Crystalline forms of inhibitors of menin / MLL interaction

Abstract

The present invention relates to crystalline forms of inhibitors of the menin / mixed lineage leukemia (MLL) protein-protein interaction. The present invention also relates to pharmaceutical compositions comprising crystalline forms of inhibitors of the menin / mixed lineage leukemia (MLL) protein-protