299] intermediate 3 - 2-((5-(2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen )-N- Second base -5- fluorine -N- Preparation of isopropylbenzamide TFA (0.5 mL, 6.4 mmol) was added to a solution of 6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylic acid tert-butyl ester (intermediate 2) (300 mg, 0.583 mmol) in DCM (5 mL), and the resulting mixture was stirred at room temperature for 3 hours. Then, a 10% NaOH solution (5 mL) was slowly added to the mixture to adjust the pH to approximately 12, and the resulting mixture was extracted with DCM (10 mL × 3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to provide the title intermediate as a white solid (220 mg, 90% yield).

[0300] compound 61 - (4-(6-(6-(2-( Ethyl ( Isopropyl ) carbamoyl )-4- fluorophenoxy )-1,2,4- three azine -5- base )-2,6- diazalozyro [3.4] pungent -2- base )-5- methylhexyl ) Preparation of tert-butyl carbamate 2-((5-(2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)- N -Ethyl-5-fluoro- N- Isopropylbenzamide (intermediate 3) (1.0 g, 2.4 mmol), (5-methyl-4-oxohexyl)carbamate tert-butyl ester (intermediate 1) (830 mg, 3.62 mmol), and ZnCl2 (660 mg, 4.84 mmol) in MeOH (15 mL) were stirred at 80 °C for 0.5 h. Then NaBH3CN (310 mg, 4.93 mmol) was added and the resulting mixture was stirred at 80 °C for 6 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to give a crude product, which was further purified by preparative HPLC using Waters Xbridge Prep OBD (column: C18 150 × 40 mm 10 μm; eluent: 45% to 75% v / v ACN / H2O (0.05% ammonia)) to provide a colorless oily title compound (700 mg, 46% yield).

[0301] compound 62 and 63 - (R)-(4-(6-(6-(2-( Ethyl ( Isopropyl ) carbamoyl )-4- fluorophenoxy )- 1,2,4- Triazine -5- base )-2,6- diazalozyro [3.4] pungent -2- base )-5- methylhexyl ) tert-butyl carbamate and (S)-(4- (6-(6-(2-( Ethyl ( Isopropyl ) carbamoyl )-4- fluorophenoxy )-1,2,4- Triazine -5- base )-2,6- diazalozyro [3.4] pungent -2- base )-5- methylhexyl ) Preparation of tert-butyl carbamate (4-6-(6-(2-( Ethyl ( Isopropyl ) carbamoyl )-4- fluorophenoxy )-1,2,4- Triazine -5- base )-2,6- diazalozyro [3.4] pungent -2- base )-5- tert-butyl methylhexylcarbamate (compound 61) (200 mg, 0.319 mmol) was purified by SFC on a DAICL CHIRALPAK IG (column: 250 × 30 mm 10 μm; isocratic elution: EtOH (containing 0.1% of 25% ammonia): supercritical CO2, 40%: 60% (v / v)) to provide the title compounds (compound 62) (85 mg, 42% yield) and (compound 63) (80 mg, 40% yield), both of which are pale yellow oils.

[0302] compound 64 - (R)-2-((5-(2-(6- amino -2- Methylhexyl -3- base )-2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen )-N- Ethyl -5- fluorine -N- Isopropylbenzamide Towards( R4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-2,6-diazaspiro[3.4]oct-2-yl)-5-methylhexyl)carbamate (compound 62) (550 mg, 0.876 mmol) was slowly added to a solution of TFA (4 mL) in DCM, and the resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was diluted in DCM (40 mL), and the pH was adjusted to approximately 12 by means of an aqueous solution of NaOH (2 M, 16 mL). The aqueous layer was extracted with DCM (10 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to provide the title compound (460 mg, crude) as a yellow solid, which could be used directly in the next step without further purification.

[0303] compound 11 - (R)-N- Ethyl -5- fluorine -N- Isopropyl -2-((5-(2-(6-((2- Methoxyethyl ) amino )- 2- Methylhexyl -3- base )-2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen ) benzamide Will( R )-2-((5-(2-(6-amino-2-methylhexyl-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)- N -Ethyl-5-fluoro- N A mixture of isopropylbenzamide (compound 64) (120 mg, crude), 1-bromo-2-methoxyethane (32 mg, 0.23 mmol), Cs₂CO₃ (222 mg, 0.681 mmol), and NaI (102 mg, 0.680 mmol) in DMF (1 mL) was stirred by microwave irradiation at 80 °C for 1 hour. After cooling to room temperature, the mixture was diluted with H₂O (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with H2O (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to provide a crude product. This crude product was purified by HPLC on a Phenomenex Gemini-NX (column: 150 × 30 mm 5 μm; eluent: 51% to 71% (v / v) ACN / H2O (10 mM NH4HCO3)) and further purified by SFC on a DAICL CHIRALCEL OD-H (column: 250 × 30 mm 5 μm; eluent: supercritical CO2 EtOH (0.1% v / v ammonia) solution 25 / 25, v / v) to provide the title compound as a yellow solid (5.13 mg, 96% purity).

[0304] LC-MS (ESI) (Method 1): R t= 2.997 min, measured m / z value is 586.3 [M+H] + .

[0305] compound A - (R)-N- Ethyl -5- fluorine -N- Isopropyl -2-((5-(2-(6-((2- Methoxyethyl )( methyl ) amino )-2- Methylhexyl -3- base )-2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen ) benzamide Will( R )- N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-(((2-methoxyethyl)amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide (compound 11) (40.0 mg, 0.068 mmol), formaldehyde (55.4 mg, 0.683 mol, 37% aqueous solution) and AcOH (8.2 mg, 0.137 mmol) in anhydrous MeOH (2 mL) were stirred at 45 °C for 1 hour. Then, NaBH3CN (8.6 mg, 0.137 mmol) was added to the mixture, and the resulting mixture was stirred at 45 °C for another 1 hour. After cooling to room temperature, the reaction mixture was treated with saturated NaHCO3 (40 mL) aqueous solution to adjust the pH to approximately 8 and further extracted with DCM (20 mL × 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. This crude product was purified by preparative HPLC on a Boston Prime column (C18 150×30mm 5µm, mobile phase A: H2O (0.04% ammonia + 10mM NH4HCO3), mobile phase B: ACN, flow rate: 25mL / min, gradient conditions B / A from 50% to 80% (50% B to 80% B)) to provide a yellow oily title compound (9.62mg, 99.10% purity, 23.3% yield).

[0306] Example 2 - ( R )- N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)) (amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3,4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide Synthesis and preparation method B of (compound A) intermediate 7 - 4-(( tert-Butoxycarbonyl )( methyl ) amino ) Preparation of butyric acid Boc₂O (4.69 g, 21.5 mmol) was added dropwise to a solution of 4-(methylamino)butyrate (3.0 g, 19.5 mmol) and TEA (7.78 mL, 58.6 mmol) in MeOH (30 mL). The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (100 mL), washed with cooled 0.1 N HCl (70 mL × 2), H₂O (50 mL × 2), and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated to provide a colorless, oily title intermediate (1.80 g, crude product).

[0307] intermediate 8 - (4-( methoxy ( methyl ) amino )-4- Oxybutyl )( methyl ) Preparation of tert-butyl carbamate Add to a solution of 4-((tert-butoxycarbonyl)(methyl)amino)butyric acid (intermediate 7) (1.80 g, crude product) in CHCl3 (30 mL) N , O -Dimethylhydroxylamine hydrochloride (960 mg, 9.84 mmol), HOBt (1.24 g, 9.18 mmol), and NMM (2.80 mL, 25.1 mmol). Then, EDCI (2.23 g, 11.6 mmol) was added, and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with DCM (100 mL), washed with 1N HCl (30 mL × 3), saturated NaHCO3 aqueous solution (30 mL × 3), and brine (30 mL), dried over Na2SO4, filtered, and concentrated under vacuum to provide a colorless, oily title intermediate (1.70 g, crude product).

[0308] intermediate 9 - methyl (5- methyl -4- Oxyhexyl ) Preparation of tert-butyl carbamate To a solution of (4-(methoxy(methyl)amino)-4-oxobutyl)(methyl)carbamate (intermediate 8) (200 mg, crude product) cooled at -70 °C under N2 atmosphere, isopropyllithium (3.2 mL, 2.24 mmol, 0.7 M in pentane) was added dropwise. The resulting mixture was stirred at -70 °C for 2 hours. The mixture was quenched with saturated aqueous NH4Cl solution (15 mL) and extracted with EtOAc (30 mL × 2). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was further purified by FCC (PE / EtOAc = 10:1) to provide a colorless oily title intermediate (60 mg).

[0309] compound 60 - (4-(6-(6-(2-( Ethyl ( Isopropyl ) carbamoyl )-4- fluorophenoxy )-1,2,4- three azine -5- base )-2,6- diazalozyro [3.4] pungent -2- base )-5- methylhexyl )( methyl ) Preparation of tert-butyl carbamate To 2-((5-(2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)- N -Ethyl-5-fluoro- N Isopropylbenzamide (intermediate 3) (600 mg, 1.45 mmol) and methyl (5-methyl-4-oxohexyl)carbamate tert-butyl ester (intermediate 9) (330 mg, 1.37 mmol) in a solution of MeOH (50 mL) were mixed with ZnCl2 (789 mg, 5.79 mmol). The resulting mixture was stirred at 80 °C for 2 hours. Then NaBH3CN (729 mg, 11.6 mmol) was added, and the reaction mixture was stirred at 80 °C overnight. After cooling to room temperature, the mixture was concentrated under reduced pressure to give a crude residue, which was diluted with DCM (50 mL), quenched with a saturated aqueous solution of NH4Cl (50 mL), and extracted with DCM (50 mL × 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was further purified by FCC (DCM / MeOH = 10:1) to provide the title compound as a white solid (400 mg, 42% yield).

[0310] compound 67 -N- Ethyl -5- fluorine -N- Isopropyl -2-((5-(2-(2- methyl -6-( methylamino ) Self -3- base )- 2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen ) Benzamide hydrochloride To a solution of (4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]oct-2-yl)-5-methylhexyl)(methyl)carbamate (compound 60) (1 g, 1.56 mmol) in DCM (10 mL), a solution of 4 M HCl in dioxane (5 mL, 20 mmol) was added, and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to provide the title compound (960 mg, crude, HCl salt), which could be used directly in the next step without further purification.

[0311] compound A - (R)-N- Ethyl -5- fluorine -N- Isopropyl -2-((5-(2-(6-((2- Methoxyethyl )( methyl ) amino )-2- Methylhexyl -3- base )-2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen ) benzamide Towards N -Ethyl-5-fluoro- N-Isopropyl-2-((5-(2-(2-methyl-6-(methylamino)hex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide hydrochloride (compound 67) (480 mg, crude), K2CO3 (700 mg, 5.07 mmol), and NaI (400 mg, 2.67 mmol) were added to a mixture in DMF (5 mL) with 1-bromo-2-methoxyethane (230 mg, 1.65 mmol). The resulting mixture was stirred overnight at 50 °C. After cooling to room temperature, the reaction mixture was quenched with H2O (30 mL) and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4, filtered, and concentrated to give a crude residue. The residue was purified by FCC (DCM / MeOH = 10:1) to provide a yellow oily substance. N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (compound 68) (250 mg, 48% yield).

[0312] First of all N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (compound 68) (960 mg, combined from several batches obtained by method B) was separated by SFC using a DAICELCHIRALPAK IG (column: 250 × 30 mm 10 μm; mobile phase: A: supercritical CO2, B: EtOH (0.1% ammonia), A:B = 40:60, at 60 mL / min) and by preparative HPLC using a Boston Prime (column: 150 × 30 mm 5 μm, mobile phase A: H2O (10 mM)). (NH4HCO3), mobile phase B: ACN, flow rate: 25 mL / min, gradient conditions B / A from 55% to 85%) for further purification to provide a colorless oily title compound (270 mg).

[0313] 1 H NMR (400MHz, methanol-) d4): δ = 8.40 (s, 1H), 7.47–7.32 (m, 1H), 7.30–7.10 (m, 2H), 4.24–4.01 (m, 2H), 3.89–3.60 (m, 3H), 3.48 (br s, 3H), 2.63–2.51 (m, 2H), 2.43–2.32 (m, 2H), 2.29–2.07 (m, 6H), 1.86–1.72 (m, 1H), 1.62–1.44 (m, 2H), 1.39–1.02 (m, 10H), 0.99–0.66 (m, 9H). Some protons are hidden by solvent peaks and have not been reported.

[0314] LCMS (ESI) (Method 2): R t = 1.965 min, measured m / z value is 600.3 [M+H] + .

[0315] SFC (Method 11): R t = 4.904 min.

[0316] Example 3 - ( R )- N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)) (amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3,4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide Synthesis-Preparation Method C of (Compound A) intermediate 227 - (R)-(1-(2,2- dimethyl -4,6- Dioxane -1,3- Dioxane -5- base )-3- First Keating -2- base ) Preparation of tert-butyl carbamate A DCM solution (607 kg) pre-cooled at -10°C to 0°C, consisting of Boc-L-valine (44.9 kg), 2,2-dimethyl-1,3-dioxane-4,6-dione (32.9 kg), and DMAP (35.5 kg), was added over 3 hours to a DCM solution (613 kg) containing DCC (55.5 kg) and aged at -10°C to 0°C for 16 hours. A 10% citric acid aqueous solution (449 kg) was added while maintaining the temperature below 10°C. The resulting slurry was aged at 0°C to 10°C for 2 hours and then filtered. The filter cake was washed with DCM (91 kg). The filtrate was separated, and the organic layer was washed with a 10% citric acid aqueous solution (twice, 450 kg) and a 10% NaCl aqueous solution (449 kg). Acetic acid (75.0 kg) was added to the organic phase (1200 kg) while maintaining the temperature between -10°C and 0°C. Sodium borohydride (18.0 kg) was added in batches over 5 hours while maintaining the temperature between -10°C and 0°C. The resulting mixture was then aged at -10°C to 0°C for 16 hours. The mixture was then heated to 15°C to 25°C and aged for 2 hours. The mixture was then washed with a 14% NaCl aqueous solution (450 kg), followed by a second wash with a 14% NaCl aqueous solution (432 kg), and finally washed with water (444 kg). The organic phase was concentrated under reduced pressure to 2–4 volumes. Isopropanol (143 kg) was added to the residue and concentrated under reduced pressure to 4–5 volumes. After cooling to -10°C to 0°C and aging for 8 hours, the resulting slurry was filtered, washed with IPA (38 kg), and dried to provide a white solid title intermediate (46.7 kg, 69% yield).

