(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 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 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.

[0002] The compounds may be useful for treatment and / or prevention in mammals, pharmaceutical compositions comprising such compounds, and uses as menin / MLL protein / protein interaction inhibitors useful for treating diseases, such as, but not limited to, cancers, including leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes. [Background technology]

[0003] Chromosomal rearrangements affecting mixed lineage leukemia genes (MLL, MLL1, KMT2A) result in aggressive acute leukemia across all age groups that remains largely incurable, highlighting the urgent need for novel therapeutic approaches. Acute leukemias with these chromosomal translocations of MLL manifest as lymphocytic, myeloid or biphenotypic disease 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 inducible loss-of-function alleles of Mll1 demonstrated that Mll1 plays an essential role in hematopoietic stem cell (HSC) maintenance and B cell development, but its histone methyltransferase activity is dispensable 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 are associated with leukemia formation / progression (Meyer et al., Leukemia 2013.27,2165-2176). Interestingly, the SET (Su(var)3-9, enhancer of zeste, and trithorax) domain of MLL is not retained in the chimeric protein but is replaced by the fusion partner (Thiel et al., Bioessays 2012.34,771-80). Recruitment of chromatin-modifying enzymes such as Dot1L and / or pTEFb complex by the fusion partner results in enhanced transcription and transcriptional elongation of MLL target genes, including most notably HOXA genes (e.g., HOXA9) and the HOX cofactor MEIS1. Aberrant expression of these genes then blocks hematopoietic differentiation and enhances proliferation.

[0006] Menin, encoded by the multiple endocrine neoplasia type 1 (MEN1) gene, is ubiquitously expressed and mainly localized in the nucleus. It has been found to interact with numerous proteins and is therefore involved in a variety of cellular processes. The best understood function of menin is its role as an oncogenic cofactor of MLL fusion proteins. Menin interacts with two motifs, MBM1 (menin binding motif 1) and MBM2, within the N-terminal fragment of MLL that is retained in all fusion proteins (Thiel et al., Bioessays 2012.34,771-80). The menin / MLL interaction creates a new interaction surface for lens epithelium-derived growth factor (LEDGF). While MLL directly binds to LEDGF, menin is essential for stable interaction between MLL and LEDGF and 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, many 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 leukocyte formation in myeloid progenitor cells ectopically expressing MLL fusions (Chen et al., Proc Natl Acad Sci 2006.103,1018-23). Similarly, genetic disruption of the menin / MLL fusion interaction by loss-of-function mutations abolishes the oncogenic properties of MLL fusion proteins, blocks leukemia development in vivo, and releases the differentiation block of MLL-transformed leukemic blasts. These studies also showed that menin is required for the maintenance of HOX gene expression by MLL fusion proteins (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 have also demonstrated efficacy in preclinical models of AML (Borkin et al., Cancer Cell 2015.27,589-602; Cierpicki and Grembecka, Future Med Chem 2014.6,447-462). Together with the observation that menin is not an essential cofactor of MLL1 during normal hematopoiesis (Li et al., Blood 2013.122,2039-2046), these data validate interfering with the menin / MLL interaction as a promising new therapeutic approach for the treatment of MLL-rearranged leukemias and other cancers with an active HOX / MEIS1 gene signature. For example, internal tandem duplications (PTDs) within the 5' region of the MLL gene represent another major abnormality found primarily in de novo and secondary AML as well as myelodysplastic syndromes. Although the molecular mechanism and biological function of MLL-PTD are not fully understood, new therapeutic targeting strategies affecting menin / MLL interaction may also prove effective in treating MLL-PTD-associated leukemia. Furthermore, castration-resistant prostate cancer has been shown to be dependent on menin / MLL interaction (Malik et al., Nat Med 2015.21,344-52).

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

[0008] Several references describe inhibitors targeting the menin-MLL interaction: WO 2011029054, J Med Chem 2016, 59, 892-913 describe the preparation of thienopyrimidine and benzodiazepine derivatives, WO 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 WO 2014,6,447-462 provides an overview of small molecule and peptidomimetic compounds, and WO 2016195776 provides a review of small molecule and peptidomimetic compounds including furo[2,3-d]pyrimidines, 9H-purines, [1,3]oxazolo[5,4-d]pyrimidines, [1,3]oxazolo[4,5-d]pyrimidines, [1,3]thiazolo[5,4-d]pyrimidines, thieno[2,3-b]pyridines and thieno[2,3-d]pyridines. Pyrimidine derivatives are described, such as 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 in WO 2016197027, and thienopyrimidine and thienopyridine compounds in WO 2016040330. Piperidines are described as menin inhibitors in WO 2017192543. Inhibitors of menin-MLL interaction are described in WO 2017112768, WO 2017207387, WO 2017214367, WO 2018053267 and WO 2018024602. WO2017161002 and WO2017161028 describe inhibitors of menin-MLL.WO2018050686, WO2018050684 and WO2018109088 describe inhibitors of menin-MLL interaction. WO2018226976 describes methods and compositions for inhibiting the interaction of menin with MLL protein. WO2018175746 provides methods for treating hematological malignancies and Ewing's sarcoma. WO2018106818 and WO2018106820 provide methods for promoting proliferation of pancreatic cells. WO2018153312 discloses azaspiro compounds related to the field of medicinal chemistry. WO2017132398 discloses a method comprising contacting leukemia cells exhibiting NPM1 mutation with a pharmacological inhibitor of the interaction between MLL and menin. WO2019060365 describes replacement inhibitors of menin-MLL. WO2020069027 describes the treatment of hematological malignancies with inhibitors of menin. Krivtsov et al., Cancer Cell 2019. No. 6 Vol. 36, 660-673 describes menin-MLL inhibitors. Summary of the Invention [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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide besylate (benzenesulfonate salt):

[0010] [ka] and solvates thereof. One of ordinary skill in the art will understand that "and solvates thereof" refers to the besylate salt 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. Thus, the present invention also encompasses the besylate salt of (RN-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide, and a solvate of the besylate salt of (RN-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.

[0011] In particular, 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 or a hydrate thereof.

[0012] In particular, 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 bisbesylate or a solvate thereof.

[0013] In particular, 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 bisbesylate or a hydrate thereof.

[0014] In particular, 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 bisbesylate 0.5 to 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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate 2.0 equivalent hydrate.

[0016] More particularly, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate.

[0017] More specifically, 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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate 0.5-2.0 equivalent hydrate.

[0018] More particularly, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate 2.0 equivalent hydrate.

[0019] The besylate salt 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 or its solvates are outstanding with regard to 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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate or a solvate thereof.

[0021] The present invention also provides pharmaceutical compositions comprising, consisting of, and / or consisting essentially of a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, and / or a pharma-ceutically acceptable diluent and (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 bisbesylate or a solvate thereof.

[0022] Also provided is a pharmaceutical composition comprising, consisting of, and / or consisting essentially 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 bisbesylate or a solvate thereof and a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, and / or a pharma-ceutically acceptable diluent.

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

[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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbesylate or a solvate thereof in the preparation of a medicament, wherein the medicament is prepared for treating a disease, for example, but not limited to, cancer, such as leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

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

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

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

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

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

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

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

[0032] In one embodiment, compounds according to the invention may be suitable for QD dosing (once per day).

[0033] The present invention also relates to the use of a compound according to the invention in combination with an additional pharmaceutical agent for use in the treatment or prevention of cancer, including but not limited to leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

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

[0035] The present invention also relates to products comprising a compound according to the invention and an additional pharmaceutical agent as a combined preparation for simultaneous, separate or sequential use in the treatment or prevention of cancer, including but not limited to leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

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

[0037] In another embodiment, 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 for use as a medicament.

[0038] In another embodiment, 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 bisbesylate and solvates thereof for use as a medicament.

[0039] In another embodiment, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide besylate hydrate for use as a medicament.

[0040] In another embodiment, the present invention relates to crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate for use as a medicament.

[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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate and solvates thereof.

[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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate. [Brief description of the drawings]

[0043] The Summary of the Invention, as well as the Detailed Description which follows, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments of the invention. However, the invention is not limited to the specific disclosure of the drawings. Of the drawings: [Figure 1] FIG. 2 is an X-ray powder diffraction (XRPD) pattern 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 bisbesylate hydrate crystalline form A. [Diagram 2]Efficacy studies in the Molm-14 subcutaneous (sc) model. [Diagram 3] Efficacy studies in disseminated OCI-AML3 models. [Figure 4] 1 is a powder X-ray diffraction (XRPD) pattern of intermediate 234b. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present disclosure may be more fully understood by reference to the following description, including the following glossary and concluding examples. It is also understood that certain features of the compounds, crystalline form A, compositions and methods of the present disclosure that are described herein in the context of separate embodiments for clarity may be provided in combination in a single embodiment. Conversely, various features of the compounds, crystalline form A, compositions and methods of the present disclosure that are described in the context of a single embodiment for brevity may also be provided separately or in any subcombination.

[0045] Some of the quantitative expressions given herein are not modified by the term "about". Regardless of whether the term "about" is explicitly used, all amounts given herein are meant to refer to their actual indicated values, and are understood to also refer to approximations of such indicated values ​​that would be reasonably estimated based on ordinary skill in the art, including approximations of such indicated values ​​by experimental and / or measurement conditions.

[0046] Throughout the description and claims of this specification, the terms "comprise" and "contain" and variations thereof, such as "comprising" and "comprises", mean "including but not limited to" and are not intended to (and do not) exclude other elements.

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

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

[0049] The term "room temperature" (RT) refers to a temperature of about 15°C to about 30°C, particularly about 20°C to about 30°C. Preferably, room temperature is a temperature of about 25°C.

[0050] When a crystalline form is identified using one or more XRPD peaks given as angles two-theta (2-theta), each 2-theta value is understood to mean the 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 term "compound of the (present) invention" or "compound according to the (present) invention" is meant to include (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, or any subgroup thereof.

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

[0054] The (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 or solvate thereof 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 invention, (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 or a solvate thereof is present in substantially pure form.

[0055] Crystalline Form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbesylate hydrate can be obtained in substantially pure form, with the mole percent of impurities in the isolated crystalline form 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 invention, (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 bisbesylate hydrate is present in substantially pure form.

[0056] Also provided herein is crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate in 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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide, such as other crystalline forms, other salt forms, or solvates thereof. At least a particular weight percentage can be (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 bisbesylate hydrate crystalline form A. Particular weight percentages include 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% and 99.9%.

[0057] Provided herein is a process for preparing the crystalline forms described herein, comprising the step of recrystallizing Compound A, the recrystallization comprising: a) adding compound A or a hydrate or solvate thereof to a mixture of a suitable solvent in the presence of benzenesulfonic acid and adjusting the temperature to a range of about 20° C. to the solvent reflux temperature; b) seeding with crystalline form A; and c) obtaining a precipitate of the crystalline form described herein.

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

[0059] In particular, a suitable solvent mixture in the process described in the previous paragraph is a mixture of isopropanol, water and IPAc. In particular, the temperature used in the process is about 25° C.

[0060] Also

[0061] [ka] A crystalline form of the citrate salt is also provided, wherein the crystalline form produces an X-ray powder diffraction pattern that includes peaks at 5.82, 10.09 and 18.42 degrees 2-theta ± 0.2 degrees 2-theta, specifically, 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 2-theta ± 0.2 degrees 2-theta.

[0062] Also, the following intermediates:

[0063] [ka] is provided as a pharma- ceutically acceptable salt or solvate thereof.

[0064] One of ordinary skill in the art will appreciate that "or a solvate thereof" refers to a pharma- ceutically acceptable salt of an intermediate, and thus encompasses solvates of pharma- ceutically acceptable salts.

[0065] Also, the following intermediates:

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

[0067] Pharmaceutically acceptable salts include acid addition salts and base addition salts.Such salts can be formed by conventional means, for example, by reacting free acid form or free base form with one or more equivalents of suitable base or acid, optionally in a solvent or in a medium in which the salt is insoluble, and then removing the solvent or medium using standard techniques (for example, in vacuum, by lyophilization, or by filtration).Salts can also be prepared by exchanging the counterion of the compound of the present disclosure in the form of a salt with another counterion, for example, by using a suitable ion exchange resin.

[0068] Suitable acids include, for example, inorganic acids such as hydrohalic acids, e.g., hydrochloric or hydrobromic acids, sulfuric, nitric, phosphoric acids, and the like, or organic acids such as, for example, 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 the like. Conversely, the above salt forms can be converted to the free base form by treatment with an appropriate base.

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

[0070] The term solvate includes solvent addition forms. Examples of such solvent addition forms are, for example, hydrates, alcoholates, and the like.

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

[0072] [ka]

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

[0074] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or State government or the appropriate agency of a country other than the United States, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, or more specifically, in humans.

[0075] The term "subject" refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or study.

[0076] The term "therapeutically effective amount," as used herein, means that amount of an active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician, including alleviation or reversal of the symptoms of the disease or disorder being treated.

[0077] The term "composition" is intended to encompass a product comprising specified ingredients in specified amounts, and any product that results directly or indirectly from a combination of the specified ingredients in the specified amounts.

[0078] As used herein, unless otherwise noted, the terms "affect" or "affected" (when 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 manifestations of said disease, syndrome, condition, or disorder and / or preventing the onset of one or more symptoms or manifestations of said disease, syndrome, condition, or disorder.

[0079] As used herein, the terms "treatment" and "treating" are intended to refer to any process that may slow, interrupt, halt or stop the progression of a disorder, or ameliorate one or more symptoms thereof, but does not necessarily indicate the complete elimination of all symptoms.

[0080] Experimental Part Synthesis of Crystalline Form A of (R)-N-Ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate

[0081] [Table 1-1]

[0082] [Table 1-2]

[0083] [Table 1-3]

[0084] Those skilled in the art will understand that, even if not explicitly mentioned in the experimental protocols below, typically after column chromatography purification, the desired fractions were collected and the solvent was evaporated.

[0085] When stereochemistry is not indicated, this is meant to be a mixture of stereoisomers unless otherwise indicated or clear from the context.

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

[0087] As will be understood by those skilled in the art, the compounds and intermediates synthesized using the protocols shown may exist as solvates, e.g., hydrates, and / or may contain residual solvents or trace impurities. Compounds or intermediates isolated in the form of salts or solvates (e.g., hydrates) may be of integer stoichiometry, i.e., mono- or di-salts, or of intermediate stoichiometry. When an intermediate or compound is shown as an "HCl salt" without indicating the number of equivalents of HCl, this means that the number of equivalents of HCl was not confirmed. When an intermediate or compound is shown as a "hydrate" without indicating the number of equivalents of H2O, this means that the number of equivalents of H2O was not confirmed.

[0088] For convenience, (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 (free base) is referred to as "Compound A" in the experimental part below.

[0089] Example 1 - Synthesis 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) - 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) in THF (60 mL) cooled to -70 °C, isopropylmagnesium bromide solution (19 mL, 55 mmol, 2.9 M in 2-methyltetrahydrofuran) was slowly added, and the resulting mixture was slowly warmed to room temperature and stirred for 12 h. The mixture was poured into saturated aqueous NH4Cl (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was further purified by FCC (PE / EtOAc = 1:0 to 100:1) to give the title intermediate (3.7 g, 60% yield) as a yellow oil.

[0092] Preparation of intermediate 13 tert-Butyl 6-(3,6-dichloro-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate

[0093] [ka]

[0094] To a solution of 3,5,6-trichloro-1,2,4-triazine (10.0 g, 54.2 mmol) and TEA (15.2 mL, 109 mmol) in DCM (100 mL) cooled 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 h. The mixture was diluted with water (20 mL) and extracted with DCM (30 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel FCC (PE / EtOAc = 1:0 to 3:1) to give the title intermediate (12.0 g, 58% yield) 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 dry DCM (150 mL) 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 h. 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 (EtOAc / PE = 0% to 20%) to give the title intermediate (12.0 g, 96% yield) as a white solid.

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

[0099] [ka]

[0100] To a solution of N-ethyl-5-fluoro-N-isopropyl-2-methoxybenzamide (intermediate 27) (12.0 g, 50.1 mmol) in dry DCM (100 mL) cooled to -78 °C, BBr3 (14.4 mL, 152 mmol) was added slowly, and the resulting mixture was slowly warmed to room temperature and stirred for 8 h. The mixture was cooled again to -78 °C, and MeOH (5 mL) was added dropwise to quench the reaction. The resulting mixture was slowly warmed to room temperature, and the pH value was adjusted to about 8 by adding saturated aqueous NaHCO3. The aqueous layer was extracted with DCM (50 mL × 3), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by FCC (EtOAc / PE = 0%-20%) to give the title intermediate (9.0 g, 78% yield) as a white solid.

[0101] Alternative preparation of intermediate 28

[0102] [ka]

[0103] A mixture of 5-fluoro-2-hydroxy-benzoic acid (14.0 kg, 89.68 mol, 1.0 equiv) in THF (168 L, 12 vol) was adjusted to 15-25 °C and 1,1-carbonyldiimidazole (17.45 kg, 107.62 mol, 1.2 equiv) was added over 1 h. After the addition, the mixture was stirred at 15-25 °C for 18 h. After this time, N-ethylpropan-2-amine (14.85 kg, 170.39 mol, 1.9 equiv) was added to the mixture over 2 h at 15-25 °C. The resulting mixture was further aged at 15-25 °C for 18-24 h. The pH was adjusted to pH 4-5 using 10% aqueous H2SO4 (140 kg, 10 vol) 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 at 15-25° C. over 3 h. The mixture was then cooled to 0-10° C. and stirred for an additional 6 h. The resulting slurry was filtered and the cake was washed with a tert-butyl methyl ether (MTBE):n-heptane mixture (25 kg of a 2:3 vol / vol mixture of MTBE:n-heptane, 2.5 vol). The cake wash was repeated two more times and the resulting solid was dried in vacuum at 50° C. to give intermediate 28 (16.5 kg, purity: 99.1%, yield: 80.4%).

[0104] Preparation of intermediate 14 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

[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) in THF (120 mL) was stirred at 25 °C for 8 h. 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 give the crude product, which was purified by FCC (PE / EtOAc = 1:0 to 3:1) to give the title intermediate (14.0 g, 73% yield) as a green solid.

[0107] Preparation of intermediate 2 tert-Butyl 6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate Synthesis method A of intermediate 2

[0108] [ka]

[0109] To a mixture of tert-butyl 6-(3-chloro-6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4 triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (intermediate 14) (20 g, 36.4 mmol), NaBH4 (2.48 g, 65.7 mmol) and TMEDA (8.54 g, 73.5 mmol) in THF (500 mL) was added Pd(dppf)Cl2×DCM (1.70 g, 2.08 mmol) under N2 atmosphere. After addition, the reaction mixture was stirred at 25° C. for 14 h. The reaction mixture was filtered, the filtrate was concentrated and the residue was purified by silica gel FCC (EtOAc) to give the title intermediate (15 g, 93% purity, 74% yield) as a brown solid.

[0110] Synthesis method B of intermediate 2

[0111] [ka]

[0112] To a solution of tert-butyl 6-(3-chloro-6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4 triazin-5-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (intermediate 14) (22.0 g, 40.1 mmol), TEA (15 mL) in MeOH (100 mL) was added Pd / C (wet, 5.0 g, 10%). The resulting mixture was stirred at 25° C. under H atmosphere (30 psi) for 8 h. The reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo to give 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)-1,2,4-triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0114] [ka]

[0115] To a solution of 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) was added TFA (0.5 mL, 6.4 mmol) and the resulting mixture was stirred at room temperature for 3 h. Then 10% NaOH (5 mL) solution was added slowly to the mixture to adjust the pH value to about 12, and the resulting mixture was extracted with DCM (10 mL×3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give the title intermediate (220 mg, 90% yield) as a white solid.

[0116] Preparation of compound 61 tert-Butyl (4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-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-triazin-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) in MeOH (15 mL) was stirred at 80° C. for 0.5 h. NaBH3CN (310 mg, 4.93 mmol) was then added and the resulting mixture was stirred at 80° C. for 6 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to give the crude product, which was further purified by preparative HPLC using a Waters Xbridge Prep OBD (column: C18 150×40 mm 10 um, eluent: ACN / H2O (0.05% ammonia) 45%-75% v / v) to give the title compound (700 mg, 46% yield) as a colorless oil.

[0119] Preparation of Compounds 62 and 63 tert-Butyl (R)-(4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-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)-1,2,4-triazin-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-triazin-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 a DAICEL CHIRALPAK IG (column: 250×30 mm 10 um, isocratic elution: EtOH (containing 0.1% of 25% ammonia): supercritical CO2, 40%:60% (v / v)) to obtain the title compound (compound 62) (85 mg, 42% yield) and (compound 63) (80 mg, 40% yield), both as pale yellow oils.

