Combinations of menin-MLL1 inhibitors, DNA insertors, and pyrimidine analogs for treating hematopoietic disorders

Abstract

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 insertion agent and a pyrimidine analog, and a method for treating a subject diagnosed with cancer using the combination.

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 intercalating agent, and a pyrimidine analog, and a method of treating a subject diagnosed with cancer using the combination.

Background Art

[0002] Cancer is a major cause of death worldwide. Of the 10 million cancer deaths recorded by GLOBOCAN in 2020, 7.1% were due to hematological disorders. Thus, there is an urgent need for new treatments for hematological disorders, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and acute lymphoblastic leukemia (ALL), as further detailed below.

[0003] AML is a common hematological malignancy whose incidence rises from 3:100,000 in young adults to over 20:100,000 in older adults. Overall survival (OS) is 40-50% in patients under 60 years of age, but only 5% in patients over 60. The majority of 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 as a result of age and comorbidities. Standard treatment 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 state-of-the-art treatments, the median OS is only about 10 months. In all types of AML, disease relapse is common despite initial treatment response and is the most common cause of death. Standard chemotherapy and allogeneic stem cell transplantation (if used) often fail to eradicate all tumor-proliferating cells and cannot select chemotherapy-resistant leukemic proliferating subclones. Patients refractory to salvage therapy are treated palliatively due to the extremely limited current treatment options. The median survival time for these patients is two months. Furthermore, newly diagnosed moderate- or high-risk MDS patients and those who relapse after standard care have a poor prognosis and a high risk of progression to AML. Therefore, there is an urgent need for new therapies for relapsed / refractory (R / R) AML and MDS patients, newly diagnosed AML patients ineligible for induction chemotherapy based on age and comorbidities, and newly diagnosed moderate / high / very-high-risk MDS patients.

[0004] Allergic leukemia (ALL) is a hematological malignancy that spreads through impaired differentiation, proliferation, and accumulation of lymphoid progenitor cells in the bone marrow and / or extramedullary regions. ALL accounts for 12% of all leukemia cases and is the most common type of acute leukemia in children, with a projected global incidence of 1–4.75 per 100,000 people. ALL accounts for approximately 20% of adult leukemia cases. Despite high complete remission (CR) rates (80–90%) with current therapies, the majority of adult ALL patients experience relapse. The 5-year overall survival rate is approximately 30–40% in adult and elderly patients. Therefore, there is an urgent need for new therapies for cancer, specifically relapsed / refractory ALL, and more specifically for adult and especially elderly patients.

[0005] Compounds A, A1, A2, and A3 described herein are disclosed in International Application No. CN2020 / 137266 (published as International Publication No. 2021 / 121327 on June 24, 2021), which is incorporated herein by reference in its entirety, along with the corresponding synthetic schemes and analytical characteristics.

[0006] (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide besylate is published in International Publication No. 2022 / 262796, which is incorporated herein by reference in its entirety, along with the corresponding synthetic scheme and analytical characterization. [Brief explanation of the drawing]

[0007] [Figure 1]This is the X-ray powder diffraction (XRPD) pattern of crystalline form A of compound A4:(R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide bis-besylate hydrate. [Figure 2A] This is a contour plot of maxR showing the effect of compound A4 in combination with idarubicin on the proliferation of MOLM-13 cells in vitro. [Figure 2B] This is a contour plot of maxR showing the effect of compound A4 in combination with idarubicin on the proliferation of OCI-AML3 cells in vitro. [Figure 2C] This is a contour plot of maxR showing the effect of compound A3 combined with cytarabine + idarubicin on the proliferation of MOLM-13 cells in vitro. [Figure 2D] This is a contour plot of maxR showing the effect of compound A3 combined with cytarabine + idarubicin on the proliferation of OCI-AML3 cells in vitro. [Figure 2E] This is a contour plot of maxR showing the effect of compound A3 combined with cytarabine + daunorubicin on the proliferation of MOLM-13 cells in vitro. [Figure 2F] This is a contour plot of maxR showing the effect of compound A3 combined with cytarabine + daunorubicin on the proliferation of OCI-AML3 cells in vitro. [Modes for carrying out the invention]

[0008] The present invention relates to a combination (triple combination) of a therapeutically effective amount of a menin-MLL inhibitor, a therapeutically effective amount of a DNA insertion agent, and a pyrimidine analog as described herein.

[0009] It will be apparent to those skilled in the art that all combinations described herein are the three-drug combinations of the menin-MLL inhibitor, DNA insertion agent, and pyrimidine analog described herein.

[0010] The menin-MLL inhibitors described herein refer to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide, its pharmaceutically acceptable salts and solvates, or subgroups thereof.

[0011] One embodiment of the present invention is (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A):

[0012] [ka] The present invention relates to pharmaceutically acceptable salts or solvates thereof, as well as combinations of therapeutically effective amounts of DNA insertors and pyrimidine analogs.

[0013] In particular, compound A or a pharmaceutically acceptable salt or solvate thereof is, in embodiments, any of its subgroups described herein.

[0014] Embodiments of the present invention include compound A1:

[0015] [ka] This also relates to combinations of therapeutically effective amounts of DNA insertion agents and pyrimidine analogs.

[0016] Embodiments of the present invention include compound A2:

[0017] [ka] This also relates to combinations of therapeutically effective amounts of DNA insertion agents and pyrimidine analogs.

[0018] Embodiments of the present invention include compound A3:

[0019] [ka] This also relates to combinations of therapeutically effective amounts of DNA insertion agents and pyrimidine analogs.

[0020] Embodiments of the present invention include compound A4-a:

[0021] [ka] The present invention relates to a solvate thereof, as well as a therapeutically effective amount of a combination of a DNA insertor and a pyrimidine analog. Those skilled in the art will understand that in this embodiment (and similar embodiments), “or thereof” refers to the besylate of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide.

[0022] Embodiments of the present invention include compound A4-a:

[0023] [ka] The present invention relates to the hydrate thereof, as well as a combination of a therapeutically effective amount of DNA insertion agent and a pyrimidine analog.

[0024] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate, and combinations of therapeutically effective amounts of DNA insertion agents and pyrimidine analogs.

[0025] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate, and combinations of therapeutically effective amounts of DNA insertion agents and pyrimidine analogs.

[0026] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate, and combinations of therapeutically effective amounts of DNA insertion agents and pyrimidine analogs.

[0027] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate, and combinations of therapeutically effective amounts of DNA insertion agents and pyrimidine analogs.

[0028] Embodiments of the present invention relate to the crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate, and combinations of therapeutically effective amounts of DNA insertors and pyrimidine analogs.

[0029] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate, and combinations of therapeutically effective amounts of DNA insertors and pyrimidine analogs.

[0030] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate, and combinations of therapeutically effective amounts of DNA insertors and pyrimidine analogs.

[0031] According to the embodiments, examples of DNA insertion agents include, but are not limited to, anthracyclines (e.g., daunorubicin, doxorubicin, idarubicin).

[0032] According to one embodiment, the DNA insertion agent is an anthracycline.

[0033] According to one embodiment, the DNA insertion agent is daunorubicin.

[0034] According to one embodiment, the DNA insertion agent is doxorubicin.

[0035] According to one embodiment, the DNA insertion agent is idarubicin.

[0036] In some embodiments, the pyrimidine analog is, but is not limited to, cytarabine (ARA-C).

[0037] According to one embodiment, the pyrimidine analog is cytarabine.

[0038] According to one embodiment, the DNA intercalating agent is daunorubicin, and the pyrimidine analog is cytarabine.

[0039] According to one embodiment, the DNA intercalating agent is doxorubicin, and the pyrimidine analog is cytarabine.

[0040] According to one embodiment, the DNA intercalating agent is idarubicin, and the pyrimidine analog is cytarabine.

[0041] One embodiment of the present invention is (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A):

[0042] [ka] The present invention relates to pharmaceutically acceptable salts or solvates thereof, as well as therapeutically effective doses of combinations of doxorubicin and cytarabine.

[0043] Embodiments of the present invention include compound A1:

[0044] [ka] Furthermore, this relates to therapeutically effective doses of doxorubicin and cytarabine in combination.

[0045] Embodiments of the present invention include compound A2:

[0046] [ka] Furthermore, this relates to therapeutically effective doses of doxorubicin and cytarabine in combination.

[0047] Embodiments of the present invention include compound A3:

[0048] [ka] Furthermore, this relates to therapeutically effective doses of doxorubicin and cytarabine in combination.

[0049] Embodiments of the present invention include compound A4-a:

[0050] [ka] The present invention relates to the solvates thereof, as well as therapeutically effective doses of combinations of doxorubicin and cytarabine.

[0051] Embodiments of the present invention include compound A4-a:

[0052] [ka] The present invention relates to the hydrate thereof, as well as to a therapeutically effective dose of doxorubicin and cytarabine in combination.

[0053] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate, and combinations of doxorubicin and cytarabine in therapeutically effective amounts.

[0054] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate, and combinations thereof in therapeutically effective amounts of doxorubicin and cytarabine.

[0055] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5 to 2.0 equivalent hydrate, and combinations of therapeutically effective amounts of doxorubicin and cytarabine.

[0056] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate, and a therapeutically effective amount of doxorubicin and cytarabine in combination.

[0057] Embodiments of the present invention relate to the crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate, and to a therapeutically effective amount of combination of doxorubicin and cytarabine.

[0058] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate, and to combinations of therapeutically effective amounts of doxorubicin and cytarabine.

[0059] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate, and to a therapeutically effective amount of combination of doxorubicin and cytarabine.

[0060] One embodiment of the present invention is (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A):

[0061] [ka] The present invention relates to pharmaceutically acceptable salts or solvates thereof, as well as therapeutically effective doses of combinations of idarubicin and cytarabine.

[0062] Embodiments of the present invention include compound A1:

[0063] [ka] Furthermore, this relates to therapeutically effective doses of idarubicin and cytarabine in combination.

[0064] Embodiments of the present invention include compound A2:

[0065] [ka] Furthermore, this relates to therapeutically effective doses of idarubicin and cytarabine in combination.

[0066] Embodiments of the present invention include compound A3:

[0067] [ka] Furthermore, this relates to therapeutically effective doses of idarubicin and cytarabine in combination.

[0068] Embodiments of the present invention include compound A4-a:

[0069] [ka] The present invention relates to the solvates thereof, as well as therapeutically effective doses of idarubicin and cytarabine in combination.

[0070] Embodiments of the present invention include compound A4-a:

[0071] [ka] The present invention relates to the hydrate thereof, as well as a therapeutically effective dose of idarubicin and cytarabine in combination.

[0072] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate, and a therapeutically effective amount of combination of idarubicin and cytarabine.

[0073] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate, and a therapeutically effective amount of combination of idarubicin and cytarabine.

[0074] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate, and combinations of therapeutically effective amounts of idarubicin and cytarabine.

[0075] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate, and a therapeutically effective amount of combination of idarubicin and cytarabine.

[0076] Embodiments of the present invention relate to the crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate, and to a therapeutically effective amount of combination of idarubicin and cytarabine.

[0077] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate, and to combinations of therapeutically effective amounts of idarubicin and cytarabine.