[0317] intermediate 228 - (R)-2- Isopropyl -5- oxypyrrolidine -1- Preparation of tert-butyl formate Will( R A solution of 46.7 kg of tert-butyl carbamate (intermediate 227) in toluene (333 kg) was heated to reflux and aged for 4 hours. The mixture was cooled to ambient temperature, filtered, and washed with toluene (20 kg). The combined filtrates were concentrated to dryness under reduced pressure to provide the desired compound in an oily form (31.05 kg, 96% yield), which could be used directly without further purification.

[0318] intermediate 229 - (5R)-2- hydroxyl -5- Isopropylpyrrolidine -1- Preparation of tert-butyl formate Will( RA solution of tert-butyl 2-isopropyl-5-oxopyrrolidine-1-carboxylate (intermediate 228) (30.9 kg) in 2-MeTHF (26.7 kg) was cooled to -5°C to 5°C. A solution of LiBH4 in 2-MeTHF (1 M, 45.2 kg, 54.4 mol) was added over 3 hours, and the mixture was aged for 4 hours. A cold 5% NaHCO3 aqueous solution (163 kg) was added over 3 hours at -5°C to 5°C, and the mixture was further aged for 2 hours. The mixture was then heated to ambient temperature and further aged for 2 hours. The aqueous layer was separated, and the organic layer was washed with a 10% NaCl aqueous solution (170 kg) and water (155 kg). During the water washing, an emulsion was formed, and solid NaCl (3.1 kg) was added to achieve separation. After removing the aqueous layer, the organic layer was concentrated to dryness under reduced pressure to provide the desired compound in oil form (28.5 kg, 91% yield), which could be used directly without further purification.

[0319] intermediate 230 - (R)-(6-((2- Methoxyethyl )( methyl ) amino )-2- Methylhexyl -3- base ) carbamic acid Preparation of tert-butyl ester At 15°C to 25°C, using 2-methoxy- N Treatment with 1-methylethyl-1-amine (12.3 kg, 138.0 mol) (5) R A solution of 2-hydroxy-5-isopropylpyrrolidine-1-carboxylic acid tert-butyl ester (intermediate 229) (28.55 kg) in DCM (344 kg) was prepared, and the resulting mixture was aged for 1 hour. Sodium triacetoxyborohydride (40.12 kg) was added in portions over 5 hours while maintaining a temperature between 15°C and 25°C, and the resulting mixture was aged for 48 hours. The reaction mixture was quenched by adding 8% NaOH aqueous solution (184 kg) over 2 hours while maintaining a temperature between 15°C and 25°C, and the mixture was further aged for 2 hours. The aqueous layer was separated, and the organic layer was washed with water (169 kg). The organic layer was then concentrated to dryness under reduced pressure to provide an oily title intermediate (33.26 kg, 88% yield), which could be used directly without further purification.

[0320] intermediate 231 - (R)-N 1 -(2- Methoxyethyl )-N 1 ,5- dimethylhexane -1,4- Diamine, dihydrochloride preparation Within 3 hours, at ambient temperature, add ( ) to a 4 molar HCl solution in isopropanol (84.80 kg). RA solution of 32.38 kg of tert-butyl 6-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexyl-3-yl)carbamate (intermediate 230) in isopropanol (25.6 kg) was prepared and the mixture was aged at ambient temperature for 19 hours. Then, methyl tert-butyl ether (95.25 kg) was added over 1 hour and the mixture was aged for 2.5 hours. The resulting slurry was filtered and washed with MTBE (53 kg). The filter cake was dried to provide the title compound as a white solid (23.92 kg, 81% yield).

[0321] intermediate 232 - 1- benzyl -3-( Chloromethyl ) pyrrolidine -3- Preparation of ethyl formate Add 2.33 kg of 2.5 M lithium in hexane (1.0 equivalent) to a solution of DIPEA (952 g, 1.1 equivalent) in THF (6 L) cooled to -35°C to -25°C, while maintaining the temperature below -25°C. Aging the resulting mixture at -35°C to -25°C for another 30 minutes, then cooling to a temperature between -78°C and -60°C. Add a solution of ethyl 1-benzylpyrrolidine-3-carboxylate (2 kg, 1.0 equivalent) in THF (2 L) at -78°C to -60°C and stir for another 30 minutes. Then, pack chloroiodomethane (1.81 kg, 1.2 equivalent) at -78°C to -60°C. Aging the reaction mixture at -60°C to -40°C for 2 hours. The reaction mixture was added to an aqueous solution of citric acid (660 g in 6 L H2O) at a temperature between 0°C and 10°C, and the resulting mixture was aged at 20°C to 30°C for 20 minutes. After separation of the layers, the aqueous layer was extracted with EtOAc (6 L), and the combined organic layers were washed with brine (6 L), then heated to 50°C to 60°C. Oxalic acid (2.22 kg) was loaded at 50°C to 60°C. The resulting mixture was stirred at 50°C to 60°C for 3 hours, then cooled to 20°C to 30°C and aged overnight. The resulting solid was filtered, and the filter cake was washed with ethyl acetate (2 L). The wet filter cake was added to toluene (4 L), H2O (8 L), and K3PO4 (1.5 equivalents), and the resulting mixture was aged at 20°C to 30°C for 20 minutes. After separation of the layers, the aqueous layer was extracted with toluene (2 L). The organic layers were combined and washed twice with water (2 L). The organic phase was concentrated under reduced pressure to provide 4.2 kg of the desired compound as a toluene solution (by determination, 46% by weight, yielding 80% of the determined yield).

[0322] intermediate 233 - 1- benzyl -3-( Chloromethyl ) pyrrolidine -3- Formaldehyde preparation The reaction was carried out in a flow chemistry system as follows: A solution of ethyl 1-benzyl-3-(chloromethyl)pyrrolidine-3-carboxylate (intermediate 232) (4.4 kg) in toluene (26 L) was pumped at 26.7 mL / min and cooled to -60 °C. After cooling, it was then mixed with a cooled solution of DIBAL-H (28.1 mol) in toluene (28 L) at -60 °C at a pumping rate of 32.1 mL / min. The mixture was passed through a perfluoroalkoxy (PFA) coil reactor at -60 °C (total flow rate 58.8 mL / min, residence time 5 s). The resulting mixture was mixed with cooled MeOH (-60 °C) pumped at a rate of 15.2 mL / min. This mixture was then pumped at -60 °C to another PFA coil reactor (total flow rate 74 mL / min, residence time 5 s). The resulting mixture was collected in a receiver containing 20% ​​by weight of an aqueous solution of Rochelle's salt (20 V). The layers were separated and the organic phase was washed twice with water (2 × 44 L). The organic phase was combined with another 3.0 kg batch prepared in a similar manner and concentrated under reduced pressure to provide a 20.8 kg toluene solution of the desired compound (25.5% by weight, 85% analytical yield by HPLC), which was ready for use without further purification.

[0323] 1 ¹H NMR (300MHz, chloroform-d): δ 9.62 (s, 1H), 7.39 - 7.20 (m, 5H), 3.83 - 3.57 (m, 4H), 2.96 (d, J = 10.2Hz, 1H), 2.80 - 2.55 (m, 3H), 2.17 (ddd, J = 13.9, 7.9, 6.1Hz, 1H), 1.83 (ddd, J = 13.4, 7.8, 5.5Hz, 1H).