[0122] compound 64 (R)-2-((5-(2-(6-amino-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-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)-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)carbamate (compound 62) (550 mg, 0.876 mmol) in DCM (4 mL), TFA (4 mL) was added slowly and the resulting mixture was stirred at 25° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with DCM (40 mL) and the pH value was adjusted to about 12 using aqueous NaOH (2 M, 16 mL). The aqueous layer was extracted with DCM (100 mL×2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford 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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide

[0126] [ka]

[0127] A mixture of (R)-2-((5-(2-(6-amino-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-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), NaI (102 mg, 0.680 mmol) in DMF (1 mL) was stirred at 80 °C for 1 h by microwave irradiation. After cooling to room temperature, the mixture was diluted with HO (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with HO (10 mL), dried over NaSO, filtered and concentrated under reduced pressure to give the crude product, which was further purified by HPLC using a Phenomenex Gemini-NX (column: 150×30 mm 5 μm, eluent: ACN / HO (10 mM NHHCO) 51%-71% (v / v)) and further purified by SFC using a DAICEL CHIRALCEL OD-H (column: 250×30 mm 5 um, eluent: supercritical CO in EtOH (0.1% v / v ammonia) 25 / 25, v / v) to give the title compound (5.13 mg, 96% purity) as a yellow solid.

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

[0129] Compound A (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

[0130] [ka]

[0131] In anhydrous MeOH (2 mL) was added (RN-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide (compound 11) (40.0 mg, 0.068 mmol), formaldehyde (55.4 mg, 0.683 mol, 37% in water) and AcOH (8.2 mg, 0. The mixture was stirred at 45° C. for 1 h. Then, NaBH3CN (8.6 mg, 0.137 mmol) was added to the mixture, and the resulting mixture was stirred at 45° C. for another 1 h. After cooling to room temperature, the reaction mixture was diluted with saturated aqueous NaHCO3 (40 mL) to adjust the pH value to about 8, and further extracted with DCM (20 mL×3). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was then purified by HPLC at Boston Scientific. Purification by preparative HPLC using Prime (column: C18 150×30 mm 5 um, mobile phase A: H2O (0.04% ammonia + 10 mM NH4HCO3), mobile phase B: ACN, flow rate: 25 mL / min, gradient condition B / A from 50% to 80% (50% B to 80% B)) afforded the title compound (9.62 mg, purity 99.10%, yield 23.3%) as a yellow oil.

[0132] Example 2 - Synthesis 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) - Preparation Method B Preparation of intermediate 7 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid

[0133] [ka]

[0134] To a solution of 4-(methylamino)butanoic acid hydrochloride (3.0 g, 19.5 mmol) and TEA (7.78 mL, 58.6 mmol) in MeOH (30 mL) was added Boc2O (4.69 g, 21.5 mmol) dropwise. The mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure and the residue was diluted with EtOAc (100 mL), washed with cold 0.1 N HCl (70 mL x 2), H2O (50 mL x 2) and brine (50 mL), dried over Na2SO4, filtered and concentrated to give 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 of 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (Intermediate 7) (1.80 g, crude) in CHCl3 (30 mL) was added N,O-dimethylhydroxylamine hydrochloride (960 mg, 9.84 mmol), HOBt (1.24 g, 9.18 mmol) and NMM (2.80 mL, 25.1 mmol). Then EDCI (2.23 g, 11.6 mmol) was added and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with DCM (100 mL), washed with 1N HCl (30 mL x 3), saturated aqueous NaHCO3 (30 mL x 3) and brine (30 mL), dried over Na2SO4, filtered and concentrated in vacuo to give the title intermediate (1.70 g, crude) as a colorless oil.

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

[0139] [ka]

[0140] Isopropyllithium (3.2 mL, 2.24 mmol, 0.7 M in pentane) was added dropwise to a solution of tert-butyl (4-(methoxy(methyl)amino)-4-oxobutyl)(methyl)carbamate (intermediate 8) (200 mg, crude) in THF (5 mL) cooled to -70 °C under N2 atmosphere. The resulting mixture was stirred at -70 °C for 2 h. The mixture was quenched with saturated aqueous NH4Cl (15 mL) and extracted with EtOAc (30 mL x 2). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was further purified by FCC (PE / EtOAc = 10:1) to give 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)-1,2,4-triazin-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)-5-methylhexyl)(methyl)carbamate

[0142] [ka]

[0143] To a solution of 2-((5-(2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)-N-ethyl-5-fluoro-N-isopropylbenzamide (Intermediate 3) (600 mg, 1.45 mmol) and tert-butyl methyl(5-methyl-4-oxohexyl)carbamate (Intermediate 9) (330 mg, 1.37 mmol) in MeOH (50 mL) was added ZnCl2 (789 mg, 5.79 mmol). The resulting mixture was stirred at 80 °C for 2 h. NaBH3CN (729 mg, 11.6 mmol) was then added and the reaction mixture was stirred at 80 °C overnight. After cooling to room temperature, the mixture was concentrated under reduced pressure to give a crude residue, which was diluted with DCM (50 mL), quenched with saturated aqueous NH4Cl (50 mL), and extracted with DCM (50 mL x 3). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was further purified by FCC (DCM / MeOH=10:1) to give the title compound (400 mg, 42% yield) as a white solid.

[0144] compound 67 N-Ethyl-5-fluoro-N-isopropyl-2-((5-(2-(2-methyl-6-(methylamino)hexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide hydrochloride

[0145] [ka]

[0146] To a solution of tert-butyl (4-(6-(6-(2-(ethyl(isopropyl)carbamoyl)-4-fluorophenoxy)-1,2,4-triazin-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) was added 4 M HCl in dioxane (5 mL, 20 mmol) and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo to give 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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide

[0148] [ka]

[0149] To a mixture of N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(2-methyl-6-(methylamino)hexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide hydrochloride (compound 67) (480 mg, crude), K2CO3 (700 mg, 5.07 mmol) and NaI (400 mg, 2.67 mmol) in DMF (5 mL) was added 1-bromo-2-methoxyethane (230 mg, 1.65 mmol). The resulting mixture was stirred at 50 °C overnight. 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 layers were washed with brine (30 mL x 3), dried over Na2SO4, filtered and concentrated to give a crude residue. The residue was purified by FCC (DCM / MeOH=10:1) to give 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 68) (250 mg, 48% yield) as a yellow oil.

[0150] 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 68) (960 mg combined from several batches obtained by method B) was first purified by SFC using a DAICEL CHIRALPAK IG (column: 250×30 mm 10 um, mobile phase: A: supercritical CO2, B: EtOH (0.1% ammonia), A:B=40:60, 60 mL / min) and further purified by SFC using a Boston Prime (column: 150×30 mm 5 um, mobile phase A: H2O (10 mM NH4HCO3), mobile phase B: ACN, flow rate: 25 mL / min, gradient condition B / A). Purification by preparative HPLC using 55%-85%) afforded the title compound (270 mg) as a colourless oil.

[0151] 1 H NMR (400 MHz, methanol-d4): δ = 8.40 (s, 1H), 7.47-7.32 (m, 1H), 7.30-7.10 (m, 2H), 4.24-4.01 (m, 2H), 3.89-3.60 (m, 3H), 3.48 (br s, 3H), 2.63-2.51 (m, 2H), 2.43-2.32 (m, 2H), 2.29-2.07 (m, 6H), 1.86-1.72 (m, 1H), 1.62-1.44 (m, 2H), 1.39-1.02 (m, 10H), 0.99-0.66 (m, 9H). Some protons are hidden by the solvent peaks and are not recorded.

[0152] LCMS(ESI)(Method 2):R t = 1.965 min, m / z measured value 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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-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 at -10 to 0 °C were added to a solution of DCC (55.5 kg) in DCM (613 kg) over 3 hours and aged at -10 to 0 °C for 16 hours. 10% aqueous citric acid (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 filter cake was washed with DCM (91 kg). The filtrate was separated and the organic layer was washed with 10% aqueous citric acid (2 times with 450 kg) and 10% aqueous NaCl (449 kg). To the organic phase (1200 kg) was added acetic acid (75.0 kg) while maintaining the temperature at -10 to 0 °C. Sodium borohydride (18.0 kg) was added in portions over 5 hours while maintaining the temperature in the range of -10 to 0 °C, and the resulting mixture was then aged at -10 to 0 °C for an additional 16 hours. The mixture was warmed to 15 to 25 °C and aged for 2 hours. The mixture was then washed with 14% aqueous NaCl (450 kg), followed by a second wash with 14% aqueous NaCl (432 kg), and finally with water (444 kg). The organic phase was concentrated under reduced pressure to 2 to 4 volumes. Iso-propanol (143 kg) was added to the residue and concentrated under reduced pressure to 4 to 5 volumes. After cooling to −10 to 0° C. and aging for 8 h, the resulting slurry was filtered, washed with IPA (38 kg) and dried to give the title intermediate (46.7 kg, 69% yield) as a white solid.

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

[0158] [ka]

[0159] tert-Butyl (R)-(1-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-methylbutan-2-yl)carbamate (Intermediate 227) (46.7 kg) in toluene (333 kg) was heated to reflux and aged for 4 hours. The mixture was cooled to ambient temperature, filtered, and washed with toluene (20 kg). The combined filtrate was concentrated to dryness under reduced pressure to give the desired compound (31.05 kg, 96% yield) 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] tert-Butyl (R)-2-isopropyl-5-oxopyrrolidine-1-carboxylate (Intermediate 228) (30.9 kg) in 2-MeTHF (26.7 kg) was cooled to -5 to 5 °C. A solution of LiBH4 in 2-MeTHF (1 M, 45.2 kg, 54.4 mol) was added over 3 h and the mixture was aged for 4 h. A cold aqueous solution of 5% NaHCO3 (163 kg) was added over 3 h at -5 to 5 °C and aged for an additional 2 h. The mixture was warmed to ambient temperature and aged for an additional 2 h. The aqueous layer was separated and the organic layer was washed with 10% aqueous NaCl (170 kg) and water (155 kg). During the water wash, 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 give the desired compound (28.5 kg, 91% yield) as an oil, which was used directly without further purification.

