(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 for the treatment of diseases such as cancer.

Abstract

The present invention relates to (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 besylate and solvates thereof. The compounds may be useful for therapy and / or prophylaxis in mammals, pharmaceutical compositions comprising such compounds, and for use as menin / MLL protein / protein interaction inhibitors useful for treating diseases, such as, but not limited to, cancers, such as leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

Description

Technical Field

[0001] The present invention relates to (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 besylate and its solvates.

[0002] This compound is useful for the treatment and / or prevention in mammals, pharmaceutical compositions containing such compounds, and for use as a menin / MLL protein / protein interaction inhibitor useful for treating diseases such as, but not limited to, cancer and diabetes such as leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN).

Background Art

[0003] Chromosomal rearrangements that affect the mixed-lineage leukemia gene (MLL, MLL1, KMT2A) result in aggressive acute leukemia across all age groups and remain largely incurable diseases, arguing for an urgent need for new treatment approaches. Acute leukemias with these chromosomal translocations of MLL present as lymphoid, myeloid or biphenotypic diseases and constitute 5-10% of acute leukemias in adults and approximately 70% in infants (Marschalek, Br J Haematol 2011.152(2), 141-54, Tomizawa et al., Pediatr Blood Cancer 2007.49(2), 127-32).

[0004] MLL is a histone methyltransferase that methylates histone H3 on lysine 4 (H3K4) and functions in a multiprotein complex. The use of the inducible loss-of-function allele of Mll1 demonstrated that Mll1 plays an essential role in the maintenance of hematopoietic stem cells (HSCs) and B cell development, but its histone methyltransferase activity is not required for hematopoiesis (Mishra et al., Cell Rep 2014.7(4),1239-47).

[0005] Fusions of MLL with over 60 different partners have been reported to date and have been associated with leukemia formation / progression (Meyer et al., Leukemia 2013.27, 2165-2176). Interestingly, the SET (Su(var)3-9, zeste enhancer, and trithorax) domains of MLL are not retained in the chimeric protein but are replaced by the fusion partner (Thiel et al., Bioessays 2012.34, 771-80). Recruitment of chromatin-modifying enzymes such as the Dot1L and / or pTEFb complex by the fusion partner results in enhanced transcription and transcriptional elongation of MLL target genes, most notably the HOXA gene (e.g., HOXA9) and the HOX cofactor MEIS1. Subsequently, the abnormal expression of these genes blocks hematopoietic differentiation and promotes proliferation.

[0006] Menin, encoded by the multiple endocrine neoplasia type 1 (MEN1) gene, is ubiquitously expressed and primarily localized in the nucleus. It is known to interact with numerous proteins and is therefore involved in various cellular processes. Menin's best-understood function is its role as an oncogenic cofactor for MLL fusion proteins. Menin interacts with two motifs within the N-terminal fragment of MLL, MBM1 (menin-binding motif 1) and MBM2, which are retained in all fusion proteins (Thiel et al., Bioessays 2012.34, 771-80). The menin / MLL interaction creates a new interaction surface for lens epithelial growth factor (LEDGF). While MLL directly binds to LEDGF, menin is essential for stable interaction between MLL and LEDGF, and for gene-specific chromatin recruitment of the MLL complex via the PWWP domain of LEDGF (Cermakova et al., Cancer Res 2014.15, 5139-51; Yokoyama & Cleary, Cancer Cell 2008.8, 36-46). Furthermore, numerous genetic studies have shown that menin is strictly required for oncogenic transformation by MLL fusion proteins, suggesting the menin / MLL interaction as an attractive therapeutic target. For example, conditional deletion of Men1 prevents leukocytosis in myeloid progenitor cells ectopically expressing MLL fusions (Chen et al., Proc Natl Acad Sci 2006.103, 1018-23). Similarly, loss-of-function mutations disrupting the menin / MLL fusion interaction gene neutralize the oncogenic properties of the MLL fusion protein, block leukemia development in vivo, and release the differentiation block of MLL-transformed leukemic blasts. These studies also demonstrated that menin is required for the maintenance of HOX gene expression by the MLL fusion protein (Yokoyama et al., Cell 2005.123,207-18).Furthermore, small molecule inhibitors of the menin / MLL interaction have been developed, suggesting the druggability of this protein / protein interaction, and their efficacy in preclinical models of AML has been demonstrated (Borkin et al., Cancer Cell 2015.27, 589-602; Cierpicki and Grembecka, Future Med Chem 2014.6, 447-462). These data, along with the observation that menin is not an essential cofactor of MLL1 in normal hematopoiesis (Li et al., Blood 2013.122, 2039-2046), justify inhibiting the menin / MLL interaction as a promising new therapeutic approach for treating MLL-rearranged leukemia and other cancers with an active HOX / MEIS1 gene signature. For example, internal tandem duplication (PTD) within the 5' region of the MLL gene represents another major abnormality primarily found in de novo and secondary AML, as well as myelodysplastic syndromes. Although the molecular mechanisms and biological functions of MLL-PTD are not fully understood, novel therapeutic targeting strategies that influence menin / MLL interactions may also prove effective in treating MLL-PTD-associated leukemia. Furthermore, castration-resistant prostate cancer is known to be dependent on menin / MLL interactions (Malik et al., Nat Med 2015.21, 344-52).

[0007] The MLL protein is also known in the scientific field as the histone-lysine N-methyltransferase 2A (KMT2A) protein (UniProt accession number Q03164).

[0008] Several references describe inhibitors that target the menin-MLL interaction. International Publication No. 2011029054, J Med Chem 2016, 59, 892-913 describes the preparation of thienopyrimidine and benzodiazepine derivatives; International Publication No. 2014164543 describes thienopyrimidine and thienopyridine derivatives; Nature Chemical Biology March 2012, 8, 277-284 and Ren, J. et al. Bioorg Med Chem Lett (2016), 26(18), 4472-4476 describe thienopyrimidine derivatives; J Med Chem 2014, 57, 1543-1556 describes hydroxy and aminomethylpiperidine derivatives; Future Med Chem 2014,6,447-462 provides an overview of small molecules and peptide mimetic compounds, and International Publication No. 2016195776 includes flu[2,3-d]pyrimidine, 9H-purine, [1,3]oxazolo[5,4-d]pyrimidine, [1,3]oxazolo[4,5-d]pyrimidine, [1,3]thiazolo[5,4-d]pyrimidine, thieno[2,3-b]pyridine and thieno[2,3-d] Pyrimidine derivatives are described. International Publication No. 2016197027 describes 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine, pyrido[2,3-d]pyrimidine, and quinoline derivatives. International Publication No. 2016040330 describes thienopyrimidine and thienopyridine compounds. International Publication No. 2017192543 describes piperidine as a menin inhibitor. International Publication Nos. 2017112768, 2017207387, 2017214367, 2018053267, and 2018024602 describe inhibitors of menin-MLL interaction. International Publication Nos. 2017161002 and 2017161028 describe menin-MLL inhibitors.International Publication Nos. 2018050686, 2018050684, and 2018109088 describe inhibitors of menin-MLL interaction. International Publication No. 2018226976 describes methods and compositions for inhibiting the interaction between menin and MLL protein. International Publication No. 2018175746 provides methods for the treatment of hematological malignancies and Ewing sarcoma. International Publication Nos. 2018106818 and 2018106820 provide methods for promoting the proliferation of pancreatic cells. International Publication No. 2018153312 discloses azaspiro compounds relating to the field of medicinal chemistry. International Publication No. 2017132398 discloses a method comprising contacting leukemia cells exhibiting an NPM1 mutation with a pharmacological inhibitor of the interaction between MLL and menin. International Publication No. 2019060365 describes menin-MLL substitution inhibitors. International Publication No. 2020069027 describes the treatment of hematological malignancies with menin inhibitors. Krivtsov et al., Cancer Cell 2019. No.6 Vol.36, 660-673 also describes menin-MLL inhibitors. [Overview of the project] [Means for solving the problem]

[0009] 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)benzamide besylate (benzene sulfonate salt):

[0010] [ka] and its solvates. Those skilled in the art will understand that “and its solvate” 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. Therefore, the present invention also includes the besilate of (RN-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octane-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide, and the solvate of the besilate of (RN-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-2,6-diazaspiro[3.4]octane-6-yl)-1,2,4-triazine-6-yl)oxy)benzamide.

[0011] In particular, 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)benzamide besylate or its hydrate.

[0012] In particular, 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.

[0013] In particular, 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.

[0014] In particular, 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 0.5~2.0 equivalent hydrate.

[0015] In particular, 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.

[0016] More specifically, 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 hydrate.

[0017] More specifically, 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.

[0018] More specifically, 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.

[0019] (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 besylates or their solvates are excellent in terms of their chemical / physical stability, their physical properties, and the fact that they can be isolated as stable crystalline solids.

[0020] One embodiment of the present invention relates to a pharmaceutical composition 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)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate or a solvate thereof.

[0021] The present invention also provides a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and / or a pharmaceutically acceptable diluent, and a pharmaceutical composition 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)benzamidebisbesylate or a solvate thereof, a pharmaceutical composition comprising the same, and / or a pharmaceutical composition essentially comprising the same.

[0022] Also provided are pharmaceutical compositions 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)benzamidebisbesylate or a solvate thereof, a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and / or a pharmaceutically acceptable diluent, a pharmaceutical composition comprising them, and / or a process for producing a pharmaceutical composition essentially comprising them.

[0023] The present invention further relates to methods for treating or improving diseases, such as cancers including, but not limited to, leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes, using (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 and its solvates.

[0024] The present invention also relates to the use 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)benzamidebisbesylate or its solvate in the preparation of pharmaceuticals, wherein the pharmaceuticals are prepared to treat diseases, for example, but not limited to, cancers such as leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0025] In particular, the compounds and pharmaceutical compositions according to the present invention may be useful for the treatment or prevention of leukemia, especially nucleophosmin (NPM1) variant leukemia, such as NPM1c.

[0026] In one embodiment, the compound according to the present invention may have improved metabolic stability properties.

[0027] In one embodiment, the compound according to the present invention may have an extended in vivo half-life (T1 / 2).

[0028] In one embodiment, the compound according to the present invention may have improved oral bioavailability.

[0029] In one embodiment, the compounds according to the present invention may reduce tumor growth, for example, tumors having rearrangements / alterations of the MLL(KMT2A) gene and / or NPM1 mutations.

[0030] In one embodiment, the compound according to the present invention may have improved PD properties in vivo over a long period, such as inhibition of target gene expression, including MEIS1, and upregulation of differentiation markers for a period of at least 16 hours.

[0031] In one embodiment, the compound according to the present invention may have an improved safety profile (e.g., reduced hERG inhibition, improved cardiovascular safety).

[0032] In one embodiment, the compound according to the present invention may be suitable for QD administration (once a day).

[0033] The present invention also relates, but is not limited to, to the use of the compounds according to the present invention in combination with additional pharmaceuticals for use in the treatment or prevention of cancers such as leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0034] Furthermore, the present invention relates to a process for preparing a pharmaceutical composition according to the present invention, characterized in that a pharmaceutically acceptable carrier is homogeneously mixed with a therapeutically effective amount of the compound according to the present invention.