[0324] intermediate 234 - (R)-4-(6- benzyl -2,6- diazalozyro [3.4] pungent -2- base )-N-(2- Methoxyethyl )- N,5- Dimethylhexyl -1- Preparation of amines At 20°C to 30°C, toluene (30L) and ( R )- N 1 -(2-Methoxyethyl)- N 15-Dimethylhexane-1,4-diamine, dihydrochloride (intermediate 231) (3.47 kg) was diluted with 1-benzyl-3-(chloromethyl)pyrrolidine-3-carboxaldehyde (intermediate 233) in toluene (3.0 kg, 10 wt%), and triethylamine (2.55 kg, 25.2 mol) was added. The resulting mixture was aged at 20°C to 30°C for 2 hours. Then, sodium triacetoxyborohydride (9.0 kg) was added at 20°C to 30°C and the mixture was aged for 12 hours. The reaction mixture was cooled to 5°C to 15°C, and 25 wt% NaOH aqueous solution (25 L, approximately 16.75 equivalents) was added, maintaining the temperature below 35°C. The resulting mixture was aged at 20°C to 30°C for 25 minutes, and the layers were separated. The organic layer was washed with 15 wt% NaCl aqueous solution (10 L), and the layers were separated again. Water (18 L) was added to the organic phase. The pH of the aqueous phase was adjusted to 6-7 using a 4M HCl aqueous solution, while maintaining the internal temperature below 35°C. The organic phase was then discarded, and the aqueous phase was separated and alkalized with K2HPO4 to pH 8-9.

[0325] The resulting mixture was heated to 50°C to 55°C and aged for 3 hours. The reaction mixture was then cooled to ambient temperature and combined with two other batches (2.4 kg + 3.0 kg). The combined streams were washed three times with methyl tert-butyl ether (3 × 40 L). Additional methyl tert-butyl ether (83 L) was added to the resulting aqueous layer, and the aqueous phase was alkalized to pH 9–10 using an 8% (w / w) NaOH aqueous solution while maintaining the temperature between 15°C and 35°C. The aqueous layer was separated, and the organic layer was washed three times with water (3 × 30 L). The organic layer was then concentrated under reduced pressure to approximately 3 volumes, washed three times with methanol (3 × 30 L), and concentrated to dryness to provide the desired intermediate (12.4 kg, 90% separation yield) in a pale yellow oil, which could be used directly without further purification.

[0326] intermediate 234a (intermediate) 234 Preparation of citrate Add EtOH (80 ml) and intermediate 234 (20 g) to a round-bottom flask. Next, add a 0.5 M citric acid solution of EtOH (100 ml; 1 equivalent) to the mixture in the round-bottom flask at room temperature. Then, evaporate the mixture to dryness (Rotavap, 40 °C). Add acetonitrile (200 ml) to the residue and evaporate the mixture to dryness (Rotavap, 40 °C). Add acetonitrile (100 ml) to the residue and stir overnight on a magnetic hot plate at room temperature. Finally, filter out intermediate 234a and dry at room temperature.

[0327] intermediate 234 Crystalline form of citrate (intermediate) 234b Preparation of ) Intermediate 234a (3.72 g) was added to acetonitrile (20 ml) at room temperature and the mixture was stirred. The mixture was heated to 60 °C until the reaction mixture became homogeneous (approximately 10 min). The mixture was then cooled to 50 °C at a rate of 0.5 °C / min. Seed crystals (19 mg of intermediate 234a; 0.5 w / w %) were then added, and the mixture was aged with stirring for 3 hours and 30 minutes. The mixture was then nonlinearly cooled to 20 °C over 8 hours at an exponent of 2,3. The resulting mixture was stirred overnight, and the product was filtered off and dried (overnight at room temperature in a fume hood). After separation, intermediate 234b (2.75 g; yield 73.9%) was obtained as the crystalline form of the citrate of intermediate 234. The ratio of intermediate to citric acid obtained was 3 / 2 (NMR).

[0328] The nonlinear cooling mentioned above is performed according to the following formula: A new linear ramp begins every 30 seconds during the defined cooling duration. The ramp is calculated using the following formula: T 设定 : Setting values ​​for each new ramp T 起始值 Temperature of the mixture measured at the start of the cooling trajectory T 最终值 The final value of the cooling trajectory limit t 动作 The actual time from the start of cooling Duration: Limited cooldown duration n: exponent.

[0329] 1 H NMR (400MHz, MeOH- d 4) δ ppm 0.91 (3 H, d, J =6.88Hz) 0.98 (3 H, d, J =6.88Hz) 1.46 - 1.57 (2 H, m) 1.67 - 1.87 (2 H, m) 1.94 - 2.03 (1 H, m) 2.20 -2.29 (2 H, m) 2.62 -2.69 (2 H, m) 2.72 - 2.77 (4 H, m) 2.77 - 2.82 (2 H, m)2.90 (2 H, t, J=7.32Hz) 2.95 - 3.02 (2 H, m) 3.07 - 3.16 (2 H, m) 3.16 - 3.22(2 H, m) 3.37 (3 H, s) 3.68 - 3.72 (2 H, m) 3.83 -3.89 (2 H, m) 3.90 - 3.92(2 H, m) 3.94 - 4.06 (2 H, m) 7.32 - 7.43 (5 H, m).

[0330] intermediate 224 - (R)-N-(2- Methoxyethyl )-N,5- dimethyl -4-(2,6- diazalozyro [3.4] pungent -2- base ) Self -1- Preparation of amines Add methanesulfonic acid (MSA) (11 kg) to carbon-supported palladium hydroxide (1.2 kg) in EtOH (1.47 kg) cooled to -5°C to 5°C. R )-4-(6-benzyl-2,6-diazaspiro[3.4]oct-2-yl- N -(2-Methoxyethyl)- N 5-Dimethylhexyl-1-amine (intermediate 234) (10 kg) and EtOH (250 L). The mixture was heated to 35°C-45°C and stirred for 16-20 hours under a hydrogen atmosphere (0.27 MPa to 0.40 MPa). The mixture was filtered through diatomaceous earth (20 kg) and washed with EtOH (24 L). The filtrate was concentrated to 2-3 volumes under reduced pressure (<40°C) and then washed twice with 2-MeTHF (73 kg and 47 kg) to obtain 2-3 volumes of solution. After dilution with 2-MeTHF (65 kg), 10% sodium sulfate aqueous solution (30 kg) was added and the mixture was cooled to 0°C-10°C, followed by addition of 16% NaOH aqueous solution (50 kg) to adjust the pH to 13-14. The temperature was adjusted to 15°C-25°C and stirred for 30-60 minutes. The aqueous layer was separated and extracted twice with 2-MeTHF (47 kg × 2). The combined organic layers were concentrated under reduced pressure (<40°C) to 3-4 volumes, and 2-MeTHF (950 g) was added. After concentration under reduced pressure (<40°C) to 3-4 volumes, the resulting solution was diluted with 2-MeTHF (30 kg), dried through a 4A molecular sieve (25 kg), and washed with 2-MeTHF (30 kg). The final solution was concentrated to provide the desired compound in an oily form (6.7 kg), with a purity of 90.1% and a corrected yield of 79%.