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

[0164] [ka]

[0165] tert-Butyl (5R)-2-hydroxy-5-isopropylpyrrolidine-1-carboxylate (Intermediate 229) (28.55 kg) in DCM (344 kg) was treated with 2-methoxy-N-methylethan-1-amine (12.3 kg, 138.0 mol) at 15-25° C. and the resulting mixture was aged for 1 h. Sodium triacetoxyborohydride (40.12 kg) was added in portions over 5 h while maintaining the temperature between 15-25° C. and the resulting mixture was aged for 48 h. The reaction mixture was quenched by the addition of 8% aqueous NaOH (184 kg) over 2 h while maintaining the temperature between 15-25° C. and the mixture was aged for an additional 2 h. 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 give the title intermediate (33.26 kg, 88% yield) 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 HCl in isopropanol (84.80 kg) was added a solution of tert-butyl (R)-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)carbamate (Intermediate 230) (32.38 kg) in isopropanol (25.6 kg) over 3 hours at ambient temperature and the mixture was aged at ambient temperature for a further 19 hours. Methyl tert-butyl ether (95.25 kg) was then added over 1 hour and the mixture was aged for 2.5 hours. The resulting slurry was filtered and washed with MTBE (53 kg). The filter cake was dried to give the title compound (23.92 kg, 81% yield) as a white solid.

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

[0170] [ka]

[0171] To a solution of DIPEA (952 g, 1.1 equiv.) in THF (6 L) cooled to -35 to -25 °C was added n-BuLi (2.33 kg, 2.5 M in hexanes, 1.0 equiv.) while maintaining the temperature below -25 °C. The resulting mixture was aged at -35 to -25 °C for an additional 30 min and then cooled to -78 to -60 °C. A solution of ethyl 1-benzylpyrrolidine-3-carboxylate (2 kg, 1.0 equiv.) in THF (2 L) was added at -78 to -60 °C and stirred for an additional 30 min. Chloroiodomethane (1.81 kg, 1.2 equiv.) was then charged at -78 to -60 °C. The reaction mixture was aged at -60 to -40 °C for 2 h. The reaction mixture was added to aqueous citric acid (660 g in 6 L H2O) at a temperature of 0-10 °C and the resulting mixture was aged at 20-30 °C for another 20 min. After separation of the layers, the aqueous layer was extracted with EtOAc (6 L) and the combined organic layers were washed with brine (6 L) and then warmed to 50-60 °C. Oxalic acid (2.22 kg) was charged at 50-60 °C. The resulting mixture was stirred at 50-60 °C for 3 h and then cooled to 20-30 °C and aged overnight. The resulting solid was filtered and the cake was washed with ethyl acetate (2 L). The wet cake was added to toluene (4 L), H2O (8 L) and K3PO4 (1.5 equiv.) and the resulting mixture was aged at 20-30 °C for 20 min. After separation of the layers, the aqueous layer was extracted with toluene (2 L). The organic layers were combined and washed twice with water (2 L). The organic phase was concentrated under reduced pressure to give 4.2 kg of the desired compound as a toluene solution (assay 46% by weight, 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. A solution of ethyl 1-benzyl-3-(chloromethyl)pyrrolidine-3-carboxylate (Intermediate 232) (4.4 kg) in toluene (26 L) was pumped at 26.7 mL / min and cooled to -60°C. After cooling, it was then mixed with a cooled solution of DIBAL-H (28.1 mol) in toluene at -60°C (28 L) at a pump rate of 32.1 mL / min. The mixture was passed through a perfluoroalkoxy (PFA) coiled tube reactor at -60°C (total flow rate 58.8 mL / min, residence time 5 s). The resulting mixture was mixed with chilled MeOH (-60°C) and pumped at a rate of 15.2 mL / min. This mixed solution was pumped to another PFA coiled tube reactor at -60°C (total flow rate 74 mL / min, residence time 5 s). The resulting mixture was collected in a receiver containing 20 wt % aqueous Rochelle's salt (20V). The layers were separated and the organic phase was washed twice with water (2×44 L). The organic phase was combined with another 3.0 kg batch prepared in a similar manner and concentrated under reduced pressure to give 20.8 kg of a toluene solution of the desired compound (assayed 25.5 wt % by HPLC, assay yield 85%), which was used directly without further purification.

[0175] 1 H NMR(300MHz,Chloroform-d):δ 9.62(s,1H),7.39-7.20(m,5H),3.83-3.57(m,4H),2.96(d,J=10.2Hz,1H),2.80-2 .55(m,3H),2.17(ddd,J=13.9,7.9,6.1Hz,1H),1.83(ddd,J=13.4,7.8,5.5Hz,1H).

[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] [ka]

[0178] Toluene (30 L) and (R)-N 1 -(2-Methoxyethyl)-N 1 To a solution of 1-benzyl-3-(chloromethyl)pyrrolidine-3-carbaldehyde (Intermediate 233) in toluene (3.0 kg, 10 wt%) diluted with ,5-dimethylhexane-1,4-diamine, dihydrochloride (Intermediate 231) (3.47 kg) was added triethylamine (2.55 kg, 25.2 mol) at 20-30 °C. The resulting mixture was aged at 20-30 °C for 2 hours. Sodium triacetoxyborohydride (9.0 kg) was then charged at 20-30 °C and the mixture was aged for 12 hours. The reaction mixture was cooled to 5-15 °C and 25 wt% aqueous NaOH (25 L, ca. 16.75 equiv.) 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 15 wt % aqueous NaCl (10 L), the layers were again separated, and water (18 L) was charged to the organic phase. The pH of the aqueous phase was adjusted to 6-7 with 4 M aqueous HCl while maintaining the internal temperature below 35° C. The organic phase was then discarded and the aqueous phase was separated and basified to pH 8-9 with K2HPO4.

[0179] The resulting mixture was warmed 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 stream was washed three times with methyl tert-butyl ether (3 x 40 L). Additional methyl tert-butyl ether (83 L) was added to the resulting aqueous layer and the aqueous phase was basified to pH 9-10 with 8 wt% aqueous NaOH while maintaining the temperature at 15-35°C. The aqueous layer was separated and the organic layer was washed three times with water (3 x 30 L). The organic layer was then concentrated under reduced pressure to approximately 3 volumes, then flushed three times with methanol (3 x 30 L) and concentrated to dryness to give the desired intermediate (12.4 kg, 90% isolated yield) as a pale yellow oil, which was used directly without further purification.

[0180] Preparation of Intermediate 234a (Citrate Salt of Intermediate 234)

[0181] [ka]

[0182] EtOH (80 mL) and intermediate 234 (20 g) were added to a round-bottom flask. Then, a 0.5 M solution of citric acid in EtOH (100 mL, 1 eq.) was added to the mixture in the round-bottom flask at room temperature. The mixture was then evaporated to dryness (rotary evaporator, 40° C.). Acetonitrile (200 mL) was added to the residue and the mixture was evaporated to dryness (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 Salt 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. until the reaction mixture became homogeneous (approximately 10 min). The mixture was then cooled to 50° C. at a rate of 0.5° C. / min. Seeds were then added (19 mg of intermediate 234a, 0.5 w / w%) and the mixture was aged for 3 h 30 min with stirring. The mixture was then nonlinearly cooled exponentially to 20° C. over 8 h. The resulting mixture was stirred overnight and the product was filtered off and dried (in a hood at room temperature overnight).

[0186] After isolation, intermediate 234b (2.75 g; 73.9% yield) was obtained as a crystalline form of the citrate salt of intermediate 234. The ratio of intermediate / citric acid obtained is 3 / 2 (NMR).

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

[0188] A new linear ramp is started every 30 seconds during the specified cool down period. 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)hexan-1-amine

[0192] [ka]

[0193] To palladium hydroxide on carbon (1.2 kg) in EtOH (1.47 kg) cooled to -5 to 5°C was added 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). Mix. The mixture was warmed to 35-45°C and stirred under hydrogen atmosphere (0.27-0.40 MPa) for 16-20 h. The mixture was filtered through diatomaceous earth (20 kg) and the pad was washed with EtOH (24 L). The filtrate was concentrated under reduced pressure (below 40°C) to 2-3 volumes and then flushed twice with 2-MeTHF (73 kg and 47 kg) to give 2-3 volumes of solution. After dilution, 10% aqueous sodium sulfate solution (30 kg) was added and the mixture was cooled to 0-10 °C, followed by the addition of 16% aqueous NaOH solution (50 kg) to adjust the pH to 13-14. The temperature was adjusted to 15-25 °C and stirred for 30-60 min. The aqueous layer was separated and extracted twice with 2-MeTHF (47 kg x 2). The combined organic layers were concentrated to 3-4 volumes under reduced pressure (below 40 °C) and 2-MeTHF (950 g) was added. After concentration to 3-4 volumes under reduced pressure (below 40 °C), the resulting solution was diluted with 2-MeTHF (30 kg), dried by passing through 4A molecular sieves (25 kg) and washed with 2-MeTHF (30 kg). The final solution was concentrated to give the desired compound (6.7 kg) as an oil with an assay purity of 90.1% in 79% corrected yield.

[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] [ka]

[0196] To (R)-N-(2-methoxyethyl)-N,5-dimethyl-4-(2,6-diazaspiro[3.4]octan-2-yl)hexan-1-amine (Intermediate 224) (100 g), 2-MeTHF (430 g) and TEA (68 g) were added and the mixture was cooled to -50 to -40 °C. 3,5,6-trichloro-1,2,4-triazine (62 g) in 2-MeTHF (172 g) was added and the mixture was stirred for 1 to 3 h. The resulting mixture was warmed to -20 to -10 °C, 7% aqueous NaHCO3 was added, and the mixture was warmed to 20 to 30 °C and stirred for 30 to 60 min. The aqueous layer was removed and the organic layer was washed with 10% Na2SO4 (500 g). The organic layer was dried by passing through 4 Å molecular sieves (220 g) and washed with 2-MeTHF (180 g). The title intermediate was obtained as a 2-14.8 wt % solution in MeTHF in 90% assay yield.

[0197] Compound 393 (R)-2-((3-chloro-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)-N-ethyl-5-fluoro-N-isopropyl-benzamide Method A for synthesis of compound 393

[0198] [ka]

[0199] A mixture of 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) in anhydrous THF (15 mL) was stirred at 40° C. for 8 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the resulting residue was diluted with DCM (60 mL) and washed with H2O (20 mL x 3). × The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the crude product, which was purified by fine FCC (MeOH / DCM=0%~10%) to give a yellow oil (1.40 g), which was further separated by SFC using DAICEL CHIRALPAK AD (column: 250×50 mm, 10 μm × mobile phase: A: supercritical CO2, B: EtOH (0.1% ammonia), A:B=50:50 at 70 mL / min; column temperature: 38°C; nozzle pressure: 100 bar; nozzle temperature: 60°C; evaporator temperature: 20°C; trimmer temperature: 25°C; wavelength: 220 nm) to give the title compound (1.0 g).