[0035] The present invention also relates, but is not limited to, to products comprising the compound and additional pharmaceuticals according to the present invention as combination formulations for simultaneous, separate, or sequential use in the treatment or prevention of cancers such as leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0036] Furthermore, the present invention relates to a method for treating or preventing cell proliferation disorders in warm-blooded animals, comprising administering an effective amount of a compound as defined herein, or a pharmaceutical composition or combination as defined herein, to the animal.

[0037] In other embodiments, 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)benzamide besylate and its solvate for use as a pharmaceutical.

[0038] In other embodiments, 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 and its solvates for use as pharmaceuticals.

[0039] In other embodiments, 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)benzamide besylate hydrate for use as a pharmaceutical.

[0040] In other embodiments, 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 hydrate for use as a pharmaceutical.

[0041] The present invention also relates to the preparation 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 and its solvates.

[0042] The present invention also relates to the preparation 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)benzamidebisbesylate hydrate. [Brief explanation of the drawing]

[0043] The summary of the invention and the modes for carrying out the invention described below will be better understood in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show exemplary embodiments of the invention. However, the invention is not limited to the specific disclosures in the drawings. Of the drawings: [Figure 1] This is the X-ray powder diffraction (XRPD) pattern 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)-1,2,4-triazine-6-yl)oxy)benzamidebisbesylate hydrate. [Figure 2]Efficacy experiment in the Molm-14 subcutaneous (sc) model. [Figure 3] Efficacy experiments in a disseminated OCI-AML3 model. [Figure 4] This is the powder X-ray diffraction (XRPD) pattern of intermediate 234b. [Modes for carrying out the invention]

[0044] This disclosure can be better understood by referring to the following description, including the glossary and concluding examples. It should also be understood that certain features of the compounds, crystalline forms A, compositions, and methods of this disclosure, which are described in this specification in the context of separate embodiments for clarity, may be provided in combination in a single embodiment. Conversely, various features of the compounds, crystalline forms A, compositions, and methods of this disclosure, which are described in the context of a single embodiment for brevity, may also be provided separately or in any partial combination.

[0045] Some of the quantitative expressions expressed herein are not modified by the term “approximately.” Whether or not the term “approximately” is explicitly used, all quantities expressed herein mean their actual expressed value, and are understood to also mean approximations of such expressed value that can be reasonably estimated on the basis of ordinary skill in the art, including approximations of such expressed value by experimental and / or measurement conditions.

[0046] Throughout this specification and in the claims, the terms “comprise” and “contain,” and their variations, such as “comprising” and “comprises,” mean “including but not limited to,” and are not intended (and do not exclude) any other components.

[0047] In this disclosure, the terms “crystal morph” and “polymorph” are synonymous. Characteristic information of crystal morphs is provided herein. It should be understood that the determination of a particular morph can be achieved using any part of the characteristic information that a person skilled in the art would recognize as sufficient to establish the existence of a particular morph. For example, even a single characteristic peak may be sufficient for a person skilled in the art to recognize the existence of a particular morph.

[0048] The term "isolated form" refers to a compound that exists in a form separated from any solid mixture with another compound, a solvent system, or a biological environment. In one embodiment of the present invention, the crystalline form exists in an isolated form.

[0049] The term "room temperature" (RT) refers to temperatures between approximately 15°C and 30°C, particularly between approximately 20°C and 30°C. Preferably, room temperature is approximately 25°C.

[0050] When the crystal morphology is identified using one or more XRPD peaks given as angles 2θ (2-theta), each 2θ value is understood to mean a given value ± 0.2 degrees 2-theta, unless otherwise stated.

[0051] The term "seeding" refers to the addition of crystalline material to a solution or mixture to initiate crystallization or recrystallization.

[0052] As used herein, the terms “(the) compound of the present invention” or “(the) compound according to the present invention” mean including (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 and its solvates, or any subgroup thereof.

[0053] (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 may exist as a solvate. The “solvate” may be a solvate with water (i.e., a hydrate) or a common organic solvent.

[0054] (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 besylate or its solvate can be obtained in substantially pure form, with the mole percent of impurities in the isolated compound being less than about 5 mole percent, preferably less than about 2 mole percent, more preferably less than about 0.5 mole percent, and most preferably less than about 0.1 mole percent. In one embodiment of the present invention, (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 besylate or its solvate is present in a substantially pure form.

[0055] 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)-1,2,4-triazine-6-yl)oxy)benzamidebisbesylate hydrate can be obtained in a substantially pure form, and the mole percent of impurities in the isolated crystalline form is less than about 5 mole percent, preferably less than about 2 mole percent, more preferably less than about 0.5 mole percent, and most preferably less than about 0.1 mole percent. In one embodiment of the present invention, (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 exists in a substantially pure form.

[0056] 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)-1,2,4triazin-6-yl)oxy)benzamide bisbesylate hydrate is also provided herein as a mixture with one or more additional forms 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)-1,2,4triazin-6-yl)oxy)benzamide bisbesylate hydrate. At least certain weight percent may be 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)-1,2,4-triazine-6-yl)oxy)benzamide bisbesylate hydrate. Specific weight percents include 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9%.

[0057] This specification describes a process for preparing the crystalline form described herein, which includes the step of recrystallizing compound A, where the recrystallization is a) Adding compound A or its hydrate or solvate to a suitable solvent mixture in the presence of benzenesulfonic acid, and adjusting the temperature to a range from approximately 20°C to the solvent reflux temperature, b) A step of sowing crystal form A, c) A method is provided which includes the step of obtaining a precipitate in the crystalline form described herein.

[0058] In particular, a suitable solvent mixture for the process described in the previous paragraph is a mixture of acetone, water, and IPAc.

[0059] In particular, the appropriate solvent mixture for the process described in the previous paragraph is a mixture of isopropanol, water, and IPAc. Specifically, the temperature used in this process is approximately 25°C.

[0060] Also

[0061] [ka] The crystalline form of the citrate is also provided. Here, the crystal morphology generates an X-ray powder diffraction pattern containing peaks at 5.82, 10.09, and 18.42 degrees with a 2-theta ± 0.2 degree interval. Specifically, the X-ray powder diffraction pattern contains peaks at 5.82, 8.52, 9.20, 10.09, 11.43, 13.61, 14.94, 15.89, 17.03, and 18.42 degrees with a 2-theta ± 0.2 degree interval.

[0062] Also, the following intermediates:

[0063] [ka] It is provided as a pharmaceutically acceptable salt or solvate thereof.

[0064] Those skilled in the art will understand that “or its solvate” refers to a pharmaceutically acceptable salt of the intermediate, and therefore includes the solvate of the pharmaceutically acceptable salt.

[0065] Also, the following intermediates:

[0066] [ka] It is provided as a solvate.

[0067] 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 or free base form with one or more equivalents of a suitable base or acid in an optionally 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.

[0068] 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.

[0069] Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, cesium, magnesium, and calcium salts; 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 isoquinoline salts; benzatine, N-methyl-glucamine, hydrabamin salts; and salts of amino acids such as arginine and lysine. Conversely, base forms can be converted to free base forms by treatment with acid.

[0070] The term solvate includes solvent-added forms. Examples of such solvent-added forms include hydrates and alcoholates.

[0071] A one-step conversion from 5-fluoro-2-hydroxybenzoic acid to N-ethyl-5-fluoro-2-hydroxy-N-isopropylbenzamide (intermediate 28) is also provided.

[0072] [ka]

[0073] This reaction is carried out in a suitable solvent such as THF, toluene, acetonitrile, or 2-methyltetrahydrofuran, in the presence of the coupling agent CDI. In particular, the solvent is THF. The reaction is typically carried out in a temperature range of 0°C to reflux temperature, preferably 0°C to 50°C, more preferably 10°C to 30°C, and even more preferably 15°C to 25°C.

[0074] "Medically acceptable" means that it is approved or eligible for approval by a federal or state regulatory authority or applicable authority in a country other than the United States, or that it is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, more specifically in humans.

[0075] The term "subject" refers to an animal, preferably a mammal, most preferably a human, that is the subject of treatment, observation, or testing.

[0076] 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.

[0077] The term “composition” is intended to encompass products containing specific components in specific amounts, and any products obtained directly or indirectly from specific combinations of specific components in specific amounts.

[0078] As used herein, unless otherwise noted, the terms “effect” or “affected” (referring to a disease, syndrome, condition, or disorder affected by inhibition of a menin / MLL protein / protein interaction inhibitor) include reducing the frequency and / or severity of one or more symptoms or symptom occurrences of the above disease, syndrome, condition, or disorder, and / or preventing the onset of one or more symptoms or symptom occurrences of the above disease, syndrome, condition, or disorder.

[0079] As used herein, the terms “treatment” and “treating” are intended to refer to all processes that may result in slowing, interrupting, stopping, or halting the progression of the disorder, or improvement of one or more of its symptoms, but not necessarily the complete elimination of all symptoms.

[0080] Experiment Part 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)-1,2,4triazin-6-yl)oxy)benzamidebisbesylate hydrate in crystalline form A

[0081] [Table 1-1]

[0082] [Table 1-2]

[0083] [Table 1-3]

[0084] 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.

[0085] If the stereochemistry is not shown, this means that it is a mixture of stereoisomers, unless otherwise specified or evident from the context.

[0086] When a stereocenter is indicated by "RS," this means that a racemic mixture was obtained at the indicated center, unless otherwise specified.

[0087] As will be understood by those skilled in the art, compounds and intermediates 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 or as solvates (e.g., hydrates) may be integer stoichiometric, i.e., monostoichiometric, distoichiometric, or intermediate stoichiometric. When an intermediate or compound is indicated as "HCl salt" without indicating the equivalent number of HCl, this means that the equivalent number of HCl was not determined. When an intermediate or compound is indicated as "hydrate" without indicating the equivalent number of H2O, this means that the equivalent number of H2O was not determined.

[0088] For convenience, (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 (free base) will be shown as "Compound A" in the following experimental section.

[0089] 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 Intermediate 1 tert-butyl(5-methyl-4-oxohexyl)carbamate

[0090] [ka]

[0091] 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.

[0092] Preparation of intermediate 13 tert-butyl6-(3,6-dichloro-1,2,4triazine-5-yl)-2,6-diazaspiro[3,4]octane-2-carboxylate

[0093] [ka]

[0094] 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.

[0095] Preparation of intermediate 27 N-ethyl-5-fluoro-N-isopropyl-2-methoxybenzamide

[0096] [ka]

[0097] 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.

[0098] Preparation of intermediate 28 N-ethyl-5-fluoro-2-hydroxy-N-isopropylbenzamide

[0099] [ka]

[0100] 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.

[0101] Alternative preparation of intermediate 28

[0102] [ka]

[0103] 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%).

[0104] Preparation of 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

[0105] [ka]

[0106] 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.

[0107] Preparation of Intermediate 2 tert-butyl6-(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

[0108] [ka]

[0109] 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.

[0110] Method B for synthesizing intermediate 2

[0111] [ka]

[0112] 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.

[0113] Preparation of Intermediate 3 2-((5-(2,6-diazaspiro[3,4]octan-6-yl)-1,2,4triazine-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0114] [ka]

[0115] 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.

[0116] 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

[0117] [ka]

[0118] 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.

[0119] 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 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

[0120] [ka]

[0121] 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.

[0122] 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

[0123] [ka]

[0124] 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 (100 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.

[0125] 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

[0126] [ka]

[0127] 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.

[0128] LC-MS(ESI)(Method 1):R t = 2.997 minutes, measured value in m / z: 586.3 [M+H] + .

[0129] 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

[0130] [ka]

[0131] (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.

[0132] 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 intermediate 7 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid

[0133] [ka]

[0134] 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.

[0135] Preparation of intermediate 8 tert-butyl(4-(methoxy(methyl)amino)-4-oxobutyl)(methyl)carbamate

[0136] [ka]

[0137] 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.

[0138] Preparation of intermediate 9 tert-butylmethyl(5-methyl-4-oxohexyl)carbamate

[0139] [ka]

[0140] 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.

[0141] 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

[0142] [ka]

[0143] 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.

[0144] 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

[0145] [ka]

[0146] 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.

[0147] 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

[0148] [ka]

[0149] 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.

[0150] 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), A: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.

[0151] 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.

[0152] LCMS(ESI)(Method 2):R t = 1.965 minutes, measured value in m / z: 600.3 [M+H] + .

[0153] SFC (Method 11):R t =4.904 minutes.

[0154] 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 intermediate 227 tert-butyl(R)-(1-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-methylbutan-2-yl)carbamate

[0155] [ka]

[0156] 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. 10% citric acid aqueous solution (449 kg) was added while maintaining the temperature below 10°C. 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.

[0157] Preparation of intermediate 228 tert-butyl(R)-2-isopropyl-5-oxopyrrolidine-1-carboxylate

[0158] [ka]

[0159] 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.

[0160] Preparation of intermediate 229 tert-butyl(5R)-2-hydroxy-5-isopropylpyrrolidine-1-carboxylate

[0161] [ka]

[0162] 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.

[0163] Preparation of intermediate 230 tert-butyl(R)-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)carbamate

[0164] [ka]

[0165] 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.

[0166] Preparation of intermediate 231 (R)-N 1 -(2-methoxyethyl)-N 1 ,5-dimethylhexane-1,4-diamine, dihydrochloride

[0167] [ka]

[0168] 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.

[0169] Preparation of intermediate 232 Ethyl 1-benzyl-3-(chloromethyl)pyrrolidine-3-carboxylate

[0170] [ka]

[0171] 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%).

[0172] Preparation of intermediate 233 1-Benzyl-3-(chloromethyl)pyrrolidine-3-carbaldehyde

[0173] [ka]

[0174] The reaction was carried out in a flow chemistry system. That is, a solution prepared by dissolving ethyl 1-benzyl-3-(chloromethyl)pyrrolidine-3-carboxylate (Intermediate 232) (4.4 kg) in toluene (26 L) was pumped at a rate of 26.7 mL / min and cooled to -60°C. After cooling, this was then mixed at -60°C (28 L) with a cooled solution of DIBAL-H (28.1 mol) dissolved in toluene 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 an aqueous solution of Rochelle salt (20 wt%, 20 V). The layers were separated and the organic phase was washed twice with water (2×44 L). The organic phase was combined with another 3.0 kg batch prepared in the same manner and concentrated under reduced pressure to obtain a toluene solution of 20.8 kg of the desired compound (assay by HPLC: 25.5 wt%, assay yield 85%), which was used directly without further purification.

[0175] 1 H NMR (300 MHz, Chloroform-d): δ 9.62 (s, 1H), 7.39 - 7.20 (m, 5H), 3.83 - 3.57 (m, 4H), 2 .96 (d, J = 10.2 Hz, 1H), 2.80 - 2.55 (m, 3H), 2.17 (ddd, J = 13.9, 7.9, 6.1 Hz, 1H), 1.83 (ddd, J = 13.4, 7.8,

[0175] 5.5 Hz, 1H).

[0176] Preparation of Intermediate 234 (R)-4-(6-Benzyl-2,6-diazaspiro[3.4]octan-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexan-1-amine

[0177] [[ID=1​​​​​​

[0178] 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 water (18 L) 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. Next, the organic phase was discarded, the aqueous phase was separated, and the pH was basicized to 8-9 with K2HPO4.

[0179] 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.

[0180] Preparation of intermediate 234a (citrate of intermediate 234)

[0181] [ka]

[0182] 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.

[0183] Preparation of the crystalline form of the citrate of intermediate 234 (intermediate 234b)

[0184] [ka]

[0185] 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. Seed was then 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).

[0186] 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 to citrate was 3 / 2 (NMR).

[0187] The above nonlinear cooling was performed according to the following equation.

[0188] During the specified cooling period, a new linear ramp is started every 30 seconds. The ramp is calculated according to the following formula:

[0189]

number

[0190] 1 H NMR(400MHz,MeOH-d4)δ ppm 0.91(3 H,d,J=6.88Hz)0.98(3 H,d,J=6.88Hz)1.46-1.57(2 H,m)1.67-1.87(2 H,m)1.94-2.03(1 H,m)2.20-2.29(2 H,m)2.62-2.69(2 H,m)2.72-2.77(4 H,m)2.77-2.82(2 H,m)2.90(2 H,t,J=7.32Hz)2.95-3.02(2 H,m)3.07-3.16(2 H,m)3.16-3.22(2 H,m)3.37(3 H,s)3.68-3.72(2 H,m)3.83-3.89(2 H,m)3.90-3.92(2 H,m)3.94-4.06(2 H,m)7.32-7.43(5 H,m).

[0191] Preparation of intermediate 224 (R)-N-(2-methoxyethyl)-N,5-dimethyl-4-(2,6-diazaspiro[3.4]octan-2-yl)hexane-1-amine

[0192] [ka]

[0193] To palladium carbon hydroxide (1.2 kg) in EtOH (1.47 kg) cooled to -5 to 5°C, methanesulfonic acid (MSA) (11 kg), (R)-4-(6-benzyl-2,6-diazaspiro[3.4]octan-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexan-1-amine (Intermediate 234) (10 kg) and EtOH (250 L) were added. The mixture was warmed to 35 to 45°C and stirred for 16 to 20 hours under a hydrogen atmosphere (0.27 to 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 to 3 volumes under reduced pressure (below 40°C), and then flushed twice with 2-MeTHF (73 kg and 47 kg) to obtain a solution of 2 to 3 volumes. After dilution with 2-MeTHF (65 kg), 10% aqueous sodium sulfate solution (30 kg) was added, the mixture was cooled to 0 to 10°C, and then 16% aqueous NaOH solution (50 kg) was added to adjust the pH to 13 to 14. The temperature was adjusted to 15 to 25°C and stirred for 30 to 60 minutes. The aqueous layer was separated and extracted twice with 2-MeTHF (47 kg × 2). The combined organic layers were concentrated to 3 to 4 volumes under reduced pressure (below 40°C), and 2-MeTHF (950 g) was added. After concentration to 3 to 4 volumes under reduced pressure (below 40°C), the resulting solution was diluted with 2-MeTHF (30 kg), passed through 4A molecular sieves (25 kg) for drying, and washed with 2-MeTHF (30 kg). The final solution was concentrated to obtain the desired compound (6.7 kg) as an oily substance with an assay purity of 90.1% at a corrected yield of 79%.

[0194] Preparation of Intermediate 225 (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-dimethylhexan-1-amine

[0195]

Chemical formula

[0196] (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%.

[0197] 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

[0198] [ka]

[0199] 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).

[0200] Synthesis method A for compound 393

[0201] [ka]

[0202] 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.

[0203] 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

[0204] [ka]

[0205] (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%.

[0206] 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)

[0207] [ka]

[0208] Compound A3 A solution 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 (Compound A) (270 mg, 0.450 mmol) in 20 mL of ACN (20 mL) was added with oxalic acid (81.0 mg, 0.900 mmol). After the addition, the reaction mixture was stirred at room temperature for 1 hour. Then, the reaction mixture was concentrated, and the residue was redissolved in ACN and deionized water and freeze-dried to obtain the title compound (350 mg) as a white solid.

[0209] 1 H NMR (400 MHz, 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).

[0210] 1 H NMR (400 MHz, 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).

[0211] LCMS (ESI) (Method 2): R t = 1.969 min, m / z found 600.4 [M + H] + .

[0212] Synthesis of Example 5 - Compound A1

[0213]

Chem.

[0214] 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), to which EtOH (63 g) was added 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 20-25°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 20-30°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.)

[0215] 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).

[0216] 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).

[0217] LCMS (Method 7):R t = 0.669 minutes, measured value in m / z: 600.5 [M+H] + .

[0218] 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)

[0219] [ka]

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 1H NMR(600MHz,DMSO-d6)δ ppm 0.69(br s,2 H)0.82-0.98(m,9 H)1.07(br s,4 H)1.31-1.46(m,1 H)1.51(br d,J=2.91Hz,1 H)1.69(br d,J=3.45Hz,2 H)1.98(br s,1 H)2.06-2.45(m,2 H)2.77(br s,3 H)2.87-3.19(m,3 H)3.24(br s,1 H)3.31(s,6 H)3.64(br s,4 H)3.71-4.59(m,7 H)7.24-7.54(m,9 H)7.61(br d,J=7.27Hz,4H)8.45-8.60(m,1H)9.24(br s,1H)9.44-9.82(m,1H).

[0226] 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).

[0227] In the examples, compound A4 is a compound included in claim 1. Other compounds in the examples are for illustrative purposes only. Some intermediates (e.g., intermediate 234b) are claimed intermediates.

[0228] Analysis method used in the above experimental section The analytical information for the above compounds was generated using the analytical methods described below.

[0229] NMR method Several NMR experiments were performed using a Bruker Avance III 400 spectrometer at ambient temperature (298.6 K), with an internal deuterium lock and equipped with a BBO 400 MHz S1 5 mm probe head with a z gradient, operating at 400 MHz for protons and 100 MHz for carbon. Chemical shifts (δ) are reported in parts per million (ppm). J values ​​are expressed in Hz.

[0230] Several NMR experiments were performed at ambient temperature (298.6 K) using a Varian400-MR spectrometer with internal deuterium locking and equipped with a Varian400 4NUC PFG probe head with a z-gradient, operating at 400 MHz for protons and 100 MHz for carbon. Chemical shifts (δ) are reported in parts per million (ppm). J values ​​are expressed in Hz.

[0231] Several NMR experiments were performed using a Varian400-V NMRS spectrometer at ambient temperature (298.6 K), with an internal deuterium lock and equipped with a Varian400 ASW PFG probe head with a z-gradient, operating at 400 MHz for protons and 100 MHz for carbon. Chemical shifts (δ) are reported in parts per million (ppm). J values ​​are expressed in Hz.

[0232] Several NMR experiments were performed using a Bruker AVANCE III HD 300 spectrometer at ambient temperature (298.6 K), with internal deuterium locking and equipped with a PA BBO 300S1 BBF-HD-05 Z 5 mm probe head with a z gradient, operating at 300 MHz for protons and 75 MHz for carbon. Chemical shifts (d) are reported in parts per million (ppm). J values ​​are expressed in Hz.

[0233] LCMS (Liquid Chromatography / Mass Spectrometry) General procedure High-performance liquid chromatography (HPLC) measurements were performed using the LC pump, diode array (DAD), or UV detector and column specified for each method. Additional detectors were included where necessary (see Table 2 below).

[0234] The flow from the column was delivered to a mass spectrometer (MS) equipped with an atmospheric pressure ion source. Setting adjustment parameters (e.g., scan range, residence time, etc.) to obtain ions that enable the identification of the nominal monoisotopic molecular weight (MW) of the compound is within the scope of the skills of those skilled in the art. Data acquisition was performed using appropriate software.

[0235] The compounds are measured by their experimental retention time (R t ) and ions are described. Unless otherwise specified in the data table, the reported molecular ions are [M+H] + (protonated molecule) and / or [MH] - This corresponds to a (deprotonated molecule). If the compound cannot be directly ionized, the type of adduct is specified (i.e., [M+NH4]). + [M+HCOO] - (etc.). For molecules with multiple isotopic patterns (Br, Cl), the reported values ​​are those obtained with respect to the lowest isotopic mass. All results are obtained with experimental uncertainty generally associated with the methods used.

[0236] Below, "SQD" refers to a single quadrupole detector, "RT" to room temperature, "BEH" to a cross-linked ethylsiloxane / silica hybrid, "HSS" to high-intensity silica, and "DAD" to a diode array detector.

[0237] [Table 2]

[0238] Analysis SFC General Procedure of the SFC Method SFC measurements were performed using an analytical supercritical fluid chromatography (SFC) system consisting of a binary pump and reformer for delivering carbon dioxide (CO2), an autosampler, a column oven, and a diode array detector with a high-pressure flow cell capable of withstanding up to 400 bar. Details of the analytical SFC are shown in Table 3 below. When configured with a mass spectrometer (MS), the flow from the column was fed into the MS. Setting adjustment parameters (e.g., scanning range, residence time, etc.) to obtain ions that enable the identification of the nominal monoisotopic molecular weight (MW) of the compound is within the knowledge of those skilled in the art. Data acquisition was performed using appropriate software.

[0239] [Table 3]

[0240] Crystal form intermediate 234b The crystalline intermediate 234b can be characterized by its X-ray powder diffraction pattern.

[0241] X-ray powder diffraction (XRPD) analysis was performed using a PANalytical Aeris 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.

[0242] The samples were subjected to XRPD using the following method: Tube:Cu:K-α(λ=1.541874Å) Generator: Voltage: 45kV Current: 15mA Geometry: Bragg-Brentano Scanning method: Continuous scanning Scanning range: 4-50 degrees Step size: 0.0217 degrees Counting time: 58s Spinner rotation time: 1 second Incident beam path (iCore) Divergence slit: 1 / 4° Solar slit: 0.04rad Mask 1: 9mm Diffraction beam path (dCore) Scatter prevention slit: 9mm Irradiation length: 10mm Solar slit: 0.04rad Detector: PIXcel3D-Medipix3 1x1

[0243] Those skilled in the art will recognize that the diffraction pattern and peak positions are typically substantially independent of the diffractometer used, and whether or not a specific calibration method is employed. Typically, peak positions may differ by only about ±0.2°² theta or less. The intensity (and relative intensity) of each particular diffraction peak may also vary as a function of various factors, including, but not limited to, particle size, orientation, and sample purity.

[0244] The X-ray powder diffraction pattern includes peaks at 5.82, 10.09, and 18.42 degrees (2-theta ± 0.2 degrees).

[0245] The X-ray powder diffraction pattern includes peaks at 5.82, 8.52, 9.20, 10.09, 11.43, 13.61, 14.94, 15.89, 17.03, and 18.42 degrees, with a 2-theta ± 0.2 degrees.

[0246] Intermediate 234b can be further characterized by an X-ray powder diffraction pattern having four, five, six, seven, eight, nine or more peaks selected from those peaks.

[0247] Intermediate 234b can be further characterized substantially by the X-ray powder diffraction pattern shown in Figure 4.

[0248] 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.

[0249] 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.

[0250] The samples were 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 Divergent 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

[0251] Those skilled in the art will recognize that the diffraction pattern and peak positions are typically substantially independent of the diffractometer used, and whether or not a specific calibration method is employed. Typically, peak positions may differ by only about ±0.2°² theta or less. The intensity (and relative intensity) of each particular diffraction peak may also vary as a function of various factors, including, but not limited to, particle size, orientation, and sample purity.

[0252] 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.

[0253] 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 4.

[0254] Morphology A can be further characterized by an X-ray powder diffraction pattern containing the peaks identified in Table 4, 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.

[0255] Morphology A can be further characterized by the X-ray powder diffraction pattern substantially shown in Figure 1.

[0256] Table 4 shows the peak list and relative intensities for XRPD 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)benzamidebisbesylate hydrate (Figure 1).

[0257] [Table 4]

[0258] Pharmacology The compounds of the present invention have been found to block the interaction between menin and MLL proteins and oncogenic MLL fusion proteins. Therefore, the compounds of the present invention and pharmaceutical compositions containing such compounds may be useful in the treatment or prevention, particularly in the treatment, of diseases such as cancers, including, but not limited to, leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0259] In particular, the compounds and pharmaceutical compositions according to the present invention may be useful for the treatment or prevention of cancer. According to one embodiment, cancers that may benefit from treatment with the menin / MLL inhibitor of the present invention include leukemia, lymphoma, myeloma, or solid tumor cancers (e.g., prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma, and glioblastoma). In some embodiments, leukemia includes 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, and leukemia exhibiting a HOX / MEIS1 gene expression signature.

[0260] In particular, the compounds and pharmaceutical compositions according to the present invention may be useful for the treatment or prevention of myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN).

[0261] In particular, the compounds and pharmaceutical compositions according to the present invention may be useful for the treatment or prevention of leukemia, especially nucleophosmin (NPM1) variant leukemia, such as NPM1c.

[0262] In particular, the compounds and pharmaceutical compositions according to the present invention are for AML, especially nucleophosmin (NPM1) mutant AML (i.e., NPM1 mut More specifically, it may be useful in the treatment or prevention of abstract NPM1 mutations in AML.

[0263] In particular, the compounds and pharmaceutical compositions according to the present invention may be useful for the treatment or prevention of MLL rearrangement leukemia, especially MLL rearrangement AML or ALL.

[0264] In particular, the compounds and pharmaceutical compositions according to the present invention may be useful for the treatment or prevention of leukemia accompanied by MLL gene mutations, especially AML or ALL accompanied by MLL gene mutations.

[0265] In particular, the compounds and pharmaceutical compositions according to the present invention may be suitable for QD (once-daily) administration.

[0266] In particular, the compounds and pharmaceutical compositions according to the present invention are used to treat NPM1 gene mutations and / or mixed lineage leukemia gene (MLL, MLL1, KMT2A) alterations, mixed type leukemia (MLL), MLL-associated leukemia, MLL-positive leukemia, MLL-induced leukemia, reconstituted mixed type leukemia, MLL-related leukemia, MLL gene reconstitution / alteration or reconstitution / alteration, acute leukemia, chronic leukemia, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), insulin resistance, prediabetes, and glucose It may be useful in the treatment or prevention of blood cancers in subjects exhibiting risk of diabetes, hyperglycemia, chromosomal rearrangement on chromosome 11q23, type 1 diabetes, type 2 diabetes, and it promotes the proliferation of pancreatic cells, where pancreatic cells are beta cells, which are islet cells, and beta cell proliferation is demonstrated by increased beta cell production or insulin production, and it is intended to inhibit the menin-MLL interaction, with the MLL fusion protein target gene being HOX or MEIS1 in humans.

[0267] Therefore, the present invention relates to the compound of the present invention for use as a pharmaceutical product.

[0268] The present invention also relates to the use of the compounds of the present invention for the manufacture of pharmaceuticals.

[0269] The present invention also relates to compounds or pharmaceutical compositions according to the present invention for use in the treatment, prevention, improvement, control or reduction of the risk of disorders associated with the interaction between menin and MLL proteins and oncogenic MLL fusion proteins in mammals, including humans, wherein the treatment or prevention is affected or promoted by blocking the interaction between menin and MLL proteins and oncogenic MLL fusion proteins.

[0270] Furthermore, the present invention relates to the use of compounds according to the present invention for the manufacture of pharmaceuticals for the treatment, prevention, improvement, control or reduction of risk of disorders related to the interaction between menin and MLL proteins and oncogenic MLL fusion proteins in mammals, including humans, wherein the treatment or prevention is affected or promoted by blocking the interaction between menin and MLL proteins and oncogenic MLL fusion proteins.

[0271] The present invention also relates to compounds according to the present invention for use in the treatment or prevention of any one of the aforementioned diseases.

[0272] The present invention also relates to compounds according to the present invention for use in treating or preventing any one of the aforementioned diseases.

[0273] The present invention also relates to the use of compounds according to the present invention for the manufacture of pharmaceuticals for treating or preventing any one of the aforementioned disease conditions.

[0274] The compounds of the present invention can be administered to mammals, preferably humans, for the treatment or prevention of any one of the aforementioned diseases.

[0275] Taking into consideration the usefulness of the compounds according to the present invention, a method for treating warm-blooded animals, including humans, suffering from any one of the aforementioned diseases is provided.

[0276] The above method includes administering a therapeutically effective amount of the compound according to the present invention to a warm-blooded animal, including a human, i.e., systemic or local administration.

[0277] Accordingly, the present invention also relates to a method for treating or preventing any of the aforementioned diseases, comprising administering a therapeutically effective amount of the compound according to the present invention to a patient in need thereof.

[0278] Those skilled in the art will understand that a therapeutically effective dose of the compound of the present invention is sufficient to have therapeutic activity, and that this dose varies, among other things, depending on the type of disease, the concentration of the compound in the therapeutic formulation, and the patient's condition. The effective daily therapeutic dose may be about 0.005 mg / kg to 100 mg / kg. The amount of the compound of the present invention, also referred to herein as the active ingredient, required to achieve a therapeutic effect may vary on a case-by-case basis, for example, depending on the specific compound, the route of administration, the age and condition of the recipient, and the specific disorder or disease being treated. The treatment method may also involve administering the active ingredient in a regimen of 1 to 4 doses per day. In these treatment methods, the compound of the present invention is preferably formulated before administration.

[0279] The present invention also provides compositions for preventing or treating disorders referred to herein. The compositions comprise a therapeutically effective amount of a compound according to the present invention and a pharmaceutically acceptable carrier or diluent.

[0280] While the active ingredient can be administered alone, it is preferable to provide it as part of a pharmaceutical composition. Therefore, the present invention further provides a pharmaceutical composition comprising the compound according to the present invention together with a pharmaceutically acceptable carrier or diluent. The carrier or diluent must be "acceptable" in the sense that it is compatible with the other components of the composition and is not harmful to its recipient.

[0281] For example, the pharmaceutical composition is described in Gennaro et al. Remington's Pharmaceutical Sciences (18 th They can be prepared by any method well known in the field of pharmacy, such as those described in (see, in particular, Part 8: Pharmaceutical preparations and their Manufacture) of Mack Publishing Company, 1990.

[0282] The compounds of the present invention can be used alone or in combination with one or more additional therapeutic agents. Combination therapy includes administering a single drug formulation containing the compounds of the present invention and one or more additional therapeutic agents, as well as administering the compounds of the present invention and each additional therapeutic agent in their own separate drug formulations.

[0283] Accordingly, one embodiment of the present invention relates to a product comprising a compound according to the present invention as a first active ingredient and one or more anticancer agents as further active ingredients, as a combination preparation for simultaneous, separate, or sequential use in the treatment of a patient suffering from cancer.

[0284] One or more other drugs and compounds according to the present invention may be administered simultaneously (e.g., separately or in a single composition) or sequentially in any order. In the latter case, the two or more compounds are administered for a period of time and in a quantity and manner sufficient to ensure that a favorable or synergistic effect is achieved. It will be understood that the preferred method and order of administration, as well as the respective dosages and regimens, for each component of the combination will depend on the specific other drugs and compounds of the present invention being administered, their routes of administration, the specific condition being treated, particularly tumors, and the specific host being treated.

[0285] Pharmacology Test The following pharmacological tests describe the following compound: 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, Compound A1: (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.2HCl.xH2O(x=2~3), Compound A3: (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 salt. 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)-1,2,4triazine-6-yl)oxy)benzamidebisbesylate hydrate, crystalline form A The results from these pharmacological tests clearly demonstrate the biological activity 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.

[0286] 1) Menin / MLL homogeneous time-resolved fluorescence (HTRF) assay To an untreated white 384-well microtiter plate, 40 nL of the 200X test compound in DMSO and 4 μL of 2X terbium chelate-labeled menine (see below for preparation) in assay buffer (40 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1 mM DTT (dithiothreitol), and 0.05% Pluronic F-127) were added. After incubating the test compound and terbium chelate-labeled menine at ambient temperature for 30 minutes, 4 μL of 2X FITC-MBM 1 peptide (FITC-β-alanine-SARWRFPARPGT-NH2) ("FITC" stands for fluorescein isothiocyanate) in assay buffer was added, the microtiter plate was centrifuged at 1000 rpm for 1 minute, and the assay mixture was incubated at ambient temperature for 15 minutes. The relative amount of menine FITC-MBM1 complex present in the assay mixture is determined by measuring the homogeneous time-resolved fluorescence (HTRF) of the terbium / FITC donor / acceptor fluorophore pair using an EnVision microplate reader (excitation 337 nm / terbium fluorescence 490 nm / FITC fluorescence 520 nm) at ambient temperature. The degree of fluorescence resonance energy transfer (HTRF value) is the ratio of the fluorescence emission intensity of FITC and terbium fluorophores (F em 520nm / F em It is expressed as 490 nm. The final reagent concentration in the binding assay is 200 pM terbium chelate-labeled menine, 75 nM FITC-MBM1 peptide, and 0.5% DMSO in assay buffer. Dose-response titration of the test compound is performed using an 11-point, 4-fold serial dilution scheme, typically starting at 10 μM.

[0287] The effectiveness of the compounds was first investigated by calculating the inhibition percentage at each compound concentration according to Equation 1. Inhibition % = ((HC-LC)-(HTRF) 化合物 -LC)) / (HC-LC)) * 100 (formula 1) In the formula, LC and HC are the HTRF values ​​of the assay in the presence or absence of a saturation concentration of a compound that competes with FITC-MBM1 for binding to menine, and HTRF化合物 This is the measured HTRF value in the presence of the test compound. The HC and LC HTRF values ​​represent the average of at least 10 replicates per plate. For each test compound, the inhibition % value is plotted against the logarithm of the test compound concentration, and the IC obtained by fitting these data to Equation 2 is obtained. 50 The values ​​were plotted. Inhibition%=Bottom+(Top-Bottom) / (1+10^((logIC 50 -log[cmpd]) * h))(Formula 2) In the formula, Bottom and Top are the lower and upper asymptotes of the dose-response curve, respectively, and IC 50 is the concentration of the compound that causes 50% inhibition of the signal, and h is the Hill coefficient.

[0288] Preparation of terbium cryptate labeling of menine: Menine (a.a1-610-6xhis tag, 2.3 mg / mL in 20 mM Hepes (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), 80 mM NaCl, 5 mM DTT (dithiothreitol), pH 7.5) was labeled with terbium cryptate as follows: 200 μg of menine was buffer-exchanged with 1 × Hepes buffer. 6.67 μM menine was incubated with an 8-fold molar excess of NHS (N-hydroxysuccinimide)-terbium cryptate at room temperature for 40 minutes. Half of the labeled protein was purified from the free-labeled protein by performing the reaction on an NAP5 column using elution buffer (0.1 M Hepes, pH 7 + 0.1% BSA (bovine serum albumin)). The remaining half was eluted with 0.1 M phosphate-buffered saline (PBS), pH 7. 400 μl of each eluent was collected, divided equally, and frozen at -80°C. The final concentrations of terbium-labeled menin protein were 115 μg / mL in Hepes buffer and 85 μg / mL in PBS buffer, respectively.

[0289] Menin protein sequence (SEQ ID NO: 1): MGLKAAQKTLFPLRSIDDVVRLFAAELGREEPDLVLLSLVLGFVEHFLAVNRVIPTNVPELTFQPSPAPDPPGGLTYFPVADLSIIAALYARFTAQIRGAVDLSLYPREGGVSSRELVKKVSDVIWNSLSRSYFKDRAHIQSLFSFITGTKLDS SGVAFAVVGACQALGLRDVHLALSEDHAWVVFGPNGEQTAEVTWHGKGNEDRRGQTVNAGVAERSWLYLKGSYMRCDRKMEVAFMVCAINPSIDLHTDSLELLQLQQKLLWLLYDLGHLERYPMALGNLADLEELEPTPGRPDPLTLYHKGIAS AKTYYRDEHIYPYMYLAGYHCRNRNVREALQAWADTATVIQDYNYCREDEEIYKEFFEVANDVIPNLLKEAASLLEAGEERPGEQSQGTQSQGSALQDPECFAHLLRFYDGICKWEEGSPTPVLHVGWATFLVQSLGRFEGQVRQKVRIVSREA EAAEAEEPWGEEAREGRRRGPRRESKPEEPPPPKKPALDKGLGTGQGAVSGPPRKPPGTVAGTARGPEGGSTAQVPAPAASPPPEGPVLTFQSEKMKGMKELLVATKINSSAIKLQLTAQSQVQMKKQKVSTPSDYTLSFLKRQRKGLHHHHHH

[0290] 2a) Growth assay The antiproliferative effects of menin / MLL protein / protein interaction inhibitor test compounds were evaluated in human leukemia cell lines. The cell line MOLM-14 possesses an MLL translocation and expresses the MLL fusion protein MLL-AF9 and the wild-type protein derived from the second allele, respectively. OCI-AML3 cells with NPM1c gene mutations were also tested. MLL-reconstituted cell lines (e.g., MOLM-14) and NPM1c mutant cell lines exhibit stem cell-like HOXA / MEIS1 gene expression signatures. To exclude compounds exhibiting general cytotoxic effects, KO-52 was used as a control cell line containing two MLL(KMT2A) wild-type alleles.

[0291] MOLM-14 cells were cultured in RPMI-1640 (Sigma Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (HyClone), 2 mM L-glutamine (Sigma Aldrich), and 50 μg / mL gentamicin (Gibco). KO-52 and OCI-AML3 cell lines were grown in alpha-MEM (Sigma Aldrich) supplemented with 20% heat-inactivated fetal bovine serum (HyClone), 2 mM L-glutamine (Sigma Aldrich), and 50 μg / mL gentamicin (Gibco). Cells were maintained at 0.3 to 2.5 million cells / mL during culture, and the number of passages did not exceed 20.

[0292] To evaluate the antiproliferative effect, 200 MOLM-14 cells, 200 OCI-AML3 cells, or 300 KO-52 cells were seeded in 200 μl of medium / well in 96-well round-bottom ultra-low adhesion plates (Costar, catalog no. 7007). Cell seeding numbers were selected based on the growth curve to ensure linear growth throughout the experiment. The test compound was added at different concentrations, and the DMSO content was normalized to 0.3%. Cells were incubated at 37°C and 5% CO2 for 8 days. Spheroid-like growth was measured in real time by live-cell imaging (IncuCyteZOOM, Essenbio, 4x objective lens) with images acquired on day 8. Confluence (%) as a measure of spheroid size was determined using an integrated analysis tool.

[0293] To determine the effect of the test compound over time, confluence was calculated as a measure of spheroid size in each well. The confluence of the highest dose of the reference compound was used as the baseline (low control) for LC, and the confluence of DMSO-treated cells was used as 0% cytotoxicity (high control, HC).

[0294] Absolute IC 50 The value was calculated as a percentage change in confluence, as follows: LC = Low control: e.g., cells treated with 1 μM of the cytotoxic agent staurosporine, or e.g., cells treated with a high concentration of an alternative reference compound; HC = High Control: Average Confluence (%) (DMSO-treated cells); Effect % = 100 - (100 * (sample-LC) / (HC-LC)); and Using GraphPad Prism (version 7.00) 50 The following calculations were performed. The dose-response equation was used with a variable gradient, with the maximum value fixed at 100% and the minimum value fixed at 0%, to plot the effect % versus Log10 compound concentration.

[0295] 2b) MEIS1 mRNA expression assay MEIS1 mRNA expression during compound treatment was investigated using the Quantigene Singleplex assay (Thermo Fisher Scientific). This technique allows for direct quantification of mRNA targets using probes that hybridize to a defined target sequence, and the signal is detected using the multimode plate reader Envision (PerkinElmer). The MOLM-14 cell line was used in this experiment. Cells were seeded at 3,750 cells / well in 96-well plates in the presence of increasing concentrations of the compound. After incubation with the compound for 48 hours, the cells were lysed in lysis buffer and incubated at 55°C for 45 minutes. The cell lysates were mixed with a human MEIS1-specific capture probe or a human RPL28 (ribosomal protein L28)-specific probe as a normalization control, as well as a blocking probe. The cell lysates were then transferred to a custom assay hybridization plate (Thermo Fisher Scientific) and incubated at 55°C for 18–22 hours. After washing the plate to remove unbound material, the pre-amplifier, amplification agent, and labeled probe were added sequentially. The signal (=gene count) was measured using the Envision multimode plate reader. 50The response was calculated using dose-response modeling with appropriate software. For all non-housekeeper genes, the response was equal to the count corrected for background and relative expression. For each sample, the signal for each test gene (with background subtracted) was divided by the normalized gene signal (RPL28: with background subtracted). The fold change was calculated by dividing the normalized value of the treated sample by the normalized value of the DMSO-treated sample. The fold change for each target gene was calculated using IC. 50 It was used in the calculation.

[0296] The results are summarized in Table 5 below.

[0297] [Table 5]

[0298] 3) Mouse PK (In vivo T) 1 / 2 (and oral bioavailability) The in vivo pharmacokinetics (PK) of the test substance, formulated in HP-β-CD 20% (w:vol) solution or pyrogen-free water, were evaluated after intravenous administration (0.5 or 1.0 mg / kg intravenously at 2.5 mL / kg) or oral administration (5 mg / kg orally at 10 mL solution / kg) to fasted male CD-1 mice (6-8 weeks old).