[0331] intermediate 225 - (R)-4-(6-(3,6- dichloro -1,2,4- Triazine -5- base )-2,6- diazalozyro [3.4] pungent - 2- base )-N-(2- Methoxyethyl )-N,5- Dimethylhexyl -1- Preparation of amines Towards( R)- N -(2-Methoxyethyl)- N 2-MeTHF (430 g) and TEA (68 g) were added to 5-dimethyl-4-(2,6-diazaspiro[3.4]oct-2-yl)hex-1-amine (intermediate 224) (100 g), and the mixture was cooled to -50°C to -40°C. 3,5,6-trichloro-1,2,4-triazine (62 g) was added to 2-MeTHF (172 g), and the mixture was stirred for 1 hour to 3 hours. The resulting mixture was heated to -20°C to -10°C, and a 7% NaHCO3 aqueous solution was added. The mixture was then heated to 20°C to 30°C and stirred for 30 minutes to 60 minutes. The aqueous layer was removed, and the organic layer was washed with 10% Na2SO4 (500 g). The organic layer was dried through a 4 Å molecular sieve (220 g) and washed with 2-MeTHF (180 g). The title intermediate was provided in 90% determined yield as a 14.8 wt% solution in 2-MeTHF.

[0332] compound 393 - (R)-2-((3- chlorine -5-(2-(6-((2- Methoxyethyl )( methyl ) amino )-2- Methylhexyl - 3- base )-2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen )-N- Ethyl -5- fluorine -N- Isopropyl - benzene formamide compound 393 Synthesis method A : Will N -Ethyl-5-fluoro-2-hydroxy- N - Isopropylbenzamide (intermediate 28) (1.10 g, 4.88 mmol), ( R )-4-(6-(3,6-dichloro-1,2,4-triazine-5-yl)-2,6-diazaspiro[3,4]oct-2-yl)- N -(2-Methoxyethyl)- NA mixture of 5-dimethylhexyl-1-amine (intermediate 225) (1.70 g, 3.82 mmol) and DBU (750 mg, 4.93 mmol) in anhydrous THF (15 mL) was stirred at 40 °C for 8 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the resulting residue was diluted with DCM (60 mL) and washed with H2O (20 mL × 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by FCC (MeOH / DCM = 0% to 10%) to provide a yellow oil (1.40 g), which was further separated by SFC on a DAICL CHIRALPAK AD (column: 250 × 50 mm, 10 μm; mobile phase: A: supercritical CO2, B: EtOH (0.1% ammonia), A:B = 50:50, at 70 mL / min; column temperature: 38 °C; nozzle pressure: 100 bar; nozzle temperature: 60 °C; evaporator temperature: 20 °C; potentiometer (Trimmer) temperature: 25 °C; wavelength: 220 nm) to provide the title compound (1.0 g).

[0333] compound 393 Synthesis method A : At 20℃ to 30℃, towards ( R )-4-(6-(3,6-dichloro-1,2,4-triazine-5-yl)-2,6-diazaspiro[3,4]oct-2-yl)- N -(2-Methoxyethyl)- N 2-MeTHF solution of 5-dimethylhexyl-1-amine (intermediate 225) (676 g of a 14.8 wt% solution in 2-MeTHF, 100 g of corrected intermediate 225) and N -Ethyl-5-fluoro-2-hydroxy- N - Tetramethylguanidine (31 g) was added to a solution of isopropylbenzamide (intermediate 28) (50.6 g) in 2-MeTHF (40 g), and the mixture was stirred for 40 to 48 hours. 7% NaHCO3 aqueous solution (500 g) was added, and the mixture was stirred for 30 to 60 minutes. The aqueous layer was removed, and the organic layer was washed twice with 4% NaOH aqueous solution (2 × 500 g) and once with 10% Na2SO4 aqueous solution (500 g). The organic layer was concentrated under reduced pressure (<40 °C) to 2.2–3.0 volumes and washed three times with MeOH (1 × 790 g and 2 × 395 g) until both 2-MeTHF and water content were ≤1.0% to provide the desired compound with an assay yield of 86% (as a 60.1% by weight solution in methanol).

[0334] compound A - (R)-N- Ethyl -5- fluorine -N- Isopropyl -2-((5-(2-(6-((2- Methoxyethyl )( methyl ) amino )-2- Methylhexyl -3- base )-2,6- diazalozyro [3.4] pungent -6- base )-1,2,4- Triazine -6- base ) Oxygen ) benzamide Will( R )-2-((3-chloro-5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)- N -Ethyl-5-fluoro- N A methanol solution of isopropylbenzamide (compound 393) (163.93 g in a 60.1 wt% solution in MeOH, 100 g of corrected compound 393), carbon-supported palladium (10 g), and MeOH (316 g) was stirred for 18 hours at 20-30°C under a hydrogen atmosphere (0.20 MPa-0.30 MPa). The mixture was filtered through diatomaceous earth (75 g), and the filter cake was washed with MeOH (158 g). The filtrate was concentrated under reduced pressure (≤40°C) to about 3 volumes, then washed with isopropyl acetate (IPAc, 870 g) and concentrated to 3 volumes. The mixture was then diluted with IPAc (696 g) and 20% Na2CO3 aqueous solution (500 g) was added. The mixture was stirred for 30-60 minutes. The aqueous layer was removed. The organic layer was washed with water (500 g) and then concentrated under reduced pressure at <45°C to about 3 volumes. The title intermediate was provided in approximately 90% determined yield as a 48.1% by weight solution in IPAc.

[0335] Example 4 - ( R )- N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)) (amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3,4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide Synthesis of oxalate (compound A3) Compound A3 Towards( R )- N -Ethyl-5-fluoro- N -Isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide (compound A) (270 mg, 0.450 mmol) was added to a solution of oxalic acid (81.0 mg, 0.900 mmol) in 20 mL of ACN (20 mL). After addition, the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated, the residue was redissolved in ACN and deionized water, and lyophilized to provide the title compound (350 mg) as a white solid.

[0336] 1 H NMR (400MHz, methanol-) d4): δ = 8.48 (s, 1H), 7.52-7.11 (m, 3H), 4.54-3.64 (m, 12H), 3.40-3.34 (m, 5H), 3.23-3.13 (m, 2H), 2.90 (s, 3H), 2.54-2.27(m, 2H), 2.19-2.03 (m, 1H), 1.97-1.77 (m, 2H), 1.75-1.50 (m, 2H), 1.35-0.65(m, 17H).

[0337] 1 H NMR (400MHz, DMSO- d 6): δ = 8.51 (s, 1H), 7.51-7.29 (m, 3H), 4.29-3.34 (m, 12H), 3.23-2.84 (m, 7H), 2.70 (s, 3H), 2.35-2.09 (m, 2H), 2.05-1.85(m,1H), 1.81-1.58(m,2H), 1.56-1.33(m,2H), 1.18-0.60(m,17H).

[0338] LCMS (ESI) (Method 2): R t = 1.969 min, measured m / z value is 600.4 [M+H] + .

[0339] Example 5 – Synthesis of Compound A1 EtOH (63 g) was added to a solution of compound A in IPAc (360 g) (207.90 g of a 48% wt solution in IPAc, 100 g of active compound A) at 20-25°C. The solution was then treated with concentrated HCl (32.9 g) in EtOH (49.5 g) over approximately 15 minutes. The mixture was inoculated with crystalline compound A1 seed crystals (2 g, 2% seed loading) and aged for 18 hours. IPAc (870 g) was slowly added over 4 hours at a temperature between 20-25°C, and the slurry was stirred again for 18 hours. After cooling to approximately 5°C, the product was filtered, washed with IPAc (522 g), and vacuum dried at 20-30°C to provide a weakly crystalline compound A1 as a white solid (91.0% yield, 115.4 g). (Note: The small amount of seed material used in the reaction was obtained on a small scale using a similar reaction protocol.) Recrystallization: A solution of weakly crystalline compound A1 (100 g), EtOH (166 g), purified water (21.5 g), and IPAc (178 g) was stirred at 20°C to 30°C for 0.5 h to 2 h to obtain a clear solution. Additional IPAc (522 g) was added dropwise over 1 h to 2 h, and the mixture was then inoculated with crystalline compound A1 seed crystals (2 g, 2% seed loading). The mixture was then aged for 18 h to 20 h, with IPAc (348 g) slowly added over 12 h at 20°C to 30°C, and the slurry was stirred again for 55 h to 60 h. The product was filtered, washed with IPAc (158 g), and vacuum dried at 20°C to 30°C to provide a white solid compound A1 (85% yield, 85.0 g, net value).