[0200] Method A for synthesis of compound 393

[0201] [ka]

[0202] Tetramethylguanidine (31 g) was added to a 2-MeTHF solution of (R)-4-(6-(3,6-dichloro-1,2,4-triazin-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, 100 g corrected 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, and the mixture was stirred for 40-48 hours. 7% NaHCO3 aqueous solution (500 g) 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% aqueous NaOH (2×500 g) and once with 10% aqueous Na2SO4 (500 g). The organic layer was concentrated under reduced pressure (<40 °C) to 2.2-3.0 volumes and flushed three times with MeOH (1×790 g and 2×395 g) until both 2-MeTHF and water content were <1.0%, affording the desired compound as a 60.1% by weight solution in methanol in 86% assay yield.

[0203] Compound A (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

[0204] [ka]

[0205] A methanol solution of (R)-2-((3-chloro-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-N-ethyl-5-fluoro-N-isopropylbenzamide (compound 393) (163.93 g of a 60.1 wt% MeOH solution, 100 g corrected for compound 393), palladium on carbon (10 g) and MeOH (316 g) was stirred under a hydrogen atmosphere (0.20 to 0.30 MPa) at 20 to 30° C. for 18 hours. The mixture was filtered through diatomaceous earth (75 g) and the cake was washed with MeOH (158 g). The filtrate was concentrated under reduced pressure (<40°C) to approximately 3 volumes and then flushed with isopropyl acetate (IPAc, 870 g) to approximately 3 volumes. The mixture was then diluted with IPAc (696 g) and 20% aqueous Na2CO3 was added (500 g). The mixture was stirred for 30-60 min. The aqueous layer was removed. The organic layer was washed with water (500 g) and then concentrated under reduced pressure at <45°C to approximately 3 volumes. The title intermediate was obtained as a 48.1 wt% solution in IPAc in approximately 90% assay yield.

[0206] Example 4 - Synthesis 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)benzamidooxalate (Compound A3)

[0207] [ka]

[0208] Compound A3 To 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 oxalic acid (81.0 mg, 0.900 mmol). After the addition, the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated and the residue was redissolved in ACN and deionized water and lyophilized to give the title compound (350 mg) as a white solid.

[0209] 1 H NMR(400MHz,Methanol-d4):δ=8.48(s,1H),7.52-7.11(m,3H),4.54-3.64(m,12H),3.40-3.34(m,5H),3.23-3.13(m ,2H),2.90(s,3H),2.54-2.27(m,2H),2.19-2.03(m,1H),1.97-1.77(m,2H),1.75-1.50(m,2H),1.35-0.65(m,17H).

[0210] 1 H NMR (400MHz, DMSO-d6): δ=8.51(s,1H),7.51-7.29(m,3H),4.29-3.34(m,12H),3.23-2.84(m,7H),2.70( s,3H),2.35-2.09(m,2H),2.05-1.85(m,1H),1.81-1.58(m,2H),1.56-1.33(m,2H),1.18-0.60(m,17H).

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

[0212] Example 5 - Synthesis of Compound A1

[0213] [ka]

[0214] To a solution of compound A (207.90 g of a 48 wt% solution in IPAc, 100 g of active compound A) in IPAc (360 g) was added EtOH (63 g) at 20°C-25°C. The solution was then treated with concentrated HCl (32.9 g) in EtOH (49.5 g) for approximately 15 min. The mixture was seeded with crystalline compound A1 seeds (2 g, 2% seed loading) and then aged for 18 h. IPAc (870 g) was added slowly over 4 h at 20-25°C and the slurry was stirred for an additional 18 h. After cooling to approximately 5°C, the product was filtered, washed with IPAc (522 g) and dried under vacuum at 20-30°C to give weakly crystalline compound A1 as a white solid (91.0% yield, 115.4 g). (Note: the small amount of seed material used in the reaction was obtained via a similar reaction protocol on a smaller scale)

[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 h to give a clear solution. Additional IPAc (522 g) was added dropwise over 1-2 h, and the mixture was then seeded with crystalline Compound A1 seeds (2 g, 2% seed loading). The mixture was then aged for 18-20 h, IPAc (348 g) was added slowly over 12 h at 20-30 °C, and the slurry was stirred for an additional 55-60 h. The product was filtered, washed with IPAc (158 g), and dried in vacuum at 20-30 °C to give Compound A1 as a white solid (85% yield, 85.0 g net).

[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 min, m / z actual value 600.5 [M+H] + .

[0218] Example 6 - Synthesis of crystal form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate (compound A4) (water equivalent not calculated)

[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 API) 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% seeds were added and the mixture was stirred at 25° C. for 1 h. 28.8 V (2312 mL) of IPAc was then added over 8 h. The suspension was then stirred at 25° C. for 18 h. 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 appreciate that the small amount of initial seed material used in the above reactions can be obtained through similar reaction protocols on a smaller scale by waiting for spontaneous nucleation without adding seeds.

[0222] The initial seed of the besylate salt was also obtained during salt screening experiments. In these experiments, 100 mg of the 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 counter ion (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] An appropriate amount of crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate was dissolved in deuterated DMSO and diluted with 1D 1 1 H NMR spectra were recorded.

[0224] One-dimensional proton experiments were collected at 300 K on samples in deuterated DMSO using a Bruker AVANCE NEO-600 MHz NMR spectrometer equipped with a Bruker 5 mm PA BBO 600S3 BB-HD-05 Z-GRD high-resolution probe and running TOPSPIN 4.0 software.

[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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate (compound A4) (water equivalent not determined) A 95 / 5 mixture of isopropanol / water (24 mL) was charged to the flask and heated to 40° C. Benzenesulfonic acid (4.31 g, 98%) was added. This was followed by the addition of 19.3 g of a solution of Compound A in IPAc (containing 8 g of Compound A). An additional 16 mL of IPAc was added. 2% seeds were added and the mixture was stirred at 40° C. for 1 h. IPAc (115.2 mL) was then added dropwise over 8 h. The mixture was then cooled to 0° C. for 15 h. 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 h to give 11.44 g of crystalline form A bis-besylate hydrate (water equivalent not determined).

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

[0228] Analytical methods used in the experimental part above Analytical information for the above compounds was generated using the analytical methods described below.

[0229] NMR method Some NMR experiments were performed at ambient temperature (298.6 K) using a Bruker Avance III 400 spectrometer equipped with a BBO 400 MHz S1 5 mm probehead with an internal deuterium lock and z-gradients, 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] Some NMR experiments were performed at ambient temperature (298.6 K) using a Varian 400-MR spectrometer equipped with an internal deuterium lock and a Varian 400 4NUC PFG probehead with z-gradients, 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] Some NMR experiments were performed at ambient temperature (298.6 K) using a Varian400-VNMRS spectrometer equipped with a Varian400 ASW PFG probehead with an internal deuterium lock and z-gradients, 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] Some NMR experiments were performed using a Bruker AVANCE III HD 300 spectrometer at ambient temperature (298.6 K) equipped with a PA BBO 300S1 BBF-HD-05 Z 5 mm probehead with internal deuterium lock and z-gradients operating at 300 MHz for proton 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 when necessary (see Table 2 below).

[0234] The flow from the column was brought to a mass spectrometer (Mass Spectrometer, MS) configured with an atmospheric pressure ion source. It is within the knowledge of one skilled in the art to set tuning parameters (e.g., scan range, dwell time, etc.) to obtain ions that allow identification of the nominal monoisotopic molecular weight (MW) of the compound. Data collection was performed with appropriate software.

[0235] Compounds were identified based on their experimental retention times (R t ) and ions. Unless otherwise specified in the tables of data, the molecular ions reported are [M+H] + (protonated molecule) and / or [MH] - (deprotonated molecule). If the compound is not directly ionizable, the type of adduct is specified (i.e., [M+NH4] + , [M+HCOO] - , etc.). For molecules with multiple isotopic patterns (Br, Cl), the values ​​reported are those obtained for the lowest isotopic mass. All results were obtained with experimental uncertainties typically associated with the methods used.

[0236] In the following, "SQD" stands for single quadrupole detector, "RT" stands for room temperature, "BEH" stands for bridged ethylsiloxane / silica hybrid, "HSS" stands for high strength silica, and "DAD" stands for 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 configured with a binary pump and modifier to deliver carbon dioxide (CO2), an autosampler, a column oven, a diode array detector with a high pressure flow cell capable of withstanding up to 400 bar. Analytical SFC details are given in Table 3 below. When configured with a mass spectrometer (MS), the flow from the column was brought to the (MS). It is within the knowledge of one skilled in the art to set tuning parameters (e.g., scan range, residence time, etc.) to obtain ions that allow identification of the nominal monoisotopic molecular weight (MW) of the compound. Data collection was performed with appropriate software.

[0239] [Table 3]

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

[0241] X-ray powder diffraction (XRPD) analyses were performed on a PANalytical Aeris diffractometer equipped with a Cu-Kα X-ray tube using iCore and dCore tunable optics for the incident and diffracted beams, respectively. Compounds were loaded into the cavity of a 16 mm sample holder using the backloading technique.

[0242] 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 mode: Continuous scanning Scanning range: 4~50 degrees Step size: 0.0217 degrees Counting time: 58s Spinner rotation time: 1 second Injection beam path (iCore) Divergence slit: 1 / 4° Soller slit: 0.04rad Mask 1: 9mm Diffraction beam path (dCore) Anti-scattering slit: 9mm Irradiation length: 10mm Soller slit: 0.04rad Detector: PIXcel3D-Medipix3 1x1

[0243] Those skilled in the art will recognize that the diffraction patterns and peak positions are typically substantially independent of the diffractometer used and whether a particular calibration method is utilized. Typically, peak positions may vary by no more than about ±0.2° 2-theta. The respective intensities (and relative intensities) of particular diffraction peaks may also vary as a function of various factors, including, but not limited to, particle size, orientation, sample purity, etc.

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

[0245] 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 2-theta ±0.2 degrees 2-theta.

[0246] Intermediate 234b may 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 may be further characterized by an X-ray powder diffraction pattern substantially as shown in FIG.

[0248] Crystal form A Crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate can be characterized by its X-ray powder diffraction pattern.

[0249] X-ray powder diffraction (XRPD) analyses were performed on a PANalytical Empyrean diffractometer equipped with a Cu-Kα X-ray tube using iCore and dCore tunable optics for the incident and diffracted beams, respectively. Compounds were loaded into the cavity of a 16 mm sample holder using the backloading technique.

[0250] 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 mode: Continuous scanning Scanning range: 3~35 degrees Process size: 0.0131 degrees Counting time: 30s Spinner rotation time: 1 second Injection beam path (iCore) Program Divergence slit: Automatic Irradiation length: 10mm Soller slit: 0.03rad Mask 1: 14mm Mask 2: 6mm Width: 7.7mm Diffraction beam path (dCore) Anti-scatter slit: Automatic Irradiation length: 10mm Soller slit: 0.04rad Detector: PIXcel3D-Medipix3 1x1

[0251] Those skilled in the art will recognize that the diffraction patterns and peak positions are typically substantially independent of the diffractometer used and whether a particular calibration method is utilized. Typically, peak positions may vary by no more than about ±0.2° 2-theta. The respective intensities (and relative intensities) of particular diffraction peaks may also vary as a function of various factors, including, but not limited to, particle size, orientation, sample purity, etc.

[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, 20.1 degrees 2-theta ± 0.2 degrees 2-theta.

[0253] Form A may 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] Form A may be further characterized by an X-ray powder diffraction pattern comprising the peaks identified in Table 4, where the relative intensities of the peaks are greater than about 2%, preferably greater than about 5%, more preferably greater than about 10%, more preferably greater than about 15%. However, one of ordinary skill in the art will understand that the relative intensities of the peaks may vary between different samples and between different measurements of the same sample.

[0255] Form A may be further characterized by an X-ray powder diffraction pattern substantially as shown in FIG.

[0256] Table 4 shows the peak list and relative intensities for the XRPD of crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate (Figure 1).

[0257] [Table 4]

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

[0259] In particular, the compounds according to the invention and pharmaceutical compositions thereof may be useful for the treatment or prevention of cancer. According to one embodiment, cancers that may benefit from treatment with the menin / MLL inhibitors of the invention include leukemia, lymphoma, myeloma or solid tumor cancers (such as prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma and glioblastoma). In some embodiments, leukemias include acute leukemia, chronic leukemia, myelocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, hairy cell leukemia (HCL), MLL-rearranged leukemia, MLL-PTD leukemia, MLL-amplified leukemia, MLL-positive leukemia, leukemias exhibiting a HOX / MEIS1 gene expression signature, and the like.

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

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

[0262] In particular, the compounds according to the present invention and pharmaceutical compositions thereof are useful in the treatment of AML, particularly nucleophosmin (NPM1)-mutated AML (i.e., NPM1 mut Abstract: The present invention may be useful in the treatment or prevention of NPM1-mutated AML, and more particularly, in the treatment or prevention of NPM1-mutated AML.

[0263] In particular, the compounds according to the invention and pharmaceutical compositions thereof may be useful for the treatment or prevention of MLL-rearranged leukemia, in particular MLL-rearranged AML or ALL.

[0264] In particular, the compounds according to the present invention and pharmaceutical compositions thereof may be useful for treating or preventing leukemia associated with MLL gene alterations, particularly AML or ALL associated with MLL gene alterations.

[0265] In particular, the compounds according to the invention and pharmaceutical compositions thereof may be suitable for QD dosing (once a day).

[0266] In particular, the compounds and pharmaceutical compositions thereof according to the present invention are useful for treating NPM1 gene mutations and / or mixed lineage leukemia gene (MLL, MLL1, KMT2A) alterations, mixed lineage leukemia (MLL), MLL-associated leukemia, MLL-associated leukemia, MLL-positive leukemia, MLL-induced leukemia, rearranged mixed lineage leukemia, MLL-associated leukemia, MLL gene rearrangements / alterations or rearrangements / alterations, acute leukemia, chronic leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), insulin resistance, pre-diabetes, diabetes, diabetes mellitus ... The present invention may be useful for treating or preventing hematological cancers in subjects exhibiting diabetes, or risk of diabetes, hyperglycemia, chromosomal rearrangements on chromosome 11q23, type 1 diabetes, type 2 diabetes, which promotes proliferation of pancreatic cells, where the pancreatic cells are beta cells, which are pancreatic islet cells, and beta cell proliferation is evidenced by increased beta cell production or insulin production, for inhibiting menin-MLL interaction, and the MLL fusion protein target gene is HOX or MEIS1 in humans.

[0267] The present invention therefore relates to the compounds of the invention for use as a medicament.

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

[0269] The present invention also relates to a compound according to the invention or a pharmaceutical composition according to the invention for use in the treatment, prevention, amelioration, control or reduction of the risk of a disorder associated with the interaction of menin with MLL proteins and oncogenic MLL fusion proteins in a mammal, including a human, which treatment or prevention is affected or promoted by blocking the interaction of menin with MLL proteins and oncogenic MLL fusion proteins.

[0270] The present invention also relates to the use of a compound according to the invention for the manufacture of a medicament for the treatment, prevention, amelioration, control or reduction of the risk of a disorder associated with the interaction of menin with MLL proteins and oncogenic MLL fusion proteins in a mammal, including a human, which treatment or prevention is affected or promoted by blocking the interaction of menin with MLL proteins and oncogenic MLL fusion proteins.

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

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

[0273] The present invention also relates to the use of a compound according to the invention for the manufacture of a medicament for the treatment or prevention of any one of the aforementioned disease conditions.

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

[0275] In view of the utility of the compounds according to the invention, there is provided a method for treating a warm-blooded animal, including a human, suffering from any one of the aforementioned diseases.

[0276] The methods involve administration, either systemically or locally, of a therapeutically effective amount of a compound according to the invention to a warm-blooded animal, including humans.

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

[0278] Those skilled in the art will appreciate that a therapeutically effective amount of a compound of the present invention is an amount sufficient to have therapeutic activity, which amount will vary depending, among other things, on the type of disease, the concentration of the compound in the therapeutic formulation, and the condition of the patient. An effective therapeutic daily dose may be about 0.005 mg / kg to 100 mg / kg. The amount of a compound according to the present invention, also referred to herein as the active ingredient, required to achieve a therapeutic effect may vary on an individual basis, depending, for example, on the particular compound, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated. The treatment method may also include administering the active ingredient in a regimen of 1 to 4 intakes per day. In these treatment methods, the compound according to the present invention is preferably formulated prior to administration.

[0279] The present invention also provides a composition for preventing or treating the disorders mentioned herein, said composition comprising a therapeutically effective amount of a compound according to the invention and a pharma- ceutically acceptable carrier or diluent.

[0280] While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical composition. Thus, the present invention further provides a pharmaceutical composition comprising a compound according to the present invention together with a pharma- ceutically acceptable carrier or diluent. The carrier or diluent must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0281] Pharmaceutical compositions can be prepared, for example, as described in Gennaro et al., Remington's Pharmaceutical Sciences (18 th They may be prepared by any method well known in the art of pharmacy, such as those described in "Pharmaceutical preparations and their Manufacture," ed., Mack Publishing Company, 1990, especially Part 8: Pharmaceutical preparations and their Manufacture.

[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 pharmaceutical dosage formulation containing a compound according to the present invention and one or more additional therapeutic agents, as well as administering a compound according to the present invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation.

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

[0284] One or more other drugs and the compound according to the present invention can 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 within a period and in an amount and manner sufficient to ensure that an advantageous or synergistic effect is achieved. It will be understood that the preferred method and order of administration and the respective dosages and regimes for each component of the combination will depend on the specific other drugs and compounds of the present invention administered, their administration routes, the specific condition, particularly tumor, being treated, and the specific host being treated.

[0285] Pharmacology Testing In the pharmacological tests described below, the following compounds are described: Compound A: (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 A1: (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.2HCl.xH2O (x=2-3), Compound A3: (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 oxalate salt. Compound A4: Crystal form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4 triazin-6-yl)oxy)benzamide bisbesylate hydrate The results from these pharmacological studies clearly demonstrate the biological activity 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.

[0286] 1) Menin / MLL homogeneous time-resolved fluorescence (HTRF) assay To a non-treated white 384-well microtiter plate, 40 nL of 200X test compound in DMSO and 4 μL of 2X terbium chelate-labeled menin (preparation see below) 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 menin for 30 minutes at ambient temperature, 4 μL of 2X FITC-MBM 1 peptide (FITC-β-alanine-SARWRFPARPGT-NH2) ("FITC" means 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 menin-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 emission 490 nm / FITC emission 520 nm) at ambient temperature. The extent of fluorescence resonance energy transfer (HTRF value) is calculated as the ratio of the fluorescence emission intensities of the FITC and terbium fluorophores (F em 520nm / F em 490 nm). Final concentrations of reagents in the binding assay are 200 pM terbium chelate-labeled menin, 75 nM FITC-MBM1 peptide and 0.5% DMSO in assay buffer. Dose-response titration of test compounds is performed using an 11-point, four-fold serial dilution scheme, typically starting at 10 μM.

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

[0288] Preparation of terbium cryptate labeling of menin: Menin (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 menin was buffer exchanged into 1× Hepes buffer. 6.67 μM of menin was incubated with an 8-fold molar excess of NHS (N-hydroxysuccinimide)-terbium cryptate for 40 min at room temperature. Half of the labeled protein was purified from free label by running the reaction on a NAP5 column with elution buffer (0.1 M Hepes, pH 7 + 0.1% BSA (bovine serum albumin)). The other half was eluted with 0.1 M phosphate-buffered saline (PBS), pH 7. 400 μl of each eluate was collected, aliquoted, 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) Proliferation assay The anti-proliferative effects of menin / MLL protein / protein interaction inhibitor test compounds were evaluated in human leukemia cell lines. The cell line MOLM-14 has an MLL translocation and expresses the MLL fusion protein MLL-AF9 and the wild-type protein from the second allele, respectively. OCI-AML3 cells with NPM1c gene mutation were also tested. MLL-rearranged cell lines (e.g., MOLM-14) and NPM1c mutant cell lines show a stem cell-like HOXA / MEIS1 gene expression signature. To exclude compounds that show a general cytotoxic effect, 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 0.3–2.5 million cells / mL in culture and did not exceed 20 passages.

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

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

[0294] Absolute IC 50 Values ​​were calculated as percentage change in confluence as follows: LC = low control: cells treated with, for example, 1 μM of the cytotoxic agent staurosporine or cells treated with, for example, a high concentration of a surrogate reference compound; HC = high control: average confluence (%) (DMSO-treated cells); Effect%=100-(100 * (Sample-LC) / (HC-LC)); and IC was calculated using GraphPad Prism (version 7.00). 50 A dose-response equation was used to plot % effect versus Log10 compound concentration with a variable slope, maximum fixed at 100% and minimum fixed at 0%.