[0299] Using EDTA as an anticoagulant, plasma and / or whole blood samples were collected from the dorsal metatarsal vein at desired time points by continuous capillary microsampling (approximately 0.03 mL). The concentrations of compounds in the plasma and blood samples were analyzed using a qualified LC-MS / MS method. In silico analysis of key pharmacokinetic parameters was performed using WinNonlin (Phoenix®, version 6.1) or similar software.

[0300] 4) Metabolic stability in human / mouse liver microsomes Experimental Procedure The objective of this experiment is to measure the in vitro metabolic stability of the test compound in human and mouse liver microsomes and to provide quantitative information regarding turnover rates (i.e., to determine the apparent intrinsic clearance of the test).

[0301] Test parameters were prepared using a stock concentration of 10 mM DMSO. To investigate metabolic turnover, the final working solution was prepared by adding 2 μL of 10 mM DMSO stock solution for the test compound or positive control compound to 198 μL of acetonitrile (final concentration 100 μM).

[0302] The incubation was performed as follows: First, liver microsomes were thawed on ice, and a pH 7.4 master solution containing liver microsomes in 100 mM PBS (phosphate-buffered saline) was prepared. Next, the liver microsome solution was added to the incubation plate, and 10 mM NADPH (nicotinamide adenine dinucleotide phosphate) was added (MW: 833.4 g / mol, Roche Diagnostics GmbH, Germany, dissolved in phosphate buffer (100 mmol / L, pH 7.4)). The mixture was mixed for 10 seconds and the incubation plate was preheated at 37°C for 10 minutes. 5 μL of a 100 μM working solution of the test compound or positive control compound was added to the incubation plate to initiate the metabolic reaction (concentration of the final test item = 1 μM). The final reaction mixture must contain 1 mM NADPH, 0.5 mg / mL microsomal protein, and 1 μM of the test compound or positive control compound in 100 mM PBS at pH 7.4. The percentage of organic solvent in the incubation mixture is 1%, and DMSO ≤ 0.02%.

[0303] The reaction was quenched by transferring 50 μL of the incubated mixture at the selected time point to a quench plate containing 200 μL of cold methanol. After sampling at all time points, the quench plate was centrifuged at 4000 rpm for 40 minutes to precipitate the protein. A total of 90 μL of supernatant was transferred to an analysis plate, and ultrapure H2O water was added to each well for LC / MS / MS analysis. All incubation and analysis were performed in a double-row system.

[0304] Data Analysis All calculations were performed using Microsoft Excel. The gradient value k was determined by linear regression of the natural logarithm of the residual percentage of the parent drug versus the incubation time curve. The results are summarized in Table 6 below.

[0305] In vitro half-life (in vitro t 1 / 2 ) was calculated from the gradient value. In Vitro T 1 / 2 =-(0.693 / k) In Vitro T 1 / 2 (minutes) in vitro specific clearance (in vitro CL) int The conversion to μL / min / mg protein units was performed using the following formula.

[0306]

number

[0307] [Table 6] "NA" means that it was not analyzed.

[0308] 5) Protocols for pharmacodynamic (PD) activity in subcutaneous (sc or SC) xenografts of MOLM-14 or OCI-AML3 cells Test drug and control Compound A3 was formulated as 20% hydroxypropyl-β-cyclodextrin (HP-β-CD), and the amount was prepared to reach a total volume of 0.2 mL (10 mL / kg) per 20 g of animal. The dose was adjusted daily according to the individual body weight. A working stock of compound A3 was prepared once a week for each experiment and stored at room temperature. Compound A3 was administered orally (PO) daily.

[0309] Assay The in vivo pharmacodynamic (PD) activity of the compounds was evaluated in subcutaneous (SC) xenografts of MOLM-14 or OCI-AML3 cells. Nude NMRI mice (Crl:NMRI-Foxn1nu / -) with MOLM-14 or OCI-AML3 tumors were treated with the vehicle or compound three times daily. Plasma samples were collected 23 hours after the second dose, 0.5 hours after the final dose, and 16 hours after the final dose, while tumor samples were collected 16 hours after the final dose. QuantiGene Plex technology (Thermo Fisher Scientific) was used to investigate the effects of the compounds on the expression of multiple menin-MLL target genes (e.g., MEIS1, MEF2C, FLT3). Frozen tumors were homogenized and transferred to individual lysis matrix tubes in lysis buffer and incubated at 55°C for 30 minutes. Cell lysates were mixed with target-specific capture probes, Luminex beads, and blocking probes, transferred to a custom assay hybridization plate (Thermo Fisher Scientific), and incubated at 54°C for 18–22 hours. The plates were then transferred to a magnetic separation plate, washed to remove unbound material from the beads, followed by sequential hybridization with a preamplifier, amplification agent, and labeled probe, and subsequent streptavidin phycoerythrin binding. Signals from the beads were measured using a Luminex FlexMap 3D instrument. For all non-housekeeper genes, the response was equal to the count corrected for background and relative expression. For each sample, the signal of each test gene (subtracting background) was divided by the normalized gene signal (RPL19, RPL28, ATP6V1A, subtracting background). The fold change was calculated by dividing the normalized values ​​of the treated samples by the normalized values ​​of the DMSO-treated samples. The results are summarized in Tables 7 and 8 below.

[0310] [Table 7]

[0311] [Table 8]

[0312] Tables 7a and 8a show the median values ​​based on repeated experiments under optimized conditions using fresh tumor samples.

[0313] [Table 9]

[0314] [Table 10]

[0315] 6) Efficacy experiments in a MOLM-14 subcutaneous model Test drug and control Compound A3 was formulated into 20% hydroxypropyl-β-cyclodextrin (HP-β-CD), and the amount was prepared to reach a total volume of 0.2 mL (10 mL / kg) per 20 g of animal. The dose was adjusted daily according to the individual body weight. Working stocks of compound A3 were prepared once a week for each experiment and stored at 25°C.

[0316] animal Female NMRI nude mice (MOLM-14 SC) were used when they were approximately 6 to 8 weeks old and weighed approximately 25 g. All animals were able to acclimate to and recover from any shipment-related stress for at least 7 days prior to experimental use. Autoclaved water and irradiated food were freely available, and the animals were maintained in a 12-hour light-dark cycle. Cages, bedding, and water bottles were autoclaved before use and replaced weekly. Further details are shown in Table 9 below.

[0317] [Table 11]

[0318] Tumor models and cell culture methods Human AML cells MOLM-14 were cultured in the indicated complete culture medium (RPMI 1640 + 10% HI-FBS + 2 mM L-glutamine + 50 ug / mL gentamicin) at 37°C and 5% CO2. Cells were harvested during logarithmic growth and resuspended in cold (4°C) Roswell Park Memorial Institute (RPMI) 1640 in serum-free medium.

[0319] In each mouse, a total volume of 0.2 mL was injected into the right flank using a 1 cc syringe and a 27 gauge needle, containing 5 × 10⁴ units of 50% Matrigel. 6 Individual MOLM-14 cells were administered.

[0320] Experimental Design Compound A3 was administered orally (PO) daily.

[0321] Day 0 is the day when tumor cell transplantation and experiments begin.

[0322] Mice with SC MOLM-14 tumors were randomized 16 days after tumor transplantation, and tumor volume (average approximately 130 mm) was measured. 3 Participants were assigned to treatment groups according to the following criteria (n=10 / group). Treatment with the vehicle or compound A3 (30 and 100 mg / kg) was initiated on the same day and administered orally daily for 21 days. For pharmacokinetic (PK) analysis, plasma was collected 1, 2, 4, 8, and 23 hours after the last dose (n=4-5 / group / time point).

[0323] Animal monitoring SC tumor volume was measured for each animal two to three times or more per week throughout the experiment.

[0324] calculation The tumor volume is calculated using the following formula: Tumor volume (mm 3 ) = (D × d 2 In the formula / 2, "D" represents the larger diameter of the tumor measured by caliper, and "d" represents the smaller diameter. Tumor volume data was graphed as mean tumor volume ± SEM.

[0325] ΔTGI% is defined as the difference in mean tumor burden between the treatment group and the control group, and ΔTGI% = ([(TV c TVc0)(TV t TV t0 )] / (TV c TVc0))×100 (in the formula, “TV c " is the mean tumor burden of a given control group, and "TVc0" is the mean initial tumor burden of a given control group, and "TV t " is the average tumor burden of the treatment group, and "TV t0 (This was calculated as the mean initial tumor burden of the treatment group.) TGI% was defined as the difference between the two groups.

[0326] The mean tumor volume for the treatment group and the control group was calculated as follows.

[0327] TGI%=((TV c TV t ) / TV c ) × 100 (in the formula, “TV c " is the average tumor volume of the control group, and "TV t (where is the mean tumor volume of the treatment group). A TGI of 60% or more is considered biologically significant, as defined by the National Cancer Institute criteria.

[0328] Tumor regression (TR)% quantified to reflect the reduction in treatment-related tumor volume compared to baseline unrelated to the control group is defined as TR% = (1 mean (TV) t i / TV t0 i)) × 100 is calculated, and in the formula, "TV t "i" is the tumor burden of individual animals in the treatment group, and "TV t0 "i" represents the initial tumor burden in animals.

[0329] Data Analysis Tumor volume was graphed using Prism software (GraphPad version 7 or 8). Statistical significance for most experiments was assessed for the compound A3 treatment group compared to the HPβCD vehicle-treated control group on the final day of the experiment, when more than two-thirds of the mice remained in each group. A difference between groups was considered statistically significant when p ≤ 0.05.

[0330] Statistical significance of animal tumor volume was calculated using linear mixed-effects (LME) analysis in R software version 3.4.2 (using Janssen's internally developed Shiny application version 4.0), with treatment and time as fixed effects and animals as a random effect. Logarithmic transformation was performed if individual longitudinal response trajectories were not linear.

[0331] Using the information obtained from this model, we performed a comparison of tumor volume between the control group and all treatment groups. The results are shown in Figure 2.

[0332] 7) Ca 2+ ECGphysiological effects of test compounds on synchronized heartbeats in human pluripotent stem cell-derived cardiomyocytes (hSC-CM) using fluorescence assay (CTCM human). protocol The compounds were tested in 96-well plates.

[0333] The compound was tested in Cor.4U®-cardiomyocytes or iCell®-cardiomyocytes 2 at concentrations of 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM (n=4 / dose).

[0334] Alternatively, the compounds were tested primarily on iCell® cardiomyocytes 2 at concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM (n=4 / dose).

[0335] Positive control and negative control 3nM dofetilide 100 nM isoproterenol 100-300 nM nimodipine 3 μM cetirizine.

[0336] Vehicle control: Dimethyl sulfoxide (DMSO). Solution of the compound in DMSO or its solvent (final concentration of 0.1% DMSO, n=8).

[0337] Preparation of test substances and controls The tested compounds were dissolved in DMSO at 1000 times the intended concentration. A compound "mother plate" was prepared containing the test compound, as well as positive and negative controls, at 1000 times the final concentration. On the day of the experiment, these stock solutions were diluted to twice the intended concentration (in a round-bottom compound plate) using Tyrode (Sigma) supplemented with 10 mM HEPES (Gibco). The final DMSO concentration in the test solution and vehicle controls was 0.1%.

[0338] cell hSC-CM (Cor.4U® cardiomyocytes) were obtained from CDI (Ncardia, Germany). The cells were pre-plated and seeded into fibronectin-coated 96-well plates at a density suitable for monolayer formation, and maintained in a culture in a stage incubator (37°C, 5% CO2) according to the cell supplier's instructions.

[0339] We purchased a second lineage of hSC-derived cardiomyocytes called iCell® cardiomyocytes 2 from FUJIFILM Cellular Dynamics (USA). Experiments using the test drug are performed 5-7 days after the cells are plated to have a living, beating monolayer of hiPSC-derived cardiomyocytes. The beating monolayer in the 96-well plate is typically taken from two vials of frozen iCell® cardiomyocytes 2 (approximately 5 million cells / vial) and plated into three 96-well plates (approximately 50K / well).

[0340] Before the experiment began At least one hour before the start of the experiment, the normal cell medium was replaced with Tyrode solution containing calcium dye (see below).

[0341] Cal 520 dye (AAT Bioquest) was dissolved in 11 mL of Tyrode supplemented with 10 mM HEPES, warmed to 37°C, and then added to the cells.

[0342] 35 μl of cell culture medium was removed from each well and replaced with 35 μl of preheated Cal 520 dye solution. The cell plate was incubated at 37°C / 5% CO2 for 45 minutes. The cells were then incubated at 37°C for 5 minutes.

[0343] experiment Spontaneous electrical activity is recorded using Cal 520® (AAT Bioquest) calcium fluorescent dye signaling. This dye integrates total intracellular calcium activity across the wells. A bottle of Cal 520 dye (50 μg, MW: 1103 / mol) is dissolved in 50 μl of DMSO as a 0.9 mM stock solution. 50 μl of the dye stock solution is added to 10 mL of Triyodes solution to achieve a dye concentration of 4.5 μM. Subsequently, 35 μl of this dye solution is added to each well to achieve a final dye concentration of 1.58 μM. The current dye protocol for this CTCM human assay was recently established (Ivan Kopljar ​​et al, Journal of Pharmacological and toxicological methods 2018.91:80-86; Lu et al., Tox Sci 2019.170(2):345-356).

[0344] Fluorescence signal (Ca 2+ The transient phenomena (morphology) were measured using a functional drug screening system (FDSS / μCell, Hamamatsu, Japan), and the records were then analyzed offline using appropriate software, such as Notocord.

[0345] For the test run, the cell plate was loaded into FDSS / μCell, and Ca 2+Transient phenomena were measured for 4 minutes to confirm the synchronized beating of cardiomyocytes in each well. All 96 wells were measured simultaneously (sampling interval: 0.06 seconds, short exposure time: 10 ms, excitation wavelength 480 nm, emission wavelength 540 nm, FDSS / μCell heated to 37°C). If all showed synchronized beating, the 96-well plate was measured three times (to verify the synchronized beating of all 96 wells at baseline, wells that did not meet the pre-set criteria were excluded from the experiment and not treated with the compound). T=0: Control period (-5 to -1 minute) + compound addition, followed by 3 minutes. T=30: Measured 29-34 minutes after compound addition

[0346] During the compound addition process, 100 μl of each double-concentrated test solution was simultaneously pipetted into each well.

[0347] The data was analyzed offline using appropriate software, such as Notocord-Hem (version 4.3).

[0348] Ca 2+ The following parameters of the transient phenomenon morphology were measured.

[0349] Heart rate (BR) Ca 2+ Amplitude (Amp) of transient phenomena, -CTD 90 : Ca at 90% 2+ The duration of the transient phenomenon (the time it takes to reach 90% of the initial base value).

[0350] Various "arrhythmia-like" activities were also observed during the experiment. These include the following:

[0351] "Early after depolarization-like" (EAD-like) events (defined as "a very small peak in the transient waveform following the initial peak of the transient") "Ventricular tachycardia-like" (VT-like) events (defined as having a very fast heart rate), or "Ventricular fibrillation-like" (VF-like) events ("small amplitude, fast rate Ca with irregular and unmeasurable transient potentials") 2+ (Defined as "waveform") Cell "pulsation cessation" (Ca 2+ (No transient phenomena are observed.)

[0352] If compound-induced changes in calcium transient signals could not be analyzed by software, these signals were identified as BQL (Below Quality for Analysis).

[0353] Data Analysis Data measured from FDSS-μCell was copied for offline analysis, analyzed, and uploaded to SPEC-II (the inventors' operational management system) for further analysis. Values ​​of variables before and after compound administration were collected and transferred to an Excel workbook.

[0354] All values ​​(in actual units and as a percentage change from baseline) are expressed as medians (minimum and maximum values). The changes observed in each compound group relative to the corresponding baseline values ​​(in actual units) were compared to the changes in the solvent control group using the Wilcoxon-Mann-Whitney test. A two-tailed test was performed with Bonferroni correction for multiplicity adjustment. Since there were 10 treatment groups compared to the solvent group, an alpha level of 0.05 / 10 (0.005) was considered to reflect a statistically significant difference from the solvent group. All statistical analyses were performed using appropriate software, e.g., R software version 3.5.2.

[0355] Quality control of hiPSC-CM in plates: The plates were rejected if they did not meet the following criteria. Stable, regular heartbeat Amplitude of more than 500 relative units Heart rate between 25 and 80 beats / minute CTD (crash to desktop) of 300-800ms 90 .

[0356] In this experiment, the hiPSC-CM in the plate met the above criteria.

[0357] These parameters, combined with the incidence of arrhythmias or cardiac arrest, were used to calculate potential risk levels using a weighted scoring method (based on Kopljar ​​et al., Stem Cell Reports 2018.11, 1365-1377). This hazard score was calculated using CTD. 90 The hazard levels are calculated for each concentration by adding weighted points based on the tolerance interval (TI) for changes in pulse rate and amplitude (ΔΔ%) and the occurrence of pulsation arrest and early after-depolarization (EAD). As a result, one of four different hazard levels is generated for each concentration. This is done after incubation with the compound for 30 minutes. The hazard levels are as follows:

[0358] No danger: Level of impact or minor, insignificant changes within the vehicle.

[0359] Low risk: While there are meaningful effects, the risk of cardiac debt is likely low.

[0360] High risk: A relatively high risk to the heart.

[0361] Very high risk: Very high risk due to arrhythmia-like events (EADs).

[0362] The "hazard score" results identify the potential acute cardiac drug-induced effects at the free drug equivalent (since plasma proteins are not added to the wells). Hazard identification is evaluated using a scoring reference book called CTCM_Scoring_version1 (Kopljar ​​et al., Stem Cell Reports 2018.11:1365-1377), and the levels are indicated according to the following color system in Table 10.

[0363] [Table 12]

[0364] Ca measured with HiPSc-CM, listed above in different colors in the related table. 2+ Ranking of test compounds based on hazard score severity for transient phenomenon assays.

[0365] result iCell® cardiomyocyte 2 cell line was used. Positive controls and negative controls: All positive controls and negative controls The expected pharmacological effects were observed in this assay. The results are summarized in Tables 11 and 12 below.

[0366] [Table 13]

[0367] [Table 14]

[0368] For compound A1: At an effective dose of 30 mpk (mg / kg) in a mouse xenograft model, the CTCM human concentration versus free C2. max It can be estimated as follows: Margin CTCM human 10 μM vs. free Cmax > 16 (mouse, human) Margin CTCM human 30 μM vs. free Cmax > 45 (mouse, human).

[0369] 8) Membrane potassium current I in hERG-transfected cell lines Kr Effects on

[0370] [Table 15]

[0371] method Experiments were conducted using CHO cells that stably express the hERG potassium channel. The cells were grown in culture flasks at 37 °C and 5% CO2 in Ham's F12 medium supplemented with 10% heat-inactivated fetal bovine serum, hygromycin B (100 μg / mL), and geneticin (100 μg / mL). Cells were harvested for use in an automated patch clamp system to obtain a cell suspension of single cells, the QPatch (Sophion).

[0372] Solutions: The bath solution contained 145 mM NaCl, 4 mM KCl, 10 mM glucose, 10 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 2 mM CaCl2, and 1 mM MgCl2 (pH 7.4 with NaOH). The pipette solution contained 120 mM KCl, 10 mM EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid), 10 mM HEPES, 5.374 mM CaCl2, and 1.75 mM MgCl2 (pH 7.2 with KOH).

[0373] Patch clamp experiments were performed in the voltage-clamp mode, and whole-cell currents were recorded using an automated patch clamp assay utilizing the QPatch system (Sophion). The current signals were amplified, digitized, stored, and analyzed using QPatch assay software.

[0374] The holding potential was -8 mV. The hERG current (K + selective outward current) was measured as the maximum tail current at -40 mV after depolarization to +60 mV for 2 seconds. The pulse cycling rate was 15 seconds. A short pulse (90 ms) to -40 mV served as the baseline step for calculating the tail current amplitude. After establishing the whole-cell configuration and stabilization period, the solvent control (0.3% DMSO) was applied for 5 minutes, followed by the test substance at 3×10 -7 M, 3×10 -6 M, 10 -5 M and 3×10 -5The test was applied at four progressively increasing concentrations of M. Each concentration of the test substance was applied twice. The results for each concentration were measured as the average current of three consecutive voltage pulses after 5 minutes. To examine the degree of blockage, the residual current was compared to the vehicle pretreatment.

[0375] The concentration / response relationship was calculated by nonlinear least-squares fitting for each data point. The maximum half-inhibitory concentration (IC50) was calculated using a fitting routine.

[0376] Each compound was replicated on the same plate in at least 5 wells. The inhibition rate results are summarized in Table 13 below.

[0377] [Table 16]

[0378] 9) Efficacy experiments in a seed-seeding OCI-AML3 model Test drug and control Compound A3 was formulated into 20% hydroxypropyl-β-cyclodextrin (HP-β-CD), and prepared to reach a total volume of 0.2 mL (10 mL / kg) per 20 g of animal. The dose was adjusted daily according to the individual body weight. Working stocks of compound A3 were prepared once a week for each experiment and stored at 25°C.

[0379] animal Female SCID beige mice (CB17.Cg-PrkdcscidLystbg-J / Crl / -) were used at approximately 6-8 weeks of age and weighing approximately 25g. All animals were able to acclimate to and recover from any shipment-related stress for at least 7 days prior to experimental use. Autoclaved water and irradiated food were freely available, and the animals were maintained in a 12-hour light-dark cycle. Cages, bedding, and water bottles were autoclaved before use and replaced weekly. Tissue culture and cell injection reagents are summarized in Table 14 below.

[0380] [Table 17]

[0381] Tumor models and cell culture methods Human AML cell line OCI-AML3 was cultured in the indicated complete culture medium (MEM alpha + 20% HI-FBS (thermally inactivated fetal bovine serum) + 2 mM L-glutamine + 50 ug / mL gentamicin) at 37°C and 5% CO2. Cells were harvested during logarithmic growth and resuspended in cold (4°C) MEM alpha (Minimum Essential Medium) in serum-free medium.