[0078] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate, and to a therapeutically effective amount of combination of idarubicin and cytarabine.

[0079] One embodiment of the present invention is (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A):

[0080] [ka] The present invention relates to pharmaceutically acceptable salts or solvates thereof, as well as therapeutically effective doses of combinations of daunorubicin and cytarabine.

[0081] Embodiments of the present invention include compound A1:

[0082] [ka] Furthermore, this relates to therapeutically effective doses of daunorubicin and cytarabine in combination.

[0083] Embodiments of the present invention include compound A2:

[0084] [ka] Furthermore, this relates to therapeutically effective doses of daunorubicin and cytarabine in combination.

[0085] Embodiments of the present invention include compound A3:

[0086] [ka] Furthermore, this relates to therapeutically effective doses of daunorubicin and cytarabine in combination.

[0087] Embodiments of the present invention include compound A4-a:

[0088] [ka] The present invention relates to the solvates thereof, as well as therapeutically effective doses of daunorubicin and cytarabine in combination.

[0089] Embodiments of the present invention include compound A4-a:

[0090] [ka] The present invention relates to the hydrate thereof, as well as a therapeutically effective dose of daunorubicin and cytarabine in combination.

[0091] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate, and a therapeutically effective amount of combination of daunorubicin and cytarabine.

[0092] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate, and a therapeutically effective amount of daunorubicin and cytarabine in combination.

[0093] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5 to 2.0 equivalent hydrate, and a combination of therapeutically effective amounts of daunorubicin and cytarabine.

[0094] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 2.0 equivalent hydrate, and a therapeutically effective amount of combination of daunorubicin and cytarabine.

[0095] Embodiments of the present invention relate to the crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate, and to a combination of therapeutically effective amounts of daunorubicin and cytarabine.

[0096] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate, and to combinations of therapeutically effective amounts of daunorubicin and cytarabine.

[0097] Embodiments of the present invention relate to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate, and to a therapeutically effective amount of combination of daunorubicin and cytarabine.

[0098] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a combination described in any of the other embodiments as an active ingredient.

[0099] Embodiments of the present invention relate to the use of the combination for treating subjects diagnosed with hematopoietic disorders such as blood cancer, but are not limited thereto. Embodiments of the present invention relate to the use of the combination for treating subjects diagnosed with hematopoietic disorders such as hematopoietic cancer, but are not limited thereto.

[0100] Embodiments of the present invention relate to a novel method for treating subjects diagnosed with hematopoietic disorders using such a combination. Embodiments of the novel method include administering to a subject a therapeutically effective amount of a menin-MLL inhibitor described herein, and a therapeutically effective amount of a DNA insertor and a pyrimidine analog.

[0101] In one embodiment, the subject is newly diagnosed in a situation that makes them eligible for chemotherapy.

[0102] In some embodiments, the present invention describes a method for treating a subject diagnosed with a hematopoietic disorder, the method comprising administering a therapeutically effective dose of a menin-MLL inhibitor and a therapeutically effective dose of a DNA insertor and pyrimidine analog to the subject.

[0103] Additional embodiments of the present invention relate to a method for treating a subject diagnosed with cancer (for example, cancer as herein is leukemia such as myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL)), the method being: Therapeutic amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (compound A):

[0104] [ka] or its pharmaceutically acceptable salt or solvate, This includes administering a therapeutically effective amount of DNA insertion agent and pyrimidine analog to the subject.

[0105] One embodiment of the present invention is (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A):

[0106] [ka] Alternatively, a pharmaceutically acceptable salt or solvate thereof may be used in combination with DNA insertion agents and pyrimidine analogs for the treatment of cancer.

[0107] One embodiment of the present invention provides a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A):

[0108] [ka] Alternatively, a pharmaceutically acceptable salt or solvate thereof may be used in combination with a therapeutically effective amount of a DNA inserter and a pyrimidine analog for the treatment of cancer.

[0109] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0110] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0111] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0112] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0113] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0114] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0115] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0116] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a therapeutically effective amount of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0117] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0118] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with a therapeutically effective amount of doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0119] In one embodiment, the present invention relates to compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with idarubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0120] In one embodiment, the present invention relates to a therapeutically effective amount of compound A or a pharmaceutically acceptable salt or solvate thereof for use in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0121] In one embodiment, the present invention relates to compound A4-a:

[0122] [ka] Alternatively, with respect to the solvates thereof, they are used in combination with DNA insertion agents and pyrimidine analogs for the treatment of cancer.

[0123] In one embodiment, the present invention provides a therapeutically effective amount of compound A4-a:

[0124] [ka] Alternatively, with respect to the solvate thereof, it is used in combination with therapeutically effective amounts of DNA insertion agents and pyrimidine analogs for the treatment of cancer.

[0125] In one embodiment, the present invention relates to compound A4-a or its solvate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0126] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its solvate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0127] In one embodiment, the present invention relates to compound A4-a or its solvate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0128] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its solvate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0129] In one embodiment, the present invention relates to compound A4-a or its solvate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0130] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its solvate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0131] In one embodiment, the present invention relates to compound A4-a or its solvate for use in combination with daunorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0132] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its solvate for use in combination with therapeutically effective amounts of daunorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0133] In one embodiment, the present invention relates to compound A4-a or its solvate for use in combination with doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0134] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its solvate for use in combination with therapeutically effective amounts of doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0135] In one embodiment, the present invention relates to compound A4-a or its solvate for use in combination with idarubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0136] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its solvate for use in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0137] In one embodiment, the present invention relates to compound A4-a:

[0138] [ka] Alternatively, the hydrate thereof may be used in combination with DNA insertion agents and pyrimidine analogs for the treatment of cancer.

[0139] In one embodiment, the present invention provides a therapeutically effective amount of compound A4-a:

[0140] [ka] Alternatively, the hydrate thereof may be used in combination with therapeutically effective amounts of DNA insertion agents and pyrimidine analogs for the treatment of cancer.

[0141] In one embodiment, the present invention relates to compound A4-a or its hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0142] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0143] In one embodiment, the present invention relates to compound A4-a or its hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0144] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0145] In one embodiment, the present invention relates to compound A4-a or its hydrate for use in combination with DNA insertion agents and pyrimidine analogs for the treatment of acute myeloid leukemia (AML).

[0146] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0147] In one embodiment, the present invention relates to compound A4-a or its hydrate for use in combination with daunorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0148] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate for use in combination with therapeutically effective amounts of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0149] In one embodiment, the present invention relates to compound A4-a or its hydrate for use in combination with doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0150] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate for use in combination with therapeutically effective amounts of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0151] In one embodiment, the present invention relates to compound A4-a or its hydrate for use in combination with idarubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0152] In one embodiment, the present invention relates to a therapeutically effective amount of compound A4-a or its hydrate for use in combination with therapeutically effective amounts of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0153] Embodiments of the present invention relate to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of cancer.

[0154] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of cancer.

[0155] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0156] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0157] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0158] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0159] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0160] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0161] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with daunorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0162] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of daunorubicin and cytarabine to treat acute myeloid leukemia (AML).

[0163] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0164] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0165] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with idarubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0166] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0167] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of cancer.

[0168] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of cancer.

[0169] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of hematopoietic disorders.

[0170] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0171] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0172] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a therapeutically effective amount of a DNA inserter and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0173] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0174] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0175] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with daunorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0176] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its solvate for use in combination with a therapeutically effective amount of daunorubicin and cytarabine to treat acute myeloid leukemia (AML).

[0177] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0178] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a therapeutically effective amount of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0179] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with idarubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0180] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or its hydrate for use in combination with a therapeutically effective amount of idarubicin and cytarabine to treat acute myeloid leukemia (AML).

[0181] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of cancer.

[0182] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in the treatment of cancer in combination with a therapeutically effective amount of DNA insertion agent and pyrimidine analog.

[0183] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of hematopoietic disorders.

[0184] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0185] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0186] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0187] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of acute myeloid leukemia (AML).

[0188] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0189] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0190] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0191] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0192] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0193] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0194] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesilate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0195] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of cancer.

[0196] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of cancer.

[0197] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of hematopoietic disorders.

[0198] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0199] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0200] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertor and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0201] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0202] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0203] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0204] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0205] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0206] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0207] In one embodiment, the present invention relates to (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0208] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0209] In one embodiment, the present invention relates to a crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with DNA insertion agents and pyrimidine analogs in the treatment of cancer.

[0210] In one embodiment, the present invention relates to a crystalline form A (compound A4) of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of cancer.

[0211] In one embodiment, the present invention relates to a crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0212] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate in crystalline form A (compound A4) for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0213] In one embodiment, the present invention relates to a crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0214] In one embodiment, the present invention relates to a crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0215] In one embodiment, the present invention relates to a crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0216] 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-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate in crystalline form A (compound A4) for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0217] In other embodiments, the present invention relates to crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with daunorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0218] In one embodiment, the present invention relates to a crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with a therapeutically effective amount of daunorubicin and cytarabine to treat acute myeloid leukemia (AML).

[0219] In other embodiments, the present invention relates to crystalline form A (compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate for use in combination with doxorubicin and cytarabine in the treatment of acute myeloid leukemia (AML).

[0220] In one embodiment, the present invention relates to crystalline form A (Compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate hydrate for use in the treatment of acute myeloid leukemia (AML) in combination with a therapeutically effective amount of doxorubicin and cytarabine.

[0221] In other embodiments, the present invention relates to crystalline form A (Compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate hydrate for use in the treatment of acute myeloid leukemia (AML) in combination with idarubicin and cytarabine.

[0222] In one embodiment, the present invention relates to crystalline form A (Compound A4) of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate hydrate for use in the treatment of acute myeloid leukemia (AML) in combination with a therapeutically effective amount of idarubicin and cytarabine.

[0223] In one embodiment, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 0.5 to 2.0 equivalents hydrate for use in the treatment of cancer in combination with a DNA intercalator and a pyrimidine analog.

[0224] In one embodiment, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 0.5 - 2.0 equivalents hydrate for use in the treatment of cancer in combination with a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analog.

[0225] In one embodiment, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 0.5 - 2.0 equivalents hydrate for use in the treatment of hematopoietic disorders in combination with a DNA intercalating agent and a pyrimidine analog.

[0226] In one embodiment, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 0.5 - 2.0 equivalents hydrate for use in the treatment of hematopoietic disorders in combination with a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analog.

[0227] In one embodiment, the present invention relates to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5~2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0228] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5~2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0229] In one embodiment, the present invention relates to a crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5~2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0230] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of acute myeloid leukemia (AML).

[0231] In one embodiment, the present invention relates to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate for use in combination with daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0232] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of daunorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0233] In one embodiment, the present invention relates to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5~2.0 equivalent hydrate for use in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0234] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0235] In one embodiment, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate for use in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0236] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 0.5-2.0 equivalent hydrate for use in combination with a therapeutically effective amount of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0237] In one embodiment, the present invention relates to a crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of cancer.

[0238] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in the treatment of cancer in combination with a therapeutically effective amount of DNA insertion agent and pyrimidine analog.

[0239] In one embodiment, the present invention relates to a crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0240] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertion agent and a pyrimidine analog for the treatment of hematopoietic disorders.