[0295] 2b) MEIS1 mRNA expression assay MEIS1 mRNA expression upon compound treatment was examined by Quantigene Singleplex assay (Thermo Fisher Scientific). This technology allows direct quantification of mRNA targets using probes hybridizing to defined target sequences of interest, and the signal is detected using the multimode plate reader Envision (PerkinElmer). The MOLM-14 cell line was used for this experiment. Cells were seeded at 3,750 cells / well in 96-well plates in the presence of increasing concentrations of compounds. After 48 h of incubation with compounds, cells were lysed in lysis buffer and incubated at 55 °C for 45 min. 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. Cell lysates were then transferred to a custom assay hybridization plate (Thermo Fisher Scientific) and incubated at 55 °C for 18–22 h. The plate was then washed to remove unbound material, after which preamplifier, amplifier, and labeled probe were added sequentially. Signals (= gene counts) were measured using the multimode plate reader Envision. IC 50was calculated by dose-response modeling using appropriate software. For all non-housekeeper genes, the response is equal to the counts corrected for background and relative expression. For each sample, each test gene signal (background subtracted) was divided by the normalized gene signal (RPL28: background subtracted). Fold changes were calculated by dividing the normalized value of the treated sample by the normalized value of the DMSO-treated sample. The fold change of each target gene was calculated as IC 50 was used to calculate.

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

[0297] [Table 5]

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

[0299] Plasma and / or whole blood samples were collected at desired time points from the dorsal metatarsal vein by continuous capillary microsampling (approximately 0.03 mL) using EDTA as an anticoagulant. Compound concentrations in plasma and blood samples were analyzed using a qualified LC-MS / MS method. In silico analysis of main 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 test compounds in human and mouse liver microsomes and to provide quantitative information on turnover rates (i.e., to determine the apparent intrinsic clearance of the test compounds).

[0301] Test items were prepared at a stock concentration of 10 mM in DMSO. To study metabolic turnover, final working solutions were prepared by adding 2 μL of a 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 master solution containing liver microsomes in 100 mM PBS (phosphate buffered saline) was prepared at pH 7.4. Then, 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 pre-heated at 37°C for 10 minutes in the incubation plate. The metabolic reaction was started by adding 5 μL of a 100 μM working solution of the test compound or positive control compound to the incubation plate (final test item concentration = 1 μM). The reaction final mixture should contain 1 mM NADPH, 0.5 mg / mL microsomal protein and 1 μM test compound or positive control compound in 100 mM PBS at pH 7.4. The percentage of organic solvents in the incubation mixture is 1%, DMSO≦0.02%.

[0303] Reactions were quenched at selected time points by transferring 50 μL of the incubated mixture to a quench plate containing 200 μL of cold methanol. After all time points were sampled, the quench plate was centrifuged at 4000 rpm for 40 min to precipitate proteins. A total of 90 μL of supernatant was transferred to the analysis plate and ultrapure H2O water was added to each well for LC / MS / MS analysis. All incubations and analyses were performed in duplicate.

[0304] Data analysis All calculations were performed using Microsoft Excel. Slope values ​​k were determined by linear regression of the natural logarithm of the percentage parent drug remaining versus 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 1 / 2 =-(0.693 / k) In vitro 1 / 2 In vitro intrinsic clearance (in vitro CL int Conversion to units (μL / min / mg protein) was performed using the following formula:

[0306]

number

[0307] [Table 6] "NA" means not analyzed

[0308] 5) Protocol for pharmacodynamic (PD) activity in subcutaneous (sc or SC) xenografts of MOLM-14 or OCI-AML3 cells Test drugs and controls Compound A3 was formulated in 20% hydroxypropyl-β-cyclodextrin (HP-β-CD) and prepared to reach a total volume of 0.2 mL per dose (10 mL / kg) for 20 g animals. Doses were adjusted daily by individual body weight. Working stocks of Compound A3 were prepared once a week for each experiment and stored at room temperature. Compound A3 was administered daily orally (PO).

[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 / -) bearing MOLM-14 or OCI-AML3 tumors were treated with vehicle or compound three times a day. Plasma samples were collected 23 hours after the second dose, 0.5 hours after the final dose, and 16 hours after the final dose, and tumor samples were collected 16 hours after the final dose. To examine the effect of compounds on the expression of multiple menin-MLL target genes (e.g., MEIS1, MEF2C, FLT3), QuantiGene Plex technology (Thermo Fisher Scientific) was used. 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 custom assay hybridization plates (Thermo Fisher Scientific) and incubated at 54°C for 18-22 hours. The plates were then transferred to magnetic separation plates and washed to remove unbound material from the beads, followed by sequential hybridization consisting of preamplifier, amplifier and labeled probe, followed by streptavidin-phycoerythrin binding. Signal from the beads was measured on a Luminex FlexMap three-dimensional instrument. For all non-housekeeper genes, the response is equal to the counts corrected for background and relative expression. For each sample, each test gene signal (background subtracted) was divided by the normalized gene signal (RPL19, RPL28, ATP6V1A, background subtracted). Fold changes were calculated by dividing the normalized values ​​of treated samples by the normalized values ​​of 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 replicate experiments under optimized conditions using fresh tumor samples.

[0313] [Table 9]

[0314] [Table 10]

[0315] 6) Efficacy study in MOLM-14 subcutaneous model Test drugs and controls Compound A3 was formulated in 20% hydroxypropyl-β-cyclodextrin (HP-β-CD) and prepared to reach a total volume of 0.2 mL per dose (10 mL / kg) for 20 g animals. Doses were adjusted daily by 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 approximately 6-8 weeks old and weighing approximately 25 g. All animals were allowed to acclimate to and recover from any shipping-related stress for a minimum of 7 days prior to experimental use. Autoclaved water and irradiated food were provided ad libitum and animals were maintained on a 12-hour light / dark cycle. Cages, bedding, and water bottles were autoclaved prior to use and changed weekly. Further details are provided 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] Inject 5 x 10 cells in 50% Matrigel into the right flank of each mouse in a total volume of 0.2 mL using a 1 cc syringe and 27 gauge needle. 6 MOLM-14 cells were administered.

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

[0321] Day 0 is the day of tumor cell implantation and initiation of the experiment.

[0322] Mice bearing SC MOLM-14 tumors were randomized 16 days after tumor implantation and tumor volume (average ∼130 mm) was determined. 3 Mice were assigned to treatment groups according to the 10-day follow-up time (n=10 / group). Treatment with vehicle or Compound A3 (30 and 100 mg / kg) was initiated on the same day and administered orally daily for 21 days. Plasma was collected for PK analysis at 1, 2, 4, 8, and 23 hours after the last dose (n=4-5 / group / time point).

[0323] Animal monitoring SC tumor volumes were measured for each animal two to three or more times weekly throughout the experiment.

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

[0325] ΔTGI% was defined as the difference between the mean tumor burden in the treatment and control groups, ΔTGI% = ([(TV c TVc0)(TV t TV t0 )] / (TV c TVc0))×100 (in the formula, “TV c " is the average tumor burden in a given control group, "TVc0" is the average initial tumor burden in a given control group, and "TV t ” is the mean tumor burden in the treatment group, and “TV t0 The mean initial tumor burden in the treatment group was calculated as % TGI (TGI = 0.01; 1 / 10 ...

[0326] The mean tumor volumes for the treatment and control groups were calculated as follows:

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

[0328] Tumor regression (TR)% was quantified to reflect treatment-related reduction in tumor volume compared to baseline independent of the control group, as follows: TR%=(1mean(TV t i / TV t0 i)) × 100, where "TV t i” is the tumor burden of individual animals in the treatment group, and “TV t0 i" is the initial tumor burden of the animal.

[0329] 4. Data Analysis Tumor volumes were graphed using Prism software (GraphPad version 7 or 8). Statistical significance for most experiments was assessed for Compound A3-treated groups compared to HPβCD vehicle-treated controls on the last day of the experiment when more than ⅔ of the mice remained in each group. Differences between groups were considered significant when p≦0.05.

[0330] Statistical significance of animal tumor volumes 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 animal as a random effect. When individual longitudinal response trajectories were not linear, logarithmic transformation was performed.

[0331] Information derived from this model was used to perform pairwise treatment comparisons of tumor volumes in the control group or among all treatment groups, the results of which are shown in Figure 2.

[0332] 7) Ca 2+ Electrocardiophysiological effects of test compounds on synchronized beating of human pluripotent stem cell-derived cardiomyocytes (hSC-CM) using a fluorescent assay (CTCM human) protocol Compounds were tested in 96-well plates.

[0333] Compounds were tested at 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2.5 μM and 5 μM (n=4 / dose) in Cor.4U®-cardiomyocytes or iCell® cardiomyocytes2.

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

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

[0336] Vehicle control: dimethylsulfoxide (DMSO). Solutions of compounds in DMSO or their solvents (final concentration of 0.1% DMSO, n=8).

[0337] Preparation of test substances and controls Compounds to be tested were dissolved in DMSO at 1000x the intended concentration. Compound "mother plates" were made containing test compounds as well as positive and negative controls at 1000x the final concentration. On the day of the experiment, these stock solutions were diluted (in round-bottom compound plates) to 2x the intended concentration with Tyrode (Sigma) supplemented with 10 mM HEPES (Gibco). The final DMSO concentration in the test solutions and vehicle controls was 0.1%.

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

[0339] A second lineage of hSC-derived cardiomyocytes, called iCell® Cardiomyocytes 2, was purchased from FUJIFILM Cellular Dynamics (USA). Experiments with test drugs are performed 5-7 days after plating the cells on the plate to have a live, 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 (≈5 million cells / vial) and plated into three 96-well plates (approximately 50K / well).

[0340] Before the experiment begins At least 1 h before the start of the experiment, normal cell culture medium was replaced with Tyrode's solution containing a calcium dye (see below).

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

[0342] 35 μl of cell culture medium was removed from each well and replaced with 35 μl of pre-warmed Cal 520 dye solution and the cell plate was incubated for 45 minutes at 37° C. / 5% CO2. Cells were 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 well. A bottle of Cal520 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 was added to 10 mL of Tryodes solution to give a dye concentration of 4.5 μM. 35 μl of this dye solution was then added to each well to give a final dye concentration of 1.58 μM. The current dye protocol for this CTCM human assay has been 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 morphology of transients) was measured using a functional drug screening system (FDSS / μCell, Hamamatsu, Japan) and the recordings were subsequently analyzed offline using appropriate software, e.g., Notocord.