[0382] For the disseminated OCI-AML3 model, each mouse was administered 5 × 10⁶ times by IV injection with a total volume of 0.2 mL using a 26 gauge needle. 5 The cells were administered.

[0383] Experimental Design Compound A3 was administered orally (PO) daily.

[0384] Day 0 is the day when tumor cell transplantation and experiments begin.

[0385] In the efficacy study, mice with IV OCI-AML3 xenograft tumors were randomly assigned to the treatment group 3 days after tumor cell engraftment. Treatment with the vehicle or compound A3 (at 30, 50, or 100 mg / kg) was initiated on the same day and administered daily for 28 days.

[0386] Animal monitoring Animals were monitored daily for clinical signs associated with either compound toxicity or tumor burden (i.e., hind limb paralysis, lethargy, etc.).

[0387] calculation For survival assessment, results were plotted as survival rate against days after tumor transplantation. Negative clinical signs and / or weight loss of ≥20% were used as surrogate endpoints for death. Median survival was determined using Kaplan-Meier survival analysis. The percentage increase in survival (ILS) was calculated as ((median survival in the treatment group - median survival in the control group) / median survival in the control group) × 100. Animals that failed to reach the surrogate endpoint due to adverse clinical signs (e.g., ulcerated tumor, weight loss, etc.) or deaths unrelated to treatment were excluded for survival assessment. An ILS of ≥25% was considered biologically significant, as defined by NCI criteria. (Johnson JI et al. Br J Cancer. 2001. 84(10), 1424-1431).

[0388] Data Analysis Survival and weight data were graphed using Prism (version 7). Statistical significance for weight was assessed as described above. Statistical significance was evaluated using the log-rank (Mantel-Cox) test in R software version 3.4.2 for Kaplan-Meier survival plots comparing the treatment group versus the appropriate vehicle-treated control group. A p-value of ≤0.05 was considered statistically significant for the difference between groups.

[0389] survival The Kaplan-Meier survival curves are shown in Figure 3. Mice with established OCI-AML3 tumors were orally administered compound A3 at 30, 50, and 100 mg / kg in a 20% HP-β-CD formulation daily for a total of 28 days (n=9-10 / group). Compared to the vehicle-treated control group, the median survival time in the compound A3 treatment groups was 75.5 days at 30 mg / kg, 58.5 days at 50 mg / kg, and 75 days at 100 mg / kg. Treatment with compound A3 statistically significantly increased the lifespan of OCI-AML3 tumor-bearing mice by 96.1%, 51.9%, and 94.8% (at 30, 50, and 100 mg / kg dose levels) compared to the lifespan of control mice (p≦0.001). This was a biologically significant ILS based on the NCI threshold for ILS of 25% or more (Johnson JI et al. Br J Cancer. 2001. 84(10), 1424-1431).

[0390] Stability data Stability experiments were conducted on 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 bisbesilate hydrate. The bisbesilate hydrate was found to be chemically and physically stable, with no degradation observed by UHPLC and no change in solid state observed by XRD under the evaluated stress conditions.

[0391] [Table 18] The present invention includes the following embodiments. [Claim 1] (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 [ka] Or its solvate. [Claim 2] The compound according to claim 1, wherein the solvate is a hydrate. [Claim 3] The compound is 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)-1,2,4-triazine-6-yl)oxy)benzamidebisbesylate hydrate, The compound according to claim 1, wherein the crystalline form generates an X-ray powder diffraction pattern including peaks at 5.4, 7.2, 11.1, 11.9, and 21.7 degrees with a 2-theta ± 0.2 degrees. [Claim 4] The crystalline morphology according to claim 3, wherein the X-ray powder diffraction pattern may further include at least one peak selected from 2 theta ± 0.2 degrees at 13.7, 14.5, 14.7, 15.0, 16.5, 17.8, 19.0, 19.4, and 2 theta at 20.1 degrees. [Claim 5] The crystalline form according to claim 3 or claim 4, further characterized by the X-ray powder diffraction pattern substantially shown in Figure 1. [Claim 6] A pharmaceutical composition comprising a compound according to any one of claims 1 to 5, and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent. [Claim 7] A process for preparing the pharmaceutical composition according to claim 6, comprising mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of the compound according to any one of claims 1 to 5. [Claim 8] A compound according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 6, for use as a pharmaceutical. [Claim 9] A compound according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 6, for use in the prevention or treatment of cancer. [Claim 10] A compound according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 6, for use in the prevention or treatment of leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasm (MPN). [Claim 11] The compound or pharmaceutical composition according to claim 10 for use in the prevention or treatment of leukemia, wherein the leukemia is (NPM1) variant leukemia. [Claim 12] The compound or pharmaceutical composition for use according to claim 9, wherein the cancer is selected from leukemia, lymphoma, myeloma, or solid tumor cancer, such as prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma, and glioblastoma. [Claim 13] A compound or pharmaceutical composition according to claim 10 for use in the prevention or treatment of leukemia, wherein the leukemia is selected from acute leukemia, chronic leukemia, myeloid leukemia, myeloid leukemia, lymphoblastic leukemia, lymphocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), T-cell pre-lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, hairy cell leukemia (HCL), MLL rearrangement leukemia, MLL-PTD leukemia, MLL amplification leukemia, MLL-positive leukemia, and leukemia exhibiting a HOX / MEIS1 gene expression signature. [Claim 14] A method for treating or preventing a disorder selected from cancer, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 to a subject in need of treatment or prevention. [Claim 15] A process for preparing the crystalline form according to any one of claims 3 to 5, comprising the step of recrystallizing compound A, wherein the recrystallization is a) Adding compound A or its hydrate or solvate to a suitable solvent mixture in the presence of benzenesulfonic acid and adjusting the temperature to a range from approximately 20°C to the solvent reflux temperature, b) A step of sowing crystal form A, c) A process comprising the step of obtaining a precipitate in the crystalline form described in any one of claims 3 to 5. [Claim 16] The process according to claim 15, wherein the suitable solvent mixture is a mixture of acetone, water, and IPAc. [Claim 17] The process according to claim 15, wherein the suitable solvent mixture is a mixture of isopropanol, water, and IPAc. [Claim 18] The process according to claim 15, claim 16, or claim 17, wherein the temperature is approximately 25°C. [Claim 19]

change

change

Claims

1. The following formula: 【Chemistry 1】 (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 or its solvate.

2. The besilate salt or solvate thereof according to Claim 1, which is a solvate of a besilate salt.

3. The besilate salt or solvate thereof according to Claim 2, which is a hydrate of a besilate salt.

4. The besilate salt or solvate thereof according to claim 1, wherein the besilate salt is a bisbesilate salt.

5. The besilate salt or solvate thereof according to claim 4, which is a solvate of a bisbesilate salt.

6. The besilate salt or solvate thereof according to claim 5, which is a bisbesilate salt hydrate.

7. The besilate salt according to claim 1 or a solvate thereof, wherein the besilate salt is a besilate salt.

8. The besilate salt or solvate thereof according to claim 1, which is a bisbesilate salt.

9. A crystal 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)benzamidebisbesylate hydrate in crystalline form A, A crystal of crystal form A, wherein the crystal form generates an X-ray powder diffraction pattern containing peaks at 5.4, 7.2, 11.1, 11.9, and 21.7 degrees with a 2-theta ± 0.2 degrees.

10. The crystal according to claim 9, wherein the X-ray powder diffraction pattern further includes 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.

11. Figure 1 below: Table 1 The crystal according to claim 9, further characterized by the X-ray powder diffraction pattern shown.

12. A pharmaceutical composition comprising a besylate salt or a solvate thereof according to any one of claims 1 to 8, or a crystal according to any one of claims 9 to 11, and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent.

13. A process for preparing the pharmaceutical composition according to claim 12, comprising mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of a besylate or solvate thereof according to any one of claims 1 to 8 or a crystal according to any one of claims 9 to 11.

14. A pharmaceutical composition for use as a pharmaceutical, comprising a besylate salt or solvate thereof according to any one of claims 1 to 8, or a crystal according to any one of claims 9 to 11.

15. A pharmaceutical composition for use in the prevention or treatment of cancer, comprising a besylate salt or solvate thereof according to any one of claims 1 to 8, or a crystal according to any one of claims 9 to 11.

16. A pharmaceutical composition comprising a besylate or solvate thereof according to any one of claims 1 to 8, or a crystal according to any one of claims 9 to 11, for use in the prevention or treatment of leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasm (MPN).

17. The pharmaceutical composition according to claim 16 for use in the prevention or treatment of leukemia, wherein the leukemia is (NPM1) variant leukemia.

18. A pharmaceutical composition according to claim 16 for use in the prevention or treatment of leukemia, wherein the leukemia is selected from acute leukemia, chronic leukemia, myeloid leukemia, myeloid leukemia, lymphoblastic leukemia, and lymphocytic leukemia.

19. A pharmaceutical composition according to claim 16 for use in the prevention or treatment of leukemia, wherein the leukemia is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), T-cell prelymphoblastic leukemia (T-PLL), large granular lymphocytic leukemia, hairy cell leukemia (HCL), KMT2A rearrangement leukemia, KMT2A-PTD leukemia, KMT2A amplification leukemia, KMT2A-positive leukemia, and leukemia exhibiting a HOX / MEIS1 gene expression signature.

20. A pharmaceutical composition according to claim 16 for use in the prevention or treatment of leukemia, wherein the leukemia is acute leukemia.

21. A pharmaceutical composition according to claim 20 for use in the treatment of acute leukemia, wherein the acute leukemia is AML.

22. A pharmaceutical composition according to claim 20 for use in the treatment of acute leukemia, wherein the acute leukemia is ALL.

23. A pharmaceutical composition according to claim 20, wherein the acute leukemia has a KMT2A gene mutation or an NPM1 mutation.

24. A pharmaceutical composition according to claim 21, wherein the AML has a KMT2A gene mutation or an NPM1 mutation.

25. A pharmaceutical composition according to claim 22, wherein ALL has a KMT2A gene mutation or an NPM1 mutation.

26. A pharmaceutical composition according to claim 21, wherein the AML has a KMT2A gene rearrangement.

27. A pharmaceutical composition according to claim 21, wherein the AML has an NPM1 mutation.

28. A pharmaceutical composition according to claim 27, wherein the NPM1 mutation is NPM1c.

29. A pharmaceutical composition according to claim 17, wherein the (NPM1) variant leukemia is (NPM1c) variant leukemia.

30. A process for preparing crystals of crystalline form A as described in any one of claims 9 to 11, comprising the step of recrystallizing (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, wherein the recrystallization is a) Adding (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 or its hydrate or solvate to a suitable solvent mixture in the presence of benzenesulfonic acid and adjusting the temperature to a range from 20°C to the solvent reflux temperature, b) A step of sowing seed crystals of crystalline form A, c) A process comprising the step of obtaining a precipitate in the crystalline form described in any one of claims 9 to 11.

31. The process according to claim 30, wherein the mixture of suitable solvents is a mixture of acetone, water, and IPAc.

32. The process according to claim 30, wherein the suitable solvent mixture is a mixture of isopropanol, water, and IPAc.

33. The process according to claim 30, wherein the temperature is 25°C.

Images