[0241] In one embodiment, the present invention relates to a crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a DNA insertion agent and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0242] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of a DNA insertor and a pyrimidine analog for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), small lymphocytic lymphoma (SLL), or chronic lymphocytic leukemia (CLL).

[0243] In one embodiment, the present invention relates to crystalline Form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 2.0 equivalent hydrate for use in the treatment of acute myeloid leukemia (AML) in combination with a DNA intercalating agent and a pyrimidine analog.

[0244] In one embodiment, the present invention relates to crystalline Form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 2.0 equivalent hydrate for use in the treatment of acute myeloid leukemia (AML) in combination with a therapeutically effective amount of a DNA intercalating agent and a pyrimidine analog.

[0245] In other embodiments, the present invention relates to crystalline Form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 2.0 equivalent hydrate for use in the treatment of acute myeloid leukemia (AML) in combination with daunorubicin and cytarabine.

[0246] In one embodiment, the present invention relates to crystalline Form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate 2.0 equivalent hydrate for use in the treatment of acute myeloid leukemia (AML) in combination with a therapeutically effective amount of daunorubicin and cytarabine.

[0247] In other embodiments, the present invention relates to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0248] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of doxorubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0249] In other embodiments, the present invention relates to the crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0250] In one embodiment, the present invention relates to a crystalline form A of a therapeutically effective amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate 2.0 equivalent hydrate for use in combination with a therapeutically effective amount of idarubicin and cytarabine for the treatment of acute myeloid leukemia (AML).

[0251] In particular, in any of the embodiments, the DNA insertion agent is daunorubicin, and the pyrimidine analog is cytarabine.

[0252] In particular, in any of the embodiments, the DNA insertion agent is doxorubicin, and the pyrimidine analog is cytarabine.

[0253] In particular, in any of the embodiments, the DNA insertion agent is idarubicin, and the pyrimidine analog is cytarabine.

[0254] A method for treating a subject diagnosed with hematopoietic disorder, comprising (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (compound A):

[0255] [ka] Alternatively, this includes administering a pharmaceutically acceptable salt or solvate thereof, as well as a therapeutically effective dose of a combination of a DNA insertor and a pyrimidine analog to the target.

[0256] Examples of pharmaceutically acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example, by reacting a free acid form or a free base form 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, and then removing the solvent or medium using standard techniques (e.g., by vacuum, by freeze-drying, or by filtration). Salts may also be prepared, for example, by exchanging a counterion of the compound of the present disclosure in salt form with another counterion using a suitable ion exchange resin.

[0257] The pharmaceutically acceptable salts referred to above or below in this specification include therapeutically active, non-toxic acid and base salt forms that can form (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide and its solvates.

[0258] Suitable acids include, for example, hydrohalic acids, such as hydrochloric acid or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and other inorganic acids, or, for example, organic acids such as acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, oxalic acid (i.e., ethanedioic acid), malonic acid, succinic acid (i.e., butanedioic acid), maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, pamoic acid, and other organic acids. Conversely, the above salt forms can be converted to free salt forms by treatment with a suitable base.

[0259] (R)-N-Ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide and its solvates containing acidic protons can further be converted into their non-toxic metal or amine salt forms by treatment with suitable organic and inorganic bases.

[0260] Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, cesium, magnesium, calcium salts, etc., organic bases such as primary, secondary and tertiary aliphatic amines and aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline, and salts with isoquinoline, benzathine, N-methyl-glucamine, hydrabamine salts, and salts with amino acids such as arginine, lysine, etc. Conversely, the base forms can be converted into the free base forms by treatment with an acid.

[0261] All isotopes and mixtures of isotopes of any particular atom or element specified herein are contemplated within the scope of the invention whether occurring naturally in natural abundance or in isotopically enriched form or whether produced synthetically. Exemplary isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine. 2 H 3 H 11 C 13 C 14 C 13 N 15 O 17 O 18 O 32 P 33P, 35 S, 18 F, 36 Cl, 122 I, 123 I, 125 I, 131 I, 75 Br, 76 Br, 77 Br, and 82 Examples include Br. Preferably, the isotope is 2 H, 3 H, 11 C, 13 C, and 18 Selected from group F. Preferably, the isotope is 2 H, 3 H, 11 C, and 18 Selected from group F. More preferably, the isotope is 2 H, 3 H, or 13 It is C. More preferably, the isotope is 2 H or 13 It is C. More preferably, the isotope is 2 It is H. In particular, deuterium compounds and 13 C-enriched compounds are intended to be included within the scope of the present invention. In particular, deuterium compounds are intended to be included within the scope of the present invention.

[0262] Specific isotope-labeled compounds (e.g., 3 H and 14 (labeled with 1C) may be useful, for example, in substrate tissue distribution assays. Tritiated ( 3 H) and carbon-14( 14 C) Isotopes are useful due to their ease of preparation and detection. Furthermore, heavier isotopes, such as deuterium (i.e., 2 Substitution with H), etc., can lead to increased metabolic stability (e.g., increased half-life in vivo or reduced required dose), resulting in certain therapeutic benefits and therefore being preferable depending on the circumstances. For example, 15 O, 13 N, 11 C, and 18Positron-emitting isotopes such as fluorine (F) are useful in positron emission tomography (PET) studies. PET imaging in cancer finds usefulness in determining tumor location and identification, disease stage, and appropriate treatment. Human cancer cells overexpress many receptors or proteins that are potential disease-specific molecular targets. Radiolabeled tracers that bind with high affinity and specificity to such receptors or proteins on tumor cells have great potential for diagnostic imaging and targeted radionuclide therapy (Charron, Carlie L. et al. Tetrahedron Lett. 2016, 57(37), 4119-4127). Furthermore, target-specific PET radiotactic tracers can be used as biomarkers to investigate and evaluate pathology, for example, by measuring target expression and treatment response (Austin R. et al. Cancer Letters (2016), doi:10.1016 / j.canlet.2016.05.008).

[0263] As used herein, the term “hematopoietic disorder” refers to any disorder related to the production of cellular components of blood and plasma, including, but is not limited to, blood cancer.

[0264] According to one embodiment, the present invention provides the combination described herein.

[0265] According to one embodiment, the present invention provides the combination described herein for use as a pharmaceutical product.

[0266] According to one embodiment, the present invention provides the combination described herein for the manufacture of a pharmaceutical product.

[0267] According to one embodiment, the present invention provides a combination described herein for manufacturing a pharmaceutical product for treating or preventing any one of the disease conditions referred to herein.

[0268] According to one embodiment, the present invention provides a combination described herein for use in the prevention or treatment of the diseases described herein, particularly in the treatment of such diseases.

[0269] According to one embodiment, the present invention provides combinations described herein for use in the prevention or treatment, particularly the treatment, of hematopoietic disorders, such as, but not limited to, blood cancers, such as, but not limited to, lymphoma, myeloma, and leukemia.

[0270] According to one embodiment, the present invention provides the combination described herein for use in the prevention or treatment of hematopoietic disorders, particularly for treatment.

[0271] According to one embodiment, the hematopoietic disorder is selected from, but is not limited to, lymphoma, myeloma, myelodysplasia, and leukemia.

[0272] According to one embodiment, the hematopoietic disorder is myelodysplasia, including but not limited to myelodysplastic syndrome (MDS).

[0273] According to one embodiment, the hematopoietic disorder is leukemia.

[0274] According to one embodiment, the hematopoietic disorder is a leukemia selected from acute leukemia and chronic leukemia. According to one embodiment, the leukemia is acute leukemia. According to one embodiment, the leukemia is chronic leukemia.

[0275] According to one embodiment, the hematopoietic disorder is myeloid leukemia, lymphoblastic leukemia, or lymphocytic leukemia. According to one embodiment, the hematopoietic disorder is acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic leukemia (SLL), acute myeloid leukemia (AML), chronic idiopathic myelofibrosis (MF), chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), chronic neutrophilic leukemia (CNL), Hairy cell leukemia (CNL), leukemia, HCL), T-cell large granular lymphocyte leukemia Leukemia is a leukemia selected from, but not limited to, leukemia (T-LGL) and rapidly progressive NK cell leukemia. In one embodiment, AML is acute megakaryoblastic leukemia (AMKL).

[0276] According to one embodiment, leukemia is MDS, CLL, SLL, ALL, or AML. According to one embodiment, leukemia is CLL, SLL, or AML. According to one embodiment, leukemia is CLL or SLL. In some embodiments, CLL or SLL is CD20-expressing cancer. According to one embodiment, leukemia is ALL or AML. According to one embodiment, leukemia is ALL. According to one embodiment, leukemia is AML. According to one embodiment, hematopoietic disorder is Waldenström macroglobulinemia.

[0277] According to one embodiment, the hematopoietic disorder is MLL rearrangement leukemia, MLL partial tandem duplication (PTD) leukemia, MLL amplification leukemia, MLL-positive leukemia, or leukemia showing an elevated HOX / MEIS1 gene expression signature.

[0278] According to one embodiment, the leukemia is MLL rearranged leukemia and / or nucleophosmin 1 (NPM1) variant leukemia.

[0279] According to one embodiment, the hematopoietic disorder is MLL rearrangement leukemia.

[0280] According to one embodiment, the hematopoietic disorder is nucleophosmin 1 (NPM1) variant leukemia (e.g., NPM1c).

[0281] According to one embodiment, the hematopoietic disorder is myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN).

[0282] According to one embodiment, the hematopoietic disorder is acute lymphoblastic leukemia (ALL).

[0283] According to one embodiment, the hematopoietic disorder is acute myeloid leukemia (AML).

[0284] According to one embodiment, the hematopoietic disorder is small lymphocytic lymphoma (SLL) or chronic lymphocytic leukemia (CLL).

[0285] According to one embodiment, the hematopoietic disorder is SLL or CLL, where SLL or CLL is a CD20-expressing cancer.

[0286] According to one embodiment, the hematopoietic disorder is myelodysplastic syndrome (MDS).

[0287] According to one embodiment, the hematopoietic disorder is myeloproliferative neoplasm (MPN).

[0288] According to one embodiment, the hematopoietic disorder is NPM1 variant leukemia with an FLT3 mutation.

[0289] According to one embodiment, the hematopoietic disorder is FLT3-dependent leukemia.

[0290] According to one embodiment, the hematopoietic disorder is MEF2G-dependent leukemia.

[0291] According to one embodiment, the hematopoietic disorder has one or more MLL1(KMT2A) gene rearrangements or genetic changes (e.g., replication or amplification) and / or NPM1 mutations.

[0292] According to one embodiment, the hematopoietic disorder has (i) one or more MLL1(KMT2A) gene rearrangements or genetic alterations (e.g., replication or amplification) and / or NPM1 mutations, in addition to (ii) an FLT3 mutation.

[0293] According to one embodiment, the hematopoietic disorder is MLL rearrangement leukemia.

[0294] According to one embodiment, the hematopoietic disorder is acute myeloid leukemia (AML).

[0295] According to one embodiment, the hematopoietic disorder is small lymphocytic lymphoma (SLL).

[0296] According to one embodiment, the hematopoietic disorder is chronic lymphocytic leukemia (CLL).

[0297] According to one embodiment, hematopoietic disorders include acute leukemia, chronic 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 prelymphocytic 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 HOX / MEIS1 gene expression signature.