[0345] Load the cell plate into the FDSS / μCell for the test run and measure Ca 2+Transients were measured for 4 minutes to confirm the synchronous 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 warmed to 37°C). If all showed synchronous beating, the 96-well plate was measured in triplicate (wells that did not meet the pre-set criteria to verify the synchronous beating of all 96 wells at baseline were excluded from the experiment and not treated with compounds). T=0: control period (-5 to -1 min) + compound addition, followed by 3 min. T=30: Measured 29-34 minutes after compound addition

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

[0347] Data was analyzed offline using appropriate software, for example Notocord-Hem (version 4.3).

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

[0349] Beat rate (BR) Ca 2+ The amplitude of the transient (Amp), -CTD 90 : Ca at 90% 2+ Duration of the transient (time to reach 90% of the initial base value).

[0350] A variety of "arrhythmia-like" activities were also noted during the study. These included the following:

[0351] "Early afterdepolarization-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 a very fast rate), or "Ventricular fibrillation-like" (VF-like) events ("small amplitude, fast-rate Ca events with irregularities and non-measurable transients" 2+ (defined as "waveform") The “beating arrest” (Ca 2+ No transients observed).

[0352] When compound-induced changes in calcium transient signals could not be analyzed by the software, these signals were identified as BQL (below quality analysis level).

[0353] 4. Data Analysis The measured data from FDSS-μCell was copied for offline analysis, analyzed, and uploaded to SPEC-II (our operation management system) for further analysis. The values ​​of the variables before and after the administration of the compound were collected and transferred to Excel workbook.

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

[0355] Quality control of hiPSC-CMs in plates: Plates were rejected if they did not meet the following criteria: A steady, regular heartbeat Over 500 relative unit amplitudes Heart rate between 25 and 80 beats per minute CTD of 300 to 800 ms 90 .

[0356] In this experiment, the hiPSC-CMs in the plates met the above criteria.

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

[0358] No risk: Effect level or small, insignificant changes in vehicle.

[0359] Low Risk: Meaningful effect, but risk of cardiac liability is probably low.

[0360] High risk: Relatively high risk for cardiac strain.

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

[0362] The "Hazard Score" result identifies potential acute cardiac drug-induced effects at free drug equivalents (as no plasma proteins were added to the wells). The assessment of hazard identification was performed using a scoring reference book called CTCM_Scoring_version1 (Kopljar ​​et al., Stem Cell Reports 2018.11:1365-1377) and indicates the levels according to the following color system in Table 10:

[0363] [Table 12]

[0364] Ca measured in HiPSc-CMs are listed above in different colors in the associated table. 2+ Ranking of test compounds by hazard score severity for the transient assay.

[0365] result iCell® Cardiomyocytes 2 was used as the cell line Positive and negative controls: All positive and negative controls were It had the expected pharmacological effect in this assay, and the results are summarized in Tables 11 and 12 below.

[0366] [Table 13]

[0367] [Table 14]

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

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

[0370] [Table 15]

[0371] method Experiments were performed with CHO cells stably expressing the hERG potassium channel. 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). QPatch (Sophion) cells were harvested to obtain a single cell suspension for use in an automated patch clamp system.

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

[0374] The holding potential was -80 mV. hERG current (K + Selective outward currents (OCR) were measured as the maximum tail current at -40 mV after a 2 s depolarization to +60 mV. The pulse cycling rate was 15 s. A short pulse (90 ms) to -40 mV served as a baseline step for calculating tail current amplitudes. After establishing the whole-cell configuration and a stabilization period, a solvent control (0.3% DMSO) was applied for 5 min, followed by test substances at 3 × 10 -7 M, 3×10 -6 M, 10 -5 M and 3×10 -5Four increasing concentrations of M were applied. Each concentration of test substance was applied twice. The results for each concentration were measured as the mean current of three consecutive voltage pulses after 5 min. To examine the extent of blockage, the residual current was compared to vehicle pretreatment.

[0375] Concentration / response relationships were calculated by nonlinear least-squares fitting to the individual data points. Half-maximal inhibitory concentrations (IC50) were calculated by the fitting routine.

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

[0377] [Table 16]

[0378] 9) Efficacy study in disseminated OCI-AML3 model Test drugs and controls Compound A3 was formulated in 20% hydroxypropyl-β-cyclodextrin (HP-β-CD) and prepared to reach a total volume of 0.2 mL per dose (10 mL / kg) for 20 g animals. Doses were adjusted daily by 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 when approximately 6-8 weeks old and weighing approximately 25 g. All animals were allowed to acclimate to and recover from any shipping-related stress for a minimum of 7 days prior to experimental use. Autoclaved water and irradiated food were provided ad libitum and animals were maintained on a 12-h light-dark cycle. Cages, bedding, and water bottles were autoclaved prior to use and changed weekly. Tissue culture and cell injection reagents are summarized in Table 14 below.

[0380] [Table 17]

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

[0382] For the disseminated OCI-AML3 model, 5×10 5 cells were administered to each mouse by IV injection at a total volume of 0.2 mL using a 26-gauge needle.

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

[0384] Day 0 is the day of tumor cell transplantation and the start of the experiment.

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

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

[0387] Calculation For survival evaluation, the results were plotted as the survival rate against the number of days after tumor transplantation. Negative clinical signs and / or a weight loss of 20% or more were used as surrogate endpoints for death. The Kaplan-Meier survival analysis was utilized to determine the median survival period. The increased percent of survival period (ILS) was calculated as ((median survival period of the treatment group - median survival period of the control group) / median survival period of the control group) × 100. Animals that could not reach the surrogate endpoint due to harmful clinical signs (e.g., ulcerated tumor, weight loss, etc.) or deaths unrelated to the treatment were excluded from the survival evaluation. An ILS of 25% or more is considered to be biologically significant as defined by the 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 evaluated as described above. Statistical significance was evaluated for Kaplan-Meier survival plots comparing the treatment group to the appropriate vehicle-treated control using the log-rank (Mantel-Cox) test of R software version 3.4.2. A difference between groups was considered significant if the p-value was ≤ 0.05.

[0389] Survival The Kaplan-Meier survival curves are shown in Figure 3. Mice with established OCI-AML3 tumors were orally administered 30, 50, or 100 mg / kg of Compound A3 in 20% HP-β-CD formulation daily for a total of 28 days (n = 9 - 10 / group). The median survival time of the vehicle-treated control group was 38.5 days. In the Compound A3 treatment groups, the median survival times reached 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, respectively) compared to that of control mice (p ≤ 0.001). This was a biologically significant ILS according to the NCI criterion threshold of an ILS of more than 25% (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-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bis besylate hydrate. The bis besylate hydrate was found to be chemically and physically stable, with no decomposition observed by UHPLC and no solid-state changes observed by XRD under the stress conditions evaluated.

[0391]

Table 18

Claims

1. (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 【Chemical 1】 or a solvate thereof.

2. The pharmaceutical composition according to claim 1, wherein the compound is a solvate of besylate.

3. The pharmaceutical composition according to claim 2, wherein the compound is a hydrate of besylate.

4. The pharmaceutical composition according to claim 1, wherein the besylate is bisbesylate.

5. The pharmaceutical composition according to claim 4, wherein the compound is a solvate of bisbesylate.

6. The pharmaceutical composition according to claim 5, wherein the compound is a hydrate of bisbesylate.

7. wherein the compound is crystalline form A of (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide bisbesylate hydrate, and the crystalline form produces an X-ray powder diffraction pattern comprising peaks at 2 theta of 5.4, 7.2, 11.1, 11.9, and 21.7 degrees plus or minus 0.2 degrees of 2 theta. The compound according to claim 1.

8. The compound according to claim 7, wherein the X-ray powder diffraction pattern may further comprise at least one peak selected from 2 theta of 13.7, 14.5, 14.7, 15.0, 16.5, 17.8, 19.0, 19.4, and 20.1 degrees plus or minus 0.2 degrees of 2 theta.

9. The compound according to claim 7, further characterized by an X-ray powder diffraction pattern substantially as shown in FIG.

1.

10. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent.

11. A process for preparing the pharmaceutical composition according to claim 10, comprising mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of the compound according to any one of claims 1 to 9.

12. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 for use as a medicament.

13. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 for use in the prevention or treatment of cancer.

14. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 for use in the prevention or treatment of leukemia, myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN).

15. The pharmaceutical composition according to claim 14 for use in the prevention or treatment of leukemia, wherein the leukemia is (NPM1) mutant leukemia.

16. The pharmaceutical composition according to claim 14 for use in the prevention or treatment of leukemia, wherein the leukemia is selected from acute leukemia, chronic leukemia, myeloblastic leukemia, myelogenous leukemia, lymphoblastic leukemia, and lymphocytic leukemia.

17. The pharmaceutical composition according to claim 14 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 prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, hairy cell leukemia (HCL), MLL-rearranged leukemia, MLL-PTD leukemia, MLL-amplified leukemia, MLL-positive leukemia, and leukemia showing HOX / MEIS1 gene expression signature.

18. The pharmaceutical composition according to claim 14 for use in the prevention or treatment of leukemia, wherein the leukemia is acute leukemia.

19. The pharmaceutical composition according to claim 18 for use in the treatment of acute leukemia, wherein the acute leukemia is AML.

20. The pharmaceutical composition according to claim 18 for use in the treatment of acute leukemia, wherein the acute leukemia is ALL.

21. The pharmaceutical composition according to claim 18, wherein the acute leukemia has KMT2A gene modification or NPM1 mutation.

22. The pharmaceutical composition according to claim 18, wherein the acute leukemia has KMT2A gene rearrangement.

23. The pharmaceutical composition according to claim 18, wherein the acute leukemia has NPM1 mutation.

24. A process for preparing the compound of crystalline form A according to any one of claims 7 to 9, comprising the step of recrystallizing (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, wherein the recrystallization comprises a) adding (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 or a hydrate or solvate thereof to a suitable mixture of solvents in the presence of benzenesulfonic acid and adjusting the temperature to a range from about 20 °C to the reflux temperature of the solvent; b) seeding with crystalline form A; c) obtaining a precipitate of the crystalline form according to any one of claims 7 to 9.

25. The process according to claim 24, wherein the suitable mixture of solvents is a mixture of acetone, water and IPAc.

26. The process according to claim 24, wherein the suitable mixture of solvents is a mixture of isopropanol, water and IPAc.

27. The process according to claim 24, wherein the temperature is about 25 °C.

28. 【Figure 2】 A crystalline form of citrate, which produces an X-ray powder diffraction pattern comprising peaks at 2 theta of 5.82, 10.09, and 18.42 degrees plus or minus 0.2 degrees of 2 theta.

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