[0340] 1 HNMR (DMSO- d 6, 400MHz): δ = 11.60 (1H, br), 10.8 (1H, br), 8.52 (1H,s), 7.36 (3H, m), 3.97-4.20 (7H, m), 3.64-3.71 (4H, m), 3.47 (7H, m), 3.25(2H, m), 3.05(3H, m), 2.73 (3H, s), 2.10-2.45 (1H, m), 1.99 (1H, m), 1.78(2H, m), 1.55 (2H, m), 0.83-1.12 (12H, m), 0.70 (2H, m).

[0341] LCMS (Method 7): R t = 0.669 min, measured m / z value is 600.5 [M+H] + .

[0342] Example 6 - ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)) (amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3,4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide Synthesis of crystalline form A (compound A4) of bisbenzenesulfonate hydrate (equivalent water undetermined) 43.06 g of benzenesulfonic acid (2 equivalents relative to free base compound A) was added to 840 ml of acetone / water 95 / 5 v / v mixture and dissolved. 192.8 g of IPAc solution of compound A (containing 80 g of API) was added. This substance was dissolved to obtain a clear solution. Another 80 ml of IPAc was added, and the temperature was adjusted to 25°C. 2% seed crystals were added, and the mixture was stirred at 25°C for 1 hour. Then, 28.8 V (2312 ml) of IPAc was added over an 8-hour period. Afterward, the suspension was stirred at 25°C for 18 hours. The suspension was filtered and washed with 320 ml of acetone / water / IPAc 23.75 / 1.75 / 75 v / v / v mixture. 122.91 g of crystalline form A bisbenzenesulfonate hydrate (equivalent water undetermined) was obtained.

[0343] Those skilled in the art will understand that the small amount of initial seed material used in the above reaction can be obtained on a small scale without the addition of seed material via a similar reaction scheme and allowed to spontaneously nucleate.

[0344] Initial seed crystals of benzenesulfonate were also obtained during salt screening experiments. In these experiments, 100 mg of free base was weighed into a 2 mL vial, and then 200 μL of ethyl acetate or acetone was added to dissolve the free base. One equivalent of the counterion (benzenesulfonic acid) was added to the sample, and the sample was stirred at 25 °C for 3 days. The resulting suspension was centrifuged to produce initial seed crystals.

[0345] Add an appropriate amount of ( R The crystalline form A of 2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate was dissolved in deuterated DMSO and recorded as 1D. 1 H NMR spectrum.

[0346] One-dimensional proton collection experiments were performed on samples from deuterated DMSO at 300 K using a Bruker AVANCENEO-600 MHz NMR spectrometer equipped with a Bruker 5 mm PA BBO 600S3 BB-HD-05 Z-GRD high-resolution probe and running TOPSPIN 4.0 software.

[0347] 1 H NMR (600MHz, DMSO- d6) δ ppm 0.69 (br s, 2 H) 0.82 - 0.98 (m, 9 H)1.07 (br s, 4 H) 1.31 - 1.46 (m, 1 H) 1.51 (br d, J =2.91Hz, 1 H) 1.69 (br d, J =3.45Hz, 2 H) 1.98 (br s, 1 H) 2.06 - 2.45 (m, 2 H) 2.77 (br s, 3 H) 2.87 -3.19 (m, 3H) 3.24 (br s, 1 H) 3.31 (s, 6 H) 3.64 (br s, 4 H) 3.71 - 4.59 (m,7 H) 7.24 - 7.54 (m, 9 H) 7.61 (br d, J =7.27Hz, 4 H) 8.45 - 8.60 (m, 1 H) 9.24 (br s, 1 H) 9.44 - 9.82 (m, 1 H).

[0348] Example 7 - ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)) (amino)-2-methylhexyl-3-yl)-2,6-diazaspiro[3,4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide Alternative synthesis of crystalline form A (compound A4) of bisbenzenesulfonate hydrate (equivalent water undetermined) A 24 ml mixture of isopropanol / water (95 / 5) was packed into a flask and heated to 40 °C. Benzenesulfonic acid (4.31 g; 98%) was added. Subsequently, a solution of compound A in IPAc (containing 8 g of compound A) was added. Another 16 ml of IPAc was added. 2% seed crystals were added, and the mixture was stirred at 40 °C for 1 hour. Then, IPAc (115.2 ml) was added dropwise over an 8-hour period. The mixture was then cooled to 0 °C for 15 hours. The suspension was filtered, and the wet filter cake was washed with (IPA / H₂O 95 / 5) / IPAc 1 / 6 (32 ml). The wet filter cake was dried at 25 °C for 16 hours to obtain 11.44 g of crystalline form A bisbenzenesulfonate hydrate (equivalent water undetermined).

[0349] Crystal form A ( R The crystalline form A of 2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate can be characterized by X-ray powder diffraction.

[0350] X-ray powder diffraction (XRPD) analysis was performed on a PANalytical Empyrean diffractometer. This instrument is equipped with a Cu-Kα X-ray tube and uses iCore and dCore tuned optics for the incident and diffracted beams, respectively. Compounds were loaded into the cavity of a 16 mm sample holder using a reverse loading technique.

[0351] Use the following method to run the sample on XRPD: Tube: Cu: K-Alpha (λ=1.541874Ǻ) Generator: Voltage: 45 kV; Current: 40mA Geometry: Bragg-Brentano Scanning mode: Continuous scan Scanning range: 3 degrees to 35 degrees Step size: 0.0131 degrees Counting time: 30s Rotator rotation time: 1 second Incident beam path (iCore) Programmable diverging slit: Automatic Irradiation length: 10mm Sola Slit: 0.03 rad Mask 1: 14mm Mask 2: 6mm Width: 7.7mm Diffraction beam path (dCore) Anti-scattering slit: Automatic Irradiation length: 10mm Sola Slit: 0.04 rad Detector: PIXcel3D-Medipix3 1x1.

[0352] Those skilled in the art will recognize that diffraction patterns and peak positions are generally largely independent of the diffractometer used and whether a specific calibration method is employed. Typically, peak positions can differ by about ±0.2° 2θ or less. The intensity (and relative intensity) of each particular diffraction peak can also vary as a function of various factors, including but not limited to particle size, orientation, and sample purity.

[0353] The X-ray powder diffraction pattern includes peaks located at 5.4, 7.2, 11.1, 11.9, and 21.7°2θ ± 0.2°2θ. The X-ray powder diffraction pattern may also include at least one peak selected from the following: 13.7, 14.5, 14.7, 15.0, 16.5, 17.8, 19.0, 19.4, and 20.1°2θ ± 0.2°2θ.

[0354] Crystallization form A is also characterized by having four, five, six, seven, eight, nine or more peaks in its X-ray powder diffraction pattern selected from those identified in Table 2.