[0298] According to one embodiment, hematopoietic disorders are AML, specifically nucleophosmin (NPM1) mutant AML (i.e., NPM1 mut AML), more specifically, abstract NPM1 variant AML.

[0299] According to one embodiment, the hematopoietic disorder is MLL rearrangement leukemia, particularly MLL rearrangement AML or ALL.

[0300] According to one embodiment, the hematopoietic disorder includes MLL gene alterations, and in particular, the hematopoietic disorder is AML or ALL accompanied by MLL gene alterations. In certain embodiments, the MLL gene alteration is duplication. In certain embodiments, the MLL gene alteration is amplification.

[0301] According to one embodiment, the hematopoietic disorder includes NPM1 gene mutations and / or MLL1 (also known as KMT2A) gene mutations.

[0302] According to one embodiment, an MLL1 gene mutation includes, but is not limited to, MLL1 gene rearrangement, replication, or amplification.

[0303] According to one embodiment, the hematopoietic disorder is a mixed lineage leukemia (MLL), MLL-associated leukemia, MLL-positive leukemia, MLL-induced leukemia, MLL-associated leukemia, acute leukemia, chronic leukemia, myelodysplastic syndrome (MDS), or myeloproliferative neoplasm (MPN).

[0304] All embodiments described herein regarding methods for treating disorders are also applicable to use in the treatment of said disorders.

[0305] All embodiments described herein regarding use in the treatment of disorders are also applicable to methods for treating said disorders.

[0306] All embodiments described herein regarding use in the treatment of disorders are also applicable to methods for treating said disorders.

[0307] All embodiments described herein regarding use in the treatment of disorders are also applicable to use in the treatment of said disorders.

[0308] All embodiments described herein regarding methods for treating disorders are also applicable to use in methods for treating said disorders.

[0309] All embodiments described herein for use in methods for treating disorders are also applicable to methods for treating such disorders.

[0310] The term “therapeutic dose” as used herein means the amount of an active compound or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue system, animal, or human, including relief or reversal of symptoms of the disease or disorder being treated, as determined by researchers, veterinarians, physicians, or other clinicians.

[0311] All abbreviations for menin-MLL inhibitors used in the general scheme and examples are as defined in Table 1. Variable elements are as defined in the range or as specifically defined in the general scheme. [Examples]

[0312] [Table 1-1]

[0313] [Table 1-2]

[0314] [Table 1-3]

[0315] As will be understood by those skilled in the art, compounds synthesized using the indicated protocols may exist as solvates, e.g., hydrates, and / or may contain residual solvent or trace impurities. Compounds or intermediates isolated in salt form may be integer stoichiometric, i.e., monostoichiometric, distoichiometric, or intermediate stoichiometric. Where intermediates or compounds in the following experimental sections are designated as "HCl salts" without specifying the equivalent number of HCl, this means that the equivalent number of HCl could not be determined. The same principle applies, for example, to "oxalates," "formates," "besylates," or

[0316] [ka] This also applies to all other salt forms mentioned in the experimental section.

[0317] Those skilled in the art will understand, even if not explicitly mentioned in the following experimental protocol, that typically, after column chromatography purification, the desired fraction is collected and the solvent is evaporated.

[0318] If the stereochemistry is not shown, this means that it is a mixture of stereoisomers, unless otherwise specified or evident from the context.

[0319] When a stereocenter is indicated by "RS," this means that a racemic mixture was obtained at the indicated center, unless otherwise specified.

[0320] Example 1 Synthesis of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A) - Preparation method A Preparation of the intermediate 1-tert-butyl(5-methyl-4-oxohexyl)carbamate

[0321] [ka]

[0322] To a solution of tert-butyl 2-oxopyrrolidine-1-carboxylate (5.0 g, 27 mmol) and TMEDA (5.0 mL, 33 mmol) dissolved in THF (60 mL) cooled to -70°C, isopropyl magnesium bromide solution (19 mL, 55 mmol, 2.9 M in 2-methyltetrahydrofuran) was slowly added. The resulting mixture was slowly warmed to room temperature and stirred for 12 hours. The mixture was poured into saturated NH4Cl aqueous solution (50 mL) and extracted with siRNA (50 mL x 3). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product, which was further purified by FCC (PE / siRNA = 1:0 to 100:1) to obtain the title intermediate (3.7 g, yield 60%) as a yellow oil.

[0323] Preparation of the intermediate 13-tert-butyl6-(3,6-dichloro-1,2,4triazine-5-yl)-2,6-diazaspiro[3,4]octane-2-carboxylate

[0324] [ka]

[0325] To a solution prepared by dissolving 3,5,6-trichloro-1,2,4-triazine (10.0 g, 54.2 mmol) and TEA (15.2 mL, 109 mmol) in 100 mL of DCM cooled to 0°C, tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate (9.21 g, 43.4 mmol) was added, and the mixture was warmed to room temperature and stirred for 1 hour. The mixture was diluted with water (20 mL) and extracted with 30 mL x 3 of DCM. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by silica gel FCC (PE / siRNA = 1:0~3:1) to obtain the title intermediate (12.0 g, yield 58%) as a yellow solid.

[0326] Preparation of the intermediate 27-N-ethyl-5-fluoro-N-isopropyl-2-methoxybenzamide

[0327] [ka]

[0328] To a mixture of 5-fluoro-2-methoxybenzoic acid (8.00 g, 47.0 mmol) and N-ethylpropan-2-amine (8.19 g, 94.0 mmol) in 150 mL of dry DCM cooled to 0°C, HATU (21.5 g, 56.5 mmol) and DIEA (9.10 g, 70.4 mmol) were slowly added in small portions. The resulting mixture was slowly warmed to room temperature and stirred for 8 hours. The organic layer was washed with water (20 mL x 3) and dried over anhydrous Na₂SO₄. After filtration, the solvent was removed under reduced pressure, and the crude product was purified by FCC (siRNA / PE = 0%~20%) to obtain the title intermediate (12.0 g, yield 96%) as a white solid.

[0329] Preparation of the intermediate 28-N-ethyl-5-fluoro-2-hydroxy-N-isopropylbenzamide

[0330] [ka]

[0331] In a solution of N-ethyl-5-fluoro-N-isopropyl-2-methoxybenzamide (intermediate 27) (12.0 g, 50.1 mmol) dissolved in dry DCM (100 mL) cooled to -78°C, BBr3 (14.4 mL, 152 mmol) was slowly added, and the resulting mixture was slowly warmed to room temperature and stirred for 8 hours. The mixture was cooled again to -78°C, and the reaction was quenched by dropwise addition of MeOH (5 mL). The resulting mixture was slowly warmed to room temperature, and the pH was adjusted to approximately 8 by adding saturated NaHCO3 aqueous solution. The aqueous layer was extracted with DCM (50 mL x 3), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by FCC (siRNA / PE = 0%~20%) to obtain the title intermediate (9.0 g, yield 78%) as a white solid.

[0332] Alternative preparation of intermediate 28

[0333] [ka]

[0334] A mixture of THF (168 L, 12 volumes) and 5-fluoro-2-hydroxybenzoic acid (14.0 kg, 89.68 mol, 1.0 equivalent) was adjusted to 15-25°C, and 1,1-carbonyldiimidazole (17.45 kg, 107.62 mol, 1.2 equivalents) was added over 1 hour. After the addition, the mixture was stirred at 15-25°C for 18 hours. After this time, N-ethylpropan-2-amine (14.85 kg, 170.39 mol, 1.9 equivalents) was added to the mixture at 15-25°C over 2 hours. The resulting mixture was further aged at 15-25°C for 18-24 hours. The pH was adjusted to pH 4-5 using a 10% H2SO4 aqueous solution (140 kg, 10 volumes), and the layers were separated. The organic phase was concentrated to 42-56 L while maintaining the temperature below 40°C, and then n-heptane (43 kg, 4.5 vol) was added to the mixture over 3 hours at 15-25°C. The mixture was then cooled to 0-10°C and stirred for a further 6 hours. The resulting slurry was filtered, and the cake was washed with a tert-butyl methyl ether (MTBE):n-heptane mixture (25 kg of MTBE:n-heptane in a 2:3 vol / vol mixture, 2.5 vol). The cake washing was repeated two more times, and the resulting solid was dried under vacuum at 50°C to obtain intermediate 28 (16.5 kg, purity: 99.1%, yield: 80.4%).

[0335] Preparation of the intermediate 14-tert-butyl6-(3-chloro-6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate

[0336] [ka]

[0337] A mixture of tert-butyl 6-(3,6-dichloro-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (intermediate 13) (12.0 g, 33.3 mmol), N-ethyl-5-fluoro-2-hydroxy-N-isopropylbenzamide (intermediate 28) (7.5 g, 33.3 mmol), and DBU (6.1 g, 40.1 mmol) was stirred at 25°C for 8 hours. The mixture was diluted with water (30 mL) and extracted with DCM (30 mL x 3). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by FCC (PE / siRNA = 1:0~3:1) to obtain the title intermediate (14.0 g, yield 73%) as a green solid.

[0338] Preparation of the intermediate 2-tert-butyl 6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate Method A for the synthesis of intermediate 2

[0339] [ka]

[0340] To a mixture of THF (500 mL), 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) was added. Pd(dppf)Cl2×DCM (1.70 g, 2.08 mmol) was added under an N2 atmosphere. After the addition, the reaction mixture was stirred at 25°C for 14 hours. The reaction mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel FCC(siRNA) to obtain the title intermediate (15 g, purity 93%, yield 74%) as a brown solid.

[0341] Method B for synthesizing intermediate 2

[0342] [ka]

[0343] To a solution prepared by dissolving 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), Pd / C (wet, 5.0 g, 10%) was added. The resulting mixture was stirred under an H2 atmosphere (30 psi) at 25°C for 8 hours. The reaction mixture was filtered through a celite pad, and the filtrate was concentrated under vacuum to obtain the title intermediate (25.0 g, crude), which was used directly in the next step without further purification.

[0344] Preparation of the intermediate 3-2-((5-(2,6-diazaspiro[3,4]octan-6-yl)-1,2,4triazine-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0345] [ka]

[0346] To a solution prepared by dissolving tert-butyl 6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (intermediate 2) (300 mg, 0.583 mmol) in DCM (5 mL), TFA (0.5 mL, 6.4 mmol) was added, and the resulting mixture was stirred at room temperature for 3 hours. Then, 10% NaOH (5 mL) solution was slowly added to the mixture to adjust the pH to approximately 12, and the resulting mixture was extracted with DCM (10 mL x 3). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain the title intermediate (220 mg, yield 90%) as a white solid.

[0347] Preparation of compound 61-tert-butyl(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazin-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)carbamate

[0348] [ka]

[0349] A mixture of 2-((5-(2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide (intermediate 3) (1.0 g, 2.4 mmol), tert-butyl(5-methyl-4-oxohexyl)carbamate (intermediate 1) (830 mg, 3.62 mmol), and ZnCl2 (660 mg, 4.84 mmol) was stirred at 80°C for 0.5 hours. Then, NaBH3CN (310 mg, 4.93 mmol) was added, and the resulting mixture was stirred at 80°C for 6 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure to obtain the crude product, which was further purified by preparative HPLC using a Waters Xbridge Prep OBD (column: C18 150×40mm 10um, eluent: ACN / H2O (0.05% ammonia) 45%~75% v / v) to obtain the title compound (700 mg, yield 46%) as a colorless oil.

[0350] Preparation of Compounds 62 and 63-tert-butyl(R)-(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazin-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)carbamate and tert-butyl(S)-(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazin-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)carbamate

[0351] [ka]

[0352] tert-butyl(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazine-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)carbamate (compound 61) (200 mg, 0.319 mmol) was purified by SFC using DAIEL CHIRALPAK IG (column: 250 × 30 mm 10 μm, fixed composition elution: EtOH (containing 0.1% 25% ammonia): supercritical CO2, 40%:60% (v / v)) to obtain the title compounds (compound 62) (85 mg, yield 42%) and (compound 63) (80 mg, yield 40%), both as pale yellow oily substances.

[0353] Compound 64-(R)-2-((5-(2-(6-amino-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0354] [ka]

[0355] To a solution of tert-butyl(R)-(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazine-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)carbamate (compound 62) (550 mg, 0.876 mmol) dissolved in DCM (4 mL), TFA (4 mL) was slowly added, 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 with DCM (40 mL), and the pH was adjusted to approximately 12 using NaOH aqueous solution (2 M, 16 mL). The aqueous layer was extracted with DCM (10 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain the title compound (460 mg, crude) as a yellow solid, which was used directly in the next step without further purification.

[0356] Compound 11-(R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide

[0357] [ka]

[0358] A mixture of DMF (1 mL), (R)-2-((5-(2-(6-amino-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)-N-ethyl-5-fluoro-N-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) was stirred at 80°C for 1 hour by microwave irradiation. After cooling to room temperature, the mixture was diluted with H2O (10 mL) and extracted with ELISA (3 × 10 mL). The combined organic layers were washed with H2O (10 mL), dehydrated with Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. This was further purified by HPLC using a Phenomenex Gemini-NX column (150 × 30 mm 5 μm, eluent: ACN / H2O (10 mM NH4HCO3) 51%~71% (v / v)), and then purified by SFC using a DAISEL CHIRALCEL OD-H column (250 × 30 mm 5 μm, eluent: supercritical CO2 in EtOH (0.1% v / v ammonia) 25 / 25, v / v) to obtain the title compound (5.13 mg, purity 96%) as a yellow solid.

[0359] LC-MS(ESI)(Method 1):R t = 2.997 minutes, measured value in m / z: 586.3 [M+H] + .

[0360] Compound A-(R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide

[0361] [ka]

[0362] (RN-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (compound 11) (40.0 mg, 0.068 mmol), formaldehyde (55.4 mg, 0.683 mol, 37% in water) and AcOH (8.2 mg, 0. The mixture containing (137 mmol) was 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 for another 1 hour at 45°C. After cooling to room temperature, the reaction mixture was diluted with saturated NaHCO3 aqueous solution (40 mL) to adjust the pH to approximately 8, and then extracted with DCM (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product, which was then used in Boston The compound was purified by preparative HPLC using Prime (column: C18 150×30mm 5um, mobile phase A: H2O (0.04% ammonia + 10mM NH4HCO3), mobile phase B: ACN, flow rate: 25 mL / min, gradient conditions B / A: 50%~80% (50%B~80%B)) to obtain the title compound (9.62 mg, purity 99.10%, yield 23.3%) as a yellow oily substance.

[0363] Example 2 Synthesis of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A) - Preparation method B Preparation of the intermediate 7-4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid

[0364] [ka]

[0365] To a solution prepared by dissolving 4-(methylamino)butanoate (3.0 g, 19.5 mmol) and TEA (7.78 mL, 58.6 mmol) in MeOH (30 mL), Boc2O (4.69 g, 21.5 mmol) was added dropwise. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure, the residue was diluted with SiO (100 mL), washed with cooled 0.1 N HCl (70 mL x 2), H2O (50 mL x 2), and brine (50 mL), dried over Na2SO4, filtered, and concentrated to obtain the title intermediate (1.80 g, crude) as a colorless oil.

[0366] Preparation of the intermediate 8-tert-butyl(4-(methoxy(methyl)amino)-4-oxobutyl)(methyl)carbamate

[0367] [ka]

[0368] To a solution prepared by dissolving 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (intermediate 7) (1.80 g, crude) 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) were added. 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 x 3), saturated NaHCO3 aqueous solution (30 mL x 3), and brine (30 mL), dried over Na2SO4, filtered, and concentrated under vacuum to obtain the title intermediate (1.70 g, crude) as a colorless oil.

[0369] Preparation of the intermediate 9-tert-butylmethyl(5-methyl-4-oxohexyl)carbamate

[0370] [ka]

[0371] In a solution prepared by dissolving tert-butyl(4-(methoxy(methyl)amino)-4-oxobutyl)(methyl)carbamate (intermediate 8) (200 mg, crude) in THF (5 mL) cooled to -70°C under an N2 atmosphere, isopropyl lithium (3.2 mL in pentane, 2.24 mmol, 0.7 M) was added dropwise. The resulting mixture was stirred at -70°C for 2 hours. The mixture was quenched with saturated NH4Cl aqueous solution (15 mL) and extracted with SiO (30 mL x 2). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was further purified by FCC (PE / SiO = 10:1) to obtain the title intermediate (60 mg) as a colorless oil.

[0372] Preparation of compound 60-tert-butyl(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazine-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)(methyl)carbamate

[0373] [ka]

[0374] ZnCl2 (789 mg, 5.79 mmol) was added to a solution prepared by dissolving 2-((5-(2,6-diazaspiro[3,4]octan-6-yl)-1,2,4triazine-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide (intermediate 3) (600 mg, 1.45 mmol) and tert-butylmethyl(5-methyl-4-oxohexyl)carbamate (intermediate 9) (330 mg, 1.37 mmol) in MeOH (50 mL). 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 overnight at 80°C. After cooling to room temperature, the mixture was concentrated under reduced pressure to obtain a crude residue, which was diluted with DCM (50 mL), quenched with saturated NH4Cl aqueous solution (50 mL), and extracted with DCM (50 mL x 3). The combined organic layers were washed with saline solution (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was further purified by FCC (DCM / MeOH = 10:1) to obtain the title compound (400 mg, yield 42%) as a white solid.

[0375] Compound 67-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(2-methyl-6-(methylamino)hexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide hydrochloride

[0376] [ka]

[0377] To a solution prepared by dissolving tert-butyl(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4triazine-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)(methyl)carbamate (compound 60) (1 g, 1.56 mmol) in DCM (10 mL), 4 M HCl (5 mL, 20 mmol) in dioxane was added, and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum to obtain the title compound (960 mg, crude, HCl salt), which was used directly in the next step without further purification.

[0378] Compound A-(R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide

[0379] [ka]

[0380] To a mixture of DMF (5 mL), N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(2-methyl-6-(methylamino)hexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide hydrochloride (compound 67) (480 mg, crude), K2CO3 (700 mg, 5.07 mmol), and NaI (400 mg, 2.67 mmol) was added, to which 1-bromo-2-methoxyethane (230 mg, 1.65 mmol) was added. 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 x 3). The combined organic layer was washed with brine (30 mL x 3), dried over Na2SO4, filtered, and concentrated to obtain the crude residue. The residue was purified by FCC (DCM / MeOH = 10:1) to obtain N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound 68) (250 mg, yield 48%) as a yellow oil.

[0381] N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound 68) (960 mg in total from several batches obtained by Method B) was first purified by SFC using DAIEL CHIRALPAK IG (column: 250 × 30 mm 10 μm, mobile phase: A: supercritical CO2, B: EtOH (0.1% ammonia), B=40:60, 60 mL / min), and then purified by 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 The title compound (270 mg) was purified by preparative HPLC using 55% to 85% of the solution to obtain a colorless oil.

[0382] 1 ¹H 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 were hidden by solvent peaks and were not recorded.

[0383] LCMS(ESI)(Method 2):R t = 1.965 minutes, measured value in m / z: 600.3 [M+H] + .

[0384] SFC (Method 11):R t =4.904 minutes.

[0385] Example 3 Synthesis of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A) - Preparation method C Preparation of the intermediate 227-tert-butyl(R)-(1-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-methylbutan-2-yl)carbamate

[0386] [ka]

[0387] Boc-L-valine (44.9 kg), 2,2-dimethyl-1,3-dioxane-4,6-dione (32.9 kg), and DMAP (35.5 kg) in DCM (607 kg) pre-cooled to -10 to 0°C were added over 3 hours to a solution of DCC (55.5 kg) dissolved in DCM (613 kg), and aged at -10 to 0°C for 16 hours. While maintaining the temperature below 10°C, 10% citric acid aqueous solution (449 kg) was added. The resulting slurry was aged at 0 to 10°C for 2 hours and then filtered. The filtered cake was washed with DCM (91 kg). The filtrate was separated, and the organic layer was washed with 10% citric acid aqueous solution (twice at 450 kg) and 10% NaCl aqueous solution (449 kg). Acetic acid (75.0 kg) was added to the organic phase (1200 kg) while maintaining the temperature at -10 to 0°C. Sodium borohydride (18.0 kg) was added in small amounts over 5 hours while maintaining the temperature in the range of -10 to 0°C, and then the resulting mixture was aged for a further 16 hours at -10 to 0°C. The mixture was heated to 15 to 25°C and aged for 2 hours. The mixture was then washed with 14% NaCl aqueous solution (450 kg), followed by a second wash with 14% NaCl aqueous solution (432 kg), and finally washed with water (444 kg). The organic phase was concentrated to 2 to 4 volumes under reduced pressure. Iso-propanol (143 kg) was added to the residue and concentrated to 4 to 5 volumes under reduced pressure. After cooling to -10 to 0°C and aging for 8 hours, the resulting slurry was filtered, washed with IPA (38 kg), and dried to obtain the title intermediate (46.7 kg, yield 69%) as a white solid.

[0388] Preparation of the intermediate 228-tert-butyl(R)-2-isopropyl-5-oxopyrrolidine-1-carboxylate

[0389] [ka]

[0390] 46.7 kg of tert-butyl(R)-(1-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-methylbutan-2-yl)carbamate (intermediate 227) in toluene (333 kg) was heated under reflux and aged for 4 hours. The mixture was cooled to ambient temperature, filtered, and washed with toluene (20 kg). The combined filtrate was concentrated to dryness under reduced pressure to obtain the desired compound (31.05 kg, yield 96%) as an oil, which was used directly without further purification.

[0391] Preparation of the intermediate 229-tert-butyl(5R)-2-hydroxy-5-isopropylpyrrolidine-1-carboxylate

[0392] [ka]

[0393] 30.9 kg of tert-butyl(R)-2-isopropyl-5-oxopyrrolidine-1-carboxylate (intermediate 228) in 2-MeTHF (26.7 kg) was cooled to -5 to 5°C. A 2-MeTHF solution of LiBH4 (1 M, 45.2 kg, 54.4 mol) was added over 3 hours, and the mixture was aged for 4 hours. A cold aqueous solution of 5% NaHCO3 (163 kg) was added over 3 hours at -5 to 5°C, and the mixture was aged for a further 2 hours. The mixture was heated to ambient temperature and aged for a further 2 hours. The aqueous layer was separated, and the organic layer was washed with 10% aqueous NaCl solution (170 kg) and water (155 kg). During the water washing, an emulsion formed, and solid NaCl (3.1 kg) was added to separate it. After removing the aqueous layer, the organic layer was concentrated to dryness under reduced pressure to obtain the desired compound (28.5 kg, 91% yield) as an oily substance, which was used directly without further purification.