[0355] The characteristic of crystalline form A is that the X-ray powder diffraction pattern includes those peaks identified in Table 2, wherein the relative intensities of these peaks are greater than about 2%, preferably greater than about 5%, more preferably greater than about 10%, and even more preferably greater than about 15%. However, those skilled in the art will recognize that the relative intensities of the peaks can vary between different samples and between different measurements on the same sample.

[0356] The characteristic of crystalline form A can also be seen in its X-ray powder diffraction pattern, which is basically as follows: Figure 1 The description.

[0357] Table 2 provides ( R A list of peaks and relative intensities of the XPRD for the crystalline form A of )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) hydrate salt. Figure 1 ).

[0358] Table 2 – Peak list and relative intensities of XRPD for crystalline form A . Position [°2θ] Rel. Intensity [%] 5.3965 16.30 7.1906 23.69 9.2513 8.14 9.4433 7.39 11.0719 11.34 11.9144 73.29 12.3921 29.17 12.5717 22.93 12.8791 8.93 13.6790 26.58 13.8694 15.67 14.4793 38.19 14.7398 55.26 14.9599 56.99 15.8715 20.66 16.4606 22.37 17.0459 20.43 17.4421 34.59 17.8203 46.78 18.2871 30.73 18.9573 43.91 19.4485 41.00 20.1190 35.53 20.7356 18.05 21.0535 30.09 21.6801 100.00 22.0236 18.06 22.7925 29.92 23.5044 41.92 23.9959 43.96 24.5555 31.47 25.1401 25.01 25.7588 59.24 26.0910 53.05 26.6137 39.47 27.5409 24.89 28.5493 22.44 29.1699 13.97 30.1441 21.01 31.2560 14.66 31.8783 16.47 32.7054 17.11 33.2797 24.40 33.9762 15.63

[0359] Pharmacology section In vitro proliferation - menin-MLL Inhibitors (compounds) A4 )and DNA Combination of intercalating agent (Idabistar) cell lines AML cell lines MOLM-13, OCI-AML3, and MV4-11 were purchased from DSMZ. MOLM-13 cells were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. MV4-11 cells were grown in IMDM medium supplemented with 10% FBS and 1% penicillin / streptomycin. OCI-AML3 cells were cultured in 80%–90% α-MEM (containing ribose and deoxyribonucleoside) + 10%–20% FBS. All cell lines were cultured at 37°C in a 5% CO2 atmosphere.

[0360] Cell Titer Glo Measurement AML cell lines (5×10) 3Cells (100 cells / well) were seeded in 96-well plates and grown for 6 days in serum-containing (10%) medium with or without a specified concentration of inhibitor. Proliferation was analyzed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Data are the mean and standard deviation of two to four independent experiments in triplicate.

[0361] Synergistic effect calculation A biochemically intuitive generalized Loewe (BIGL) model based on R, implemented using the highest single-dose (HSA) null model. Specifically, the BIGL method was applied to calculate drug-drug interactions (Van der Borght, K., Tourny, A., Bagdziunas, R. et al. BIGL: Biochemically Intuitive Generalized Loewe null model for prediction of the expected combined effect compatible with partial agonism and antagonism. Sci Rep 7, 17935 (2017); Thas, O., Tourny, A., Verbist, B., Hawinkel, S., Nazarov, M., Mutambanengwe, K. and Bijnens, L. Statistical detection of synergy: New methods and a comparative study. Pharmaceutical Statistics (2021)).

[0362] The synergistic effect matrix results from the BIGL analysis of cell line data, with HSA as the mean model, are calculated based on cellular metabolic activity measured using Cell Titer-Glo. Bootstrap confidence intervals are indicated. Effect sizes and their confidence intervals are shown. Notably, each data point is based on a p-value and the sign of the corresponding maxR statistic, where the size of the point reflects the degree of synergistic or antagonistic effect corresponding to the tier scale. No significant mean effect is indicated if 0 is included in the interval.

[0363] result menin-MLL Inhibitors (compounds) A4 The combination of Idabi Star In MOLM-13 (KMT2A-AF9; FLT3-ITD) and OCI-AML3 (NPM1c AML) cells, the pairwise matrix combination of idarubicin and compound A4 was evaluated using a 6-day CellTiter-Glo assay. Notably, the combination of idarubicin and compound A4 did not exhibit antagonistic cytotoxicity in MOLM-13 (KMT2A-AF9; FLT3-ITD) cells, as reflected in the isoline plots. Figure 2A ) and as detailed in Table 3A below.

[0364] Table 3A - Effects of the combination of compound A4 and idarubicin on MOLM-13 cell proliferation. Note that there are no asterisks indicating additive effects; antagonistic effects are indicated by "*", and synergistic effects by "**". Antagonistic and synergistic effects are derived from significant calls based on the maxR test and their associated numbers, which are interpreted as antagonistic or synergistic effects, respectively.

[0365] Similarly, the combination of idarubicin and compound A4 showed no antagonistic cytotoxicity in OCI-AML3 (NPM1c AML) cells, as shown in the contour plot. Figure 2B (And detailed in Table 3B.)

[0366] Table 3B - Effects of the combination of compound A4 and idarubicin on the proliferation of OCI-AML3 cells. Note that there are no asterisks indicating additive effects; antagonistic effects are indicated by "*", and synergistic effects by "**". Antagonistic and synergistic effects are derived from significant calls based on the maxR test and their associated numbers, which are interpreted as antagonistic or synergistic effects, respectively.

[0367] In vitro proliferation - menin-MLL Inhibitors (compounds) A3 ) and pyrimidine analogues (cytarabine) and DNA Inlay (A combination of idarubicin or daunorubicin) cell lines AML cell lines MOLM-13 and OCI-AML3 were purchased from DSMZ. MOLM-13 cells were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. OCI-AML3 cells were cultured in 80%–90% α-MEM (ribonucleoside and deoxyribonucleoside-free) + 10%–20% FBS. All cell lines were cultured at 37°C in a 5% CO2 atmosphere.

[0368] Cell Titer Glo Measurement AML cell line (10×10) 3Cells (100 cells / well) were seeded in 96-well plates and grown for 6 days in complete medium with or without a specified concentration of inhibitor. Proliferation was analyzed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Data are the mean and standard deviation of two to four independent experiments in triplicate.

[0369] Synergistic effect calculation A biochemically intuitive generalized Loewe (BIGL) model based on R, implemented using the highest single-dose (HSA) null model. Specifically, the BIGL method was applied to calculate drug-drug interactions (Van der Borght, K., Tourny, A., Bagdziunas, R. et al. BIGL: Biochemically Intuitive Generalized Loewe null model for prediction of the expected combined effect compatible with partial agonism and antagonism. Sci Rep 7, 17935 (2017); Thas, O., Tourny, A., Verbist, B., Hawinkel, S., Nazarov, M., Mutambanengwe, K. and Bijnens, L. Statistical detection of synergy: New methods and a comparative study. Pharmaceutical Statistics (2021)).

[0370] The synergistic effect matrix results from the BIGL analysis of cell line data, with HSA as the mean model, are calculated based on cellular metabolic activity measured using Cell Titer-Glo. Bootstrap confidence intervals are indicated. Effect sizes and their confidence intervals are shown. Notably, each data point is based on a p-value and the sign of the corresponding maxR statistic, where the size of the point reflects the degree of synergistic or antagonistic effect corresponding to the tier scale. No significant mean effect is indicated if 0 is included in the interval.