[0394] Preparation of the intermediate 230-tert-butyl(R)-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)carbamate

[0395] [ka]

[0396] 28.55 kg of tert-butyl(5R)-2-hydroxy-5-isopropylpyrrolidine-1-carboxylate (intermediate 229) in DCM (344 kg) was treated with 2-methoxy-N-methylethane-1-amine (12.3 kg, 138.0 mol) at 15-25°C, and the resulting mixture was aged for 1 hour. 40.12 kg of sodium triacetoxyborohydride was added in fractions over 5 hours while maintaining the temperature between 15-25°C, and the resulting mixture was aged for 48 hours. The reaction mixture was quenched by adding 184 kg of 8% NaOH aqueous solution over 2 hours while maintaining the temperature between 15-25°C, and the mixture was aged for a further 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 obtain the title intermediate (33.26 kg, yield 88%) as an oil, which was used directly without further purification.

[0397] Intermediate 231-(R)-N 1 -(2-methoxyethyl)-N 1 Preparation of ,5-dimethylhexane-1,4-diamine dihydrochloride

[0398] [ka]

[0399] To a 4 molar solution of isopropanol (84.80 kg) dissolved in HCl, a solution of isopropanol (25.6 kg) in which tert-butyl(R)-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)carbamate (intermediate 230) (32.38 kg) was dissolved, and the mixture was added over 3 hours at ambient temperature. The mixture was then aged at ambient temperature for a further 19 hours. Next, 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 filtered cake was dried to obtain the title compound (23.92 kg, yield 81%) as a white solid.

[0400] Preparation of the intermediate 232-ethyl-1-benzyl-3-(chloromethyl)pyrrolidine-3-carboxylate

[0401] [ka]

[0402] A solution of DIPEA (952 g, 1.1 equivalent) was dissolved in THF (6 L) cooled to -35 to -25°C. n-BuLi (2.33 kg, 2.5 M in hexane, 1.0 equivalent) was added to this solution while maintaining the temperature below -25°C. The resulting mixture was aged at -35 to -25°C for a further 30 minutes, then cooled to -78 to -60°C. A solution of ethyl 1-benzylpyrrolidine-3-carboxylate (2 kg, 1.0 equivalent) dissolved in THF (2 L) was added at -78 to -60°C, and the mixture was stirred for a further 30 minutes. Chloroiodomethane (1.81 kg, 1.2 equivalents) was then added at -78 to -60°C. The reaction mixture was aged at -60 to -40°C for 2 hours. The reaction mixture was added to an aqueous citric acid solution (660g in 6L H2O) at a temperature of 0-10°C, and the resulting mixture was aged at 20-30°C for a further 20 minutes. After separating the layers, the aqueous layer was extracted with toluene (6L), the combined organic layers were washed with brine (6L), and then heated to 50-60°C. Oxalic acid (2.22kg) was added at 50-60°C. The resulting mixture was stirred at 50-60°C for 3 hours, then cooled to 20-30°C and aged overnight. The resulting solid was filtered, and the cake was washed with ethyl acetate (2L). The wet cake was added to toluene (4L), H2O (8L), and K3PO4 (1.5 equivalents), and the resulting mixture was aged at 20-30°C for 20 minutes. After separating the layers, the aqueous layer was extracted with toluene (2L). The organic layers were combined and washed twice with water (2L). The organic phase was concentrated under reduced pressure to obtain 4.2 kg of the desired compound as a toluene solution (46% by weight by assay, assay yield 80%).

[0403] Preparation of the intermediate 233-1-benzyl-3-(chloromethyl)pyrrolidine-3-carbaldehyde

[0404] [ka]

[0405] The reaction was carried out in a fluid chemical system. Specifically, a solution of ethyl 1-benzyl-3-(chloromethyl)pyrrolidine-3-carboxylate (intermediate 232) (4.4 kg) dissolved in toluene (26 L) was pumped at 26.7 mL / min and cooled to -60°C. After cooling, this was then mixed with a cooled solution of DIBAL-H (28.1 mol) dissolved in toluene at -60°C (28 L) at a pump flow rate of 32.1 mL / min. The mixture was passed through a perfluoroalkoxy (PFA) coil tube reactor at -60°C (total flow rate 58.8 mL / min, residence time 5 seconds). The resulting mixture was mixed with cooled MeOH (-60°C) and pumped at a rate of 15.2 mL / min. This mixed solution was pumped at -60°C into another PFA coil tube reactor (total flow rate 74 mL / min, residence time 5 seconds). The resulting mixture was collected in a receiver containing a 20% by weight Rochelle salt aqueous solution (20V). 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 the same manner and concentrated under reduced pressure to obtain 20.8 kg of toluene solution of the desired compound (25.5% by weight, assay yield 85% obtained by HPLC assay), which was used directly without further purification.

[0406] 1 H 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).

[0407] Preparation of the intermediate 234-(R)-4-(6-benzyl-2,6-diazaspiro[3.4]octan-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexane-1-amine

[0408] [ka]

[0409] Toluene (30L) and (R)-N 1 -(2-methoxyethyl)-N 1 Triethylamine (2.55 kg, 25.2 mol) was added at 20-30°C to a solution of 1-benzyl-3-(chloromethyl)pyrrolidine-3-carbaldehyde (intermediate 233) dissolved in toluene (3.0 kg, 10 wt%) diluted with 5-dimethylhexane-1,4-diamine, dihydrochloride (intermediate 231) (3.47 kg). The resulting mixture was aged at 20-30°C for 2 hours. Then, sodium triacetoxyborohydride (9.0 kg) was added at 20-30°C, and the mixture was aged for 12 hours. The reaction mixture was cooled to 5-15°C, and a 25 wt% NaOH aqueous solution (25 L, approximately 16.75 equivalents) was added while maintaining the temperature below 35°C. The resulting mixture was aged at 20-30°C for 25 minutes, and the layers were separated. The organic layer was washed with 10 L of 15 wt% NaCl aqueous solution, the layers were separated again, and 18 L of water was added to the organic phase. The pH of the aqueous phase was adjusted to 6-7 using 4 M HCl aqueous solution while maintaining the internal temperature below 35°C. The organic phase was then discarded, the aqueous phase was separated, and the pH was basicized to 8-9 with K2HPO4.

[0410] The resulting mixture was heated to 50-55°C and aged for 3 hours. The reaction mixture was then cooled to ambient temperature and combined with the other two batches (2.4 kg + 3.0 kg). The combined flow was washed three times with methyl tert-butyl ether (3 × 40 L). Further methyl tert-butyl ether (83 L) was added to the resulting aqueous layer, and the aqueous phase was basicized to pH 9-10 with an 8 wt% NaOH aqueous solution while maintaining a temperature of 15-35°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 to approximately 3 volumes under reduced pressure, then flushed three times with methanol (3 × 30 L), and concentrated to dryness to obtain the desired intermediate (12.4 kg, isolation yield 90%) as a pale yellow oily substance, which was used directly without further purification.

[0411] Preparation of intermediate 234a (citrate of intermediate 234)

[0412] [ka]

[0413] EtOH (80 mL) and intermediate 234 (20 g) were added to a round-bottom flask. Next, a 0.5 M solution (100 mL, 1 equivalent) of citric acid dissolved in EtOH was added to the mixture in the round-bottom flask at room temperature. The mixture was then evaporated until dry (rotary evaporator, 40°C). Acetonitrile (200 mL) was added to the residue and the mixture was evaporated until dry (rotary evaporator, 40°C). Acetonitrile (100 mL) was added to the residue and stirred overnight at room temperature using a magnetic heating plate. Finally, intermediate 234a was filtered off and dried at room temperature.

[0414] Preparation of the crystalline form of the citrate of intermediate 234 (intermediate 234b)

[0415] [ka]

[0416] 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 (approximately 10 minutes) until homogeneous. The mixture was then cooled to 50°C at a rate of 0.5°C / min. Next, seed was added (19 mg of intermediate 234a, 0.5 w / w%), and the mixture was aged for 3 hours and 30 minutes with stirring. The mixture was then cooled nonlinearly to 20°C at an exponent of 2,3 over 8 hours. The resulting mixture was stirred overnight, the product was filtered off, and the mixture was dried (overnight at room temperature in a hood). After isolation, intermediate 234b (2.75 g; yield 73.9%) was obtained as the crystalline form of the citrate of intermediate 234. The ratio of the obtained intermediate / citric acid was 3 / 2 (NMR).

[0417] The above nonlinear cooling was performed according to the following equation.

[0418] During the specified cooling period, a new linear ramp is started every 30 seconds. The ramp is calculated according to the following formula:

[0419]

number

[0420] 1 H NMR(400MHz,MeOH-d4)δ ppm 0.91(3H,d,J=6.88Hz)0.98(3H,d,J=6.88Hz)1.46-1.57(2H,m)1.67-1.87(2H,m)1.94- 2.03(1H,m)2.20-2.29(2H,m)2.62-2.69(2H,m)2.72-2.77(4H,m)2.77-2.82(2H,m)2.9 0(2H,t,J=7.32Hz)2.95-3.02(2H,m)3.07-3.16(2H,m)3.16-3.22(2H,m)3.37(3H,s)3. 68-3.72(2H,m)3.83-3.89(2H,m)3.90-3.92(2H,m)3.94-4.06(2H,m)7.32-7.43(5H,m).

[0421] Preparation of the intermediate 224-(R)-N-(2-methoxyethyl)-N,5-dimethyl-4-(2,6-diazaspiro[3.4]octan-2-yl)hexane-1-amine

[0422] [ka]

[0423] To palladium carbon hydroxide (1.2 kg) in EtOH (1.47 kg) cooled to -5~5°C, methanesulfonic acid (MSA) (11 kg), (R)-4-(6-benzyl-2,6-diazaspiro[3.4]octan-2-yl-N-(2-methoxyethyl)-N,5-dimethylhexane-1-amine (intermediate 234) (10 kg), and EtOH (250 L) were added. The mixture was heated to 35-45°C and stirred for 16-20 hours under a hydrogen atmosphere (0.27-0.40 MPa). The mixture was filtered through diatomaceous earth (20 kg) and the pad was washed with EtOH (24 L). The filtrate was concentrated to 2-3 volumes under reduced pressure (below 40°C), and then flushed twice with 2-MeTHF (73 kg and 47 kg) to obtain 2-3 volumes of solution. After dilution, 30 kg of 10% sodium sulfate aqueous solution was added, the mixture was cooled to 0-10°C, and then 50 kg of 16% NaOH aqueous solution was added to adjust the pH to 13-14. The temperature was adjusted to 15-25°C and the mixture was stirred for 30-60 minutes. The aqueous layer was separated and extracted twice with 2-MeTHF (47 kg x 2). The combined organic layers were concentrated to 3-4 volumes under reduced pressure (below 40°C), and 950 g of 2-MeTHF was added. After concentrating to 3-4 volumes under reduced pressure (below 40°C), the resulting solution was diluted with 30 kg of 2-MeTHF, dried through a 25 kg 4A molecular sieve, and washed with 30 kg of 2-MeTHF. The final solution was concentrated to obtain the desired compound (6.7 kg) as an oily substance with an assay purity of 90.1% with a 79% corrected yield.

[0424] Preparation of the intermediate 225-(R)-4-(6-(3,6-dichloro-1,2,4triazin-5-yl)-2,6-diazaspiro[3,4]octan-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexane-1-amine

[0425] [ka]

[0426] (R)-N-(2-methoxyethyl)-N,5-dimethyl-4-(2,6-diazaspiro[3.4]octan-2-yl)hexane-1-amine (intermediate 224) (100 g) was mixed with 2-MeTHF (430 g) and TEA (68 g), and the mixture was cooled to -50 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 to 3 hours. The resulting mixture was heated to -20 to -10°C, 7% NaHCO3 aqueous solution was added, and the mixture was heated to 20 to 30°C and stirred for 30 to 60 minutes. The aqueous layer was removed, and the organic layer was washed with 10% Na2SO4 (500 g). The organic layer was dried by passing it through a 4 Å molecular sieve (220 g) and washed with 2-MeTHF (180 g). The title intermediate was obtained as a solution of 2-14.8 wt% MeTHF in assay yield of 90%.

[0427] Compound 393-(R)-2-((3-chloro-5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropyl-benzamide Synthesis method A for compound 393

[0428] [ka]

[0429] A mixture of anhydrous THF (15 mL), 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-triazin-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexane-1-amine (intermediate 225) (1.70 g, 3.82 mmol), and DBU (750 mg, 4.93 mmol) 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 x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. This was purified by purified FCC (MeOH / DCM = 0%~10%) to obtain a yellow oily substance (1.40 g). This was further separated by SFC using a DAIEL CHIRALPAK AD (column: 250 × 50 mm, 10 μm; mobile phase: A: supercritical CO2, B: EtOH (0.1% ammonia), 70 mL / min with A:B = 50:50; column temperature: 38 °C; nozzle pressure: 100 bar; nozzle temperature: 60 °C; evaporator temperature: 20 °C; trimmer temperature: 25 °C; wavelength: 220 nm) to obtain the title compound (1.0 g).

[0430] Synthesis method A for compound 393

[0431] [ka]

[0432] To a 2-MeTHF solution prepared by dissolving (R)-4-(6-(3,6-dichloro-1,2,4triazin-5-yl)-2,6-diazospiro[3.4]octan-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexane-1-amine (intermediate 225) (676 g of 2-14.8 wt% MeTHF solution, corrected to 100 g for intermediate 225) and N-ethyl-5-fluoro-2-hydroxy-N-isopropylbenzamide (intermediate 28) (50.6 g) in 2-MeTHF (40 g) at 20-30°C, 31 g of tetramethylguanidine was added, and the mixture was stirred for 40-48 hours. 500 g of 7% NaHCO3 aqueous solution was added, and the mixture was stirred for 30-60 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 to 2.2–3.0 volumes under reduced pressure (below 40°C), and flushed three times with MeOH (1 × 790 g and 2 × 395 g) until both 2-MeTHF and water content were less than 1.0%, yielding the desired compound in an assay yield of 86% as a 60.1% wt methanol solution.

[0433] Compound A-(R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide

[0434] [ka]

[0435] (R)-2-((3-chloro-5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy-N-ethyl-5-fluoro-N-isopropylbenzamide (compound 393) (163.93 g of a 60.1 wt% MeOH solution, corrected to 100 g for compound 393), palladium carbon (10 g), and a methanol solution of MeOH (316 g) were stirred at 20-30°C for 18 hours under a hydrogen atmosphere (0.20-0.30 MPa). The mixture was filtered through diatomaceous earth (75 g), and the cake was washed with MeOH (158 g). The filtrate was concentrated to approximately 3 volumes under reduced pressure (below 40°C), and then flushed with isopropyl acetate (IPAc, 870 g) and concentrated to approximately 3 volumes. The mixture was then diluted with IPAc (696 g), and a 20% Na2CO3 aqueous solution was added (500 g). 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 to approximately 3 volumes under reduced pressure below 45°C. The title intermediate was obtained as a 48.1 wt% IPAc solution in an assay yield of approximately 90%.

[0436] Example 4: Synthesis of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide oxalate (compound A3)

[0437] [ka]

[0438] (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide (compound A) (270 mg, 0.450 mmol) was dissolved in 20 mL of ACN (20 mL), to which oxalic acid (81.0 mg, 0.900 mmol) was added. After the 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 freeze-dried to obtain the title compound (350 mg) as a white solid.

[0439] 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).

[0440] 1 H NMR(400MHz,DMSO-d6):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).

[0441] LCMS(ESI)(Method 2):R t = 1.969 minutes, measured value in m / z: 600.4 [M+H] + .

[0442] Example 5 - Synthesis of Compound A1

[0443] [ka]

[0444] A solution of compound A (207.90 g of a 48 wt% solution in IPAc, 100 g of active compound A) was dissolved in IPAc (360 g). EtOH (63 g) was added to this solution at 20°C to 25°C. The solution was then treated with concentrated HCl (32.9 g) in EtOH (49.5 g) for approximately 15 minutes. Crystalline compound A1 seeds (2 g, 2% seed load) were seeded into the mixture, and the mixture was aged for 18 hours. IPAc (870 g) was slowly added over 4 hours at 20 to 25°C, and the slurry was stirred for a further 18 hours. After cooling to approximately 5°C, the product was filtered, washed with IPAc (522 g), and dried under vacuum at 20 to 30°C to obtain weakly crystalline compound A1 as a white solid (yield 91.0%, 115.4 g). (Note: The small amount of seed material used in the reaction was obtained via a similar small-scale reaction protocol.)

[0445] 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 20-30°C for 0.5 to 2 hours to obtain a clear solution. Additional IPAc (522 g) was added dropwise over 1-2 hours, and then crystalline compound A1 was seeded into the mixture (2 g, 2% seed load). The mixture was then aged for 18-20 hours, IPAc (348 g) was slowly added over 12 hours at 20-30°C, and the slurry was stirred for a further 55-60 hours. The product was filtered, washed with IPAc (158 g), and dried in vacuum at 20-30°C to obtain compound A1 as a white solid (yield 85%, net 85.0 g).

[0446] 1HNMR (DMSO-d6,400MHz):δ=11.60(1H,brs),10.8(1H,brs),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).

[0447] LCMS (Method 7):R t = 0.669 minutes, measured value in m / z: 600.5 [M+H] + .

[0448] Example 6: Synthesis of crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide bisbesylate hydrate (compound A4) (water equivalent not determined)

[0449] [ka]

[0450] 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 a solution of compound A (containing 80 g of API) dissolved in IPAc was added. The material dissolved, and a clear solution was obtained. An additional 80 mL of IPAc was added, and the temperature was adjusted to 25°C. 2% seed was added, and the mixture was stirred at 25°C for 1 hour. Then, 28.8 V (2312 mL) of IPAc was added over 8 hours. After that, 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 bis-besylate hydrate (water equivalent not determined) was obtained.

[0451] Those skilled in the art will understand that the small amount of initial seed material used in the above reaction can be obtained by awaiting spontaneous nucleation without adding seed, via a similar small-scale reaction protocol.

[0452] During the salt screening experiments, the initial seed of besylates was also obtained. 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 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 obtain the initial seed.

[0453] Dissolve an appropriate amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate in deuterated DMSO, and 1D 1 The 1H NMR spectrum was recorded.

[0454] One-dimensional proton experiments at 300K on samples in deuterated DMSO were collected using a Bruker AVANCE NEO-600MHz NMR spectrometer equipped with a Bruker 5mm PA BBO 600S3 BB-HD-05 Z-GRD high-resolution probe and running TOPSPIN 4.0 software.

[0455] 1H NMR(600MHz,DMSO-d6)δ ppm 0.69(br s,2H)0.82-0.98(m,9H)1.07(br s,4H)1.31-1.46(m,1H)1.51(br d,J=2.91Hz,1H)1.69(br d,J=3.45Hz,2H)1.98(br s,1H)2.06-2.45(m,2H)2.77(br s,3H)2.87-3.19(m,3H)3.24(br s,1H)3.31(s,6H)3.64(br s,4H)3.71-4.59(m,7H)7.24-7.54(m,9H)7.61(br d,J=7.27Hz,4H)8.45-8.60(m,1H)9.24(br s,1H)9.44-9.82(m,1H).

[0456] Example 7: Alternative synthesis of crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide bisbesylate hydrate (compound A4) (water equivalent not determined) A mixture of 95 / 5 isopropanol / water (24 mL) was placed in a flask and heated to 40°C. Benzesulfonic acid (4.31 g, 98%) was added. Subsequently, a solution of 19.3 g of compound A in IPAc (containing 8 g of compound A) was added. Further, 16 mL of IPAc was added. 2% seed was added, and the mixture was stirred at 40°C for 1 hour. Then, IPAc (115.2 mL) was added dropwise over 8 hours. Next, the mixture was cooled to 0°C over 15 hours. The suspension was filtered, and the wet cake was washed with (IPA / H2O 95 / 5) / IPAc 1 / 6 (32 mL). The wet cake was dried at 25°C for 16 hours to obtain 11.44 g of crystalline form A bis-besylate hydrate (water equivalent was not determined).

[0457] Crystal form A The crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate can be characterized by its X-ray powder diffraction pattern.

[0458] X-ray powder diffraction (XRPD) analysis was performed using a PANalytical Empyrean diffractometer. This instrument features a Cu-Kα X-ray tube with iCore and dCore tunable optics for the incident and diffracted beams, respectively. The compounds were loaded into a 16 mm sample holder cavity using backloading techniques.

[0459] The sample was subjected to XRPD using the following method: Tube:Cu:K-α(λ=1.541874Å) Generator: Voltage: 45kV Current: 40mA Geometry: Bragg-Brentano Scanning method: Continuous scanning Scanning range: 3-35 degrees Process size: 0.0131 degrees Counting time: 30s Spinner rotation time: 1 second Incident beam path (iCore) Program divergence slit: automatic Irradiation length: 10mm Solar slit: 0.03rad Mask 1: 14mm Mask 2: 6mm Width: 7.7mm Diffraction beam path (dCore) Scatter prevention slit: Automatic Irradiation length: 10mm Solar slit: 0.04rad Detector: PIXcel3D-Medipix3 1x1

[0460] Those skilled in the art will recognize that diffraction patterns and peak positions are generally substantially independent, regardless of the diffractometer used and whether a specific calibration method is employed. Typically, peak positions can differ by no more than approximately ±0.2°²θ. The intensity (and relative intensity) of each specific diffraction peak can also vary as a function of various factors, including, but not limited to, particle size, orientation, and sample purity.

[0461] The X-ray powder diffraction pattern includes peaks at 5.4, 7.2, 11.1, 11.9, and 21.7 degrees 2-theta ± 0.2 degrees 2-theta. The X-ray powder diffraction pattern may further include at least one peak selected from 13.7, 14.5, 14.7, 15.0, 16.5, 17.8, 19.0, 19.4, and 20.1 degrees 2-theta ± 0.2 degrees 2-theta.