[0371] result menin-MLL Inhibitors (compounds) A3 ) and cytarabine + The combination of Idabi Star In MOLM-13 (KMT2A-AF9; FLT3-ITD) and OCI-AML3 (NPM1c AML) cells, the pairwise matrix combination of cytarabine + idarubicin and compound A3 was evaluated using a 6-day CellTiter-Glo assay. As reflected below, the triple combination of cytarabine + idarubicin and compound A3 exhibited synergistic cytotoxicity in MOLM-13 cells. Figure 2C (and Table 3C), and showed no antagonistic cytotoxicity in OCI-AML3 cells ( Figure 2D (and Table 3D).

[0372] Table 3C - Effects of the combination of compound A3 with cytarabine and idarubicin on MOLM-13 cell proliferation. Note that there are no asterisks indicating additive effects; antagonistic effects are indicated by "*", and synergistic effects by "**". Antagonistic and synergistic effects are derived from significant calls based on the maxR test and their associated numbers, which are interpreted as antagonistic or synergistic effects, respectively.

[0373] Table 3D - Effects of the combination of compound A3 with cytarabine and idarubicin on the proliferation of OCI-AML3 cells. Note that there are no asterisks indicating additive effects; antagonistic effects are indicated by "*", and synergistic effects by "**". Antagonistic and synergistic effects are derived from significant calls based on the maxR test and their associated numbers, which are interpreted as antagonistic or synergistic effects, respectively.

[0374] menin-MLL Inhibitors (compounds) A3 ) and cytarabine + Combination of daunorubicin In MOLM-13 (KMT2A-AF9; FLT3-ITD) and OCI-AML3 (NPM1c) cells, the pairwise matrix combination of cytarabine + daunorubicin and compound A3 was evaluated using a 6-day CellTiter-Glo assay. As reflected below, the triple combination of cytarabine + daunorubicin and compound A3 exhibited synergistic cytotoxicity in MOLM-13 cells. Figure 2E (and Table 3E), and showed no antagonistic cytotoxicity in OCI-AML3 cells ( Figure 2F (and Table 3F).

[0375] Table 3. Effects of compound A3 combined with cytarabine and daunorubicin on MOLM-13 cell proliferation. Note that there are no asterisks indicating additive effects; antagonistic effects are indicated by "*", and synergistic effects by "**". Antagonistic and synergistic effects are derived from significant calls based on the maxR test and their associated numbers, which are interpreted as antagonistic or synergistic effects, respectively.

[0376] Table 3 shows the effect of the combination of compound A3 with cytarabine and daunorubicin on the proliferation of OCI-AML3 cells. Note that there are no asterisks indicating additive effects; antagonistic effects are indicated by "*", and synergistic effects by "**". Antagonistic and synergistic effects are derived from significant calls based on the maxR test and their associated numbers, which are interpreted as antagonistic or synergistic effects, respectively.

[0377] In vitro proliferation - For primary NPM1c Positive AML Patient samples menin-MLL Inhibitors (compounds) A3 )and DNA A combination of an intercalating agent (daunorubicin) and a pyrimidine analog (cytarabine). Samples from patients with primary NPM1c-positive AML (N=8) were used to test the combination of compound A3 with cytarabine and daunorubicin. All samples were cultured in complete medium and seeded in 96-well plates at a density of 20,000 cells / well. The plates were then treated with either a dual combination of cytarabine and daunorubicin (initial concentration of 10 μM cytarabine plus 5 μM daunorubicin, followed by 10-fold serial dilutions at a 6-point dilution rate) or a triple combination of compound A3 with cytarabine and daunorubicin (initial concentration of 10 μM compound A3, 10 μM cytarabine plus 5 μM daunorubicin, followed by 10-fold serial dilutions at a 6-point dilution rate). The cell plates treated with the above drugs, along with controls (wells containing only medium (“medium control”) or wells containing medium containing the medium (“medium control”)), were incubated at 37°C and 5% CO2 for 6 days; no medium was added or replaced during the experimental period. ATP levels, as an alternative marker of cell viability, were assessed using the CELLTITER-GLO assay. In short, the 96-well plate was removed from the incubator and allowed to equilibrate to room temperature for 30 minutes. CELLTITER-GLO was added to the wells and mixed on a shaker for 2 minutes, then incubated at room temperature for 10 minutes to stabilize the luminescence signal, followed by quantification using a plate reader.

[0378] Data were corrected for background signal using a culture medium control, normalized using a medium control, and then log-transformed. The changes in relative light units corresponding to different drug concentrations were reported as proportional to cell viability. A variable slope model was used to generate concentration-response curves; for each drug concentration (N=24, from N=8 patient samples, each concentration evaluated in triplicate), data were expressed as mean ± standard deviation. IC50 was calculated using nonlinear regression analysis. 50 value.

[0379] Overall, no antagonistic effect was observed in samples from patients with primary NPM1c-positive AML treated with a triple combination of compound A3, cytarabine, and daunorubicin, as reflected in Table 4 below.

[0380] Table 4 - Cellular activity of cytarabine and daunorubicin + / - compound A3 in primary NPM1c-positive AML patient samples The effect of vitality 。

Claims

1. A combination of the following (triple therapy): a therapeutically effective dose of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or a pharmaceutically acceptable salt or solvate thereof; and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs.

2. The combination according to claim 1, wherein compound A or a pharmaceutically acceptable salt or solvate thereof is a benzenesulfonate (compound A4-a). Compound A4-a Or its solvates.

3. The combination according to claim 2, wherein compound A4-a or its solvate is ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis(benzenesulfonate) or its hydrate.

4. The combination according to any one of claims 1 to 3, wherein the DNA intercalator is idarubicin and the pyrimidine analog is cytarabine.

5. The combination according to any one of claims 1 to 3, wherein the DNA intercalating agent is daunorubicin and the pyrimidine analog is cytarabine.

6. The combination according to any one of claims 1 to 3, wherein the DNA intercalating agent is doxorubicin and the pyrimidine analogue is cytarabine.

7. A pharmaceutical composition comprising the combination of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. The combination of any one of claims 1 to 6 or the pharmaceutical composition of claim 7, wherein the combination or the pharmaceutical composition is used as a pharmaceutical agent.

9. The combination of any one of claims 1 to 6 or the pharmaceutical composition of claim 7, wherein the combination or the pharmaceutical composition is used for the prevention or treatment of hematopoietic dysfunction, particularly for the treatment of hematopoietic dysfunction.

10. The combination or pharmaceutical composition for use according to claim 9, wherein the hematopoietic dysfunction has (i) one or more MLL1 (KMT2A) gene rearrangements or alterations (e.g., duplication or amplification) and / or NPM1 mutations plus (ii) FLT3 mutations.

11. The combination or pharmaceutical composition for the purpose according to claim 9, wherein the hematopoietic dysfunction has (i) one or more MLL1 (KMT2A) gene rearrangements plus (ii) an FLT3 mutation.

12. The combination or pharmaceutical composition for use according to claim 9, 10 or 11, wherein the hematopoietic dysfunction is acute myeloid leukemia (AML).

13. A method for treating a subject diagnosed with hematopoietic dysfunction, the method comprising administering to the subject a combination of the following (a triple therapy): a therapeutically effective dose of ( R )-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-(((2-methoxyethyl)(methyl)amino)-2-methylhex-3-yl)-2,6-diazaspiro[3.4]oct-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (Compound A): Compound A Or a pharmaceutically acceptable salt or solvate thereof; and therapeutically effective amounts of DNA intercalating agents and pyrimidine analogs.

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