[0462] Morphology A can be further characterized by an X-ray powder diffraction pattern having 4, 5, 6, 7, 8, 9 or more peaks selected from the peaks identified in Table 2.

[0463] Morphology A can be further characterized by an X-ray powder diffraction pattern containing the peaks identified in Table 2, where the relative intensity of the peaks is greater than about 2%, preferably greater than about 5%, more preferably greater than about 10%, and more preferably greater than about 15%. However, those skilled in the art will understand that the relative intensity of the peaks may differ between different samples and between different measurements of the same sample.

[0464] Morphology A can be further characterized by the X-ray powder diffraction pattern substantially shown in Figure 1.

[0465] Table 2 shows the peak list and relative intensity for XPRD of crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4triazine-6-yl)oxy)benzamide bisbesylate hydrate salt (Figure 1).

[0466] [Table 2]

[0467] Pharmacological aspects In vitro proliferation-menin-MLL inhibitor (compound A4) combined with DNA insertion agent (idarubicin) cell line 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% alpha-MEM (containing ribonucleosides and deoxyribonucleosides) + 10-20% FBS. All cell lines were cultured at 37°C under a 5% CO2 atmosphere.

[0468] Cell titer Glo assay AML cell line (5 × 10 3 Cells (per well) were seeded in 96-well plates and grown for 6 days in serum (10%) medium in the presence or absence of the indicated inhibitor concentration. Growth was analyzed by the CellTiter Glo assay using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. Data are the mean with standard deviation of 2-4 independent experiments in technical triplicates.

[0469] Synergistic effect calculation This is an R-based biochemically intuitive generalized Loewe (BIGL) model implemented using a high-level single-drug (HSA) null model. Specifically, drug-drug interactions were calculated by applying the BIGL methodology (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., & Bijnens, L. Statistical detection of synergy: New methods and a comparative study. Pharmaceutical Statistics (2021)).

[0470] Synergistic matrix results of BIGL analysis of cell line data using HSA as the mean model calculated based on cellular metabolic activity using the Cell Titer-Glo assay. Bootstrap confidence intervals are shown. Effect sizes and their confidence intervals are shown. In particular, each data point is based on the p-value and sign of its respective maxR statistic, and the size of the dot reflects the degree of synergistic or antagonistic effect corresponding to a stepwise scale. If the interval contains 0, there is no significant mean effect.

[0471] result Menin-MLL inhibitor in combination with idarubicin (compound A4) The pairwise matrix combination of idarubicin and compound A4 was evaluated in MOLM-13 (KMT2A-AF9, FLT3-ITD) cells and OCI-AML3 (NPM1c AML) cells using the 6-day CellTiter-Glo assay format. Notably, the combination of idarubicin and compound A4 was not antagonistically cytotoxic in MOLM-13 (KMT2A-AF9, FLT3-ITD) cells, as reflected in the contour plot (Figure 2A) and detailed in Table 3A below.

[0472] [Table 3] Note that if there is no asterisk, it indicates an additive effect, and if there is an antagonistic effect, * The synergistic effect is shown as " ** This is shown as follows. Antagonisms and synergies are derived from key calls based on the maxR test and the associated numerical values ​​that are interpreted as antagonisms or synergies, respectively.

[0473] Similarly, the combination of idarubicin and compound A4 is not antagonistically cytotoxic in OCI-AML3 (NPM1c AML) cells, as reflected in the contour plot (Figure 2B) and detailed in Table 3B below.

[0474] [Table 4] Note that if there is no asterisk, it indicates an additive effect, and if there is an antagonistic effect, * The synergistic effect is shown as " ** This is shown as follows. Antagonisms and synergies are derived from key calls based on the maxR test and the associated numerical values ​​that are interpreted as antagonisms or synergies, respectively.

[0475] In vitro proliferation-menin-MLL inhibitor (compound A3) combined with a pyrimidine analog (cytarabine) and a DNA insertion agent (idarubicin or daunorubicin). cell line The 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% alpha-MEM (without ribonucleosides and deoxyribonucleosides) + 10-20% FBS. All cell lines were cultured at 37°C under a 5% CO2 atmosphere.

[0476] Cell titer Glo assay AML cell line (10×10 3 Cells (per well) were seeded in 96-well plates and grown in complete medium for 6 days in the presence or absence of the indicated inhibitor concentration. Growth was analyzed by the CellTiter Glo assay using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. Data are the mean with standard deviation of 2–4 independent experiments using technical triplicates.

[0477] Synergistic effect calculation This is an R-based biochemically intuitive generalized Loewe (BIGL) model implemented using a high-level single-drug (HSA) null model. Specifically, drug-drug interactions were calculated by applying the BIGL methodology (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., & Bijnens, L. Statistical detection of synergy: New methods and a comparative study. Pharmaceutical Statistics (2021)).

[0478] Synergistic matrix results of BIGL analysis of cell line data using HSA as the mean model calculated based on cellular metabolic activity using the Cell Titer-Glo assay. Bootstrap confidence intervals are shown. Effect sizes and their confidence intervals are shown. In particular, each data point is based on the p-value and sign of its respective maxR statistic, and the size of the dot reflects the degree of synergistic or antagonistic effect corresponding to a stepwise scale. If the interval contains 0, there is no significant mean effect.

[0479] result A menin-MLL inhibitor (compound A3) combined with cytarabine and idarubicin. The pairwise matrix combination of cytarabine + idarubicin and compound A3 was evaluated in MOLM-13 (KMT2A-AF9, FLT3-ITD) cells and OCI-AML3 (NPM1c AML) cells using the 6-day CellTiter-Glo assay format. The tripartite combination of cytarabine + idarubicin and compound A3 was synergistically cytotoxic in MOLM-13 cells (Figure 2C and Table 3C), but not antagonistically cytotoxic in OCI-AML3 cells, as reflected below (Figure 2D and Table 3D).

[0480] [Table 5] Note that if there is no asterisk, it indicates an additive effect, and if there is an antagonistic effect, * The synergistic effect is shown as " ** This is shown as follows. Antagonisms and synergies are derived from key calls based on the maxR test and the associated numerical values ​​that are interpreted as antagonisms or synergies, respectively.

[0481] [Table 6] Note that if there is no asterisk, it indicates an additive effect, and if there is an antagonistic effect, * The synergistic effect is shown as " ** This is shown as follows. Antagonisms and synergies are derived from key calls based on the maxR test and the associated numerical values ​​that are interpreted as antagonisms or synergies, respectively.

[0482] A menin-MLL inhibitor (compound A3) combined with cytarabine and daunorubicin. The pairwise matrix combination of cytarabine + daunorubicin and compound A3 was evaluated in MOLM-13 (KMT2A-AF9, FLT3-ITD) cells and OCI-AML3 (NPM1c) cells using the 6-day CellTiter-Glo assay format. The tripartite combination of cytarabine + daunorubicin and compound A3 was synergistically cytotoxic in MOLM-13 cells (Figure 2E and Table 3E), but not antagonistically cytotoxic in OCI-AML3 cells, as reflected below (Figure 2F and Table 3F).

[0483] [Table 7] Note that if there is no asterisk, it indicates an additive effect, and if there is an antagonistic effect, * The synergistic effect is shown as " ** This is shown as follows. Antagonisms and synergies are derived from key calls based on the maxR test and the associated numerical values ​​that are interpreted as antagonisms or synergies, respectively.

[0484] [Table 8] Note that if there is no asterisk, it indicates an additive effect, and if there is an antagonistic effect, * The synergistic effect is shown as " ** This is shown as follows. Antagonisms and synergies are derived from key calls based on the maxR test and the associated numerical values ​​that are interpreted as antagonisms or synergies, respectively.

[0485] In vitro proliferation-menin-MLL inhibitor (combination of compound A3 and DNA insertion agent (daunorubicin)) and pyrimidine analog (cytarabine) in samples from primary NPM1c-positive AML patients. Compound A3 was tested in combination with cytarabine and daunorubicin using samples from primary NPM1c-positive AML patients (N=8). All samples were cultured in complete medium and seeded at 20,000 cells per well in 96-well plates. They were then treated with a two-drug combination of cytarabine and daunorubicin (initial concentration of 10 μM cytarabine + 5 μM daunorubicin, followed by 6 points, 10-fold serial dilutions), or a three-drug combination of compound A3 and cytarabine + daunorubicin (initial concentration of 10 μM compound A3, 10 μM cytarabine + 5 μM daunorubicin, followed by 6 points, 10-fold serial dilutions). Wells containing only the cells with the aforementioned drug treatments and controls ("medium control") or wells containing the medium with the vehicle ("vehicle control") were incubated at 37°C, 5% CO2 for 6 days. No further additions or modifications were made to the medium throughout the experiment. ATP levels, as a surrogate marker of cell viability, were evaluated using the CELLTITER-GLO assay. Briefly, a 96-well plate was removed from the incubator and equilibrated at room temperature for 30 minutes. CELLTITER-GLO was added to the wells, mixed on a plate rack for 2 minutes, and then incubated at room temperature for 10 minutes to stabilize the luminescence signal before quantification using a plate reader.

[0486] Data were corrected for background signaling using a culture medium control, normalized to a vehicle control, and then logarithmically transformed. These results were reported as changes in relative light units in response to treatment at different drug concentrations, directly proportional to cell viability. A variable gradient model was applied to construct concentration-response curves. For each drug concentration (from N=8 patient samples to N=24, evaluated using triplicates per drug concentration), data are presented as mean ± standard deviation. IC 50 The values ​​were calculated using nonlinear regression analysis.

[0487] Overall, as reflected in Table 4 below, a lack of antagonistic activity was observed in primary NPM1c-positive AML patient samples treated with compound A3, cytarabine, and daunorubicin.

[0488] Table 9

Claims

1. Therapeutic amount of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (compound A): 【Chemistry 1】 Alternatively, a pharmaceutically acceptable salt or solvate thereof, and a therapeutically effective amount of a combination of a DNA insertor and a pyrimidine analog (triple combination).

2. Compound A or a pharmaceutically acceptable salt or solvate thereof is a besylate (compound A4-a): 【Chemistry 2】 The combination according to claim 1, or a solvate thereof.

3. Compound A4-a The combination according to claim 2, wherein the solvate is (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamidebisbesylate or its hydrate.

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

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

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

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

8. A combination according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, for use as a pharmaceutical.

9. A combination according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, for use in the prevention or treatment of hematopoietic disorders, particularly for treatment.

10. The hematopoietic disorder comprises (i) one or more MLL1 (KMT2A) gene rearrangements or genetic alterations (e.g., replication or amplification) and / or NPM1 mutations, in addition to (ii) an FLT3 mutation, according to the combination or pharmaceutical composition for use of claim 9.

11. The hematopoietic disorder is (i) having one or more MLL1 (KMT2A) gene rearrangements in addition to (ii) an FLT3 mutation, according to the combination or pharmaceutical composition for use according to claim 9.

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

13. A method for treating a subject diagnosed with hematopoietic disorder, comprising (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide (compound A): 【Transformation 3】 A method comprising administering to the subject a pharmaceutically acceptable salt or solvate thereof, as well as a therapeutically effective amount of a combination of a DNA insertor and a pyrimidine analog (a triplicate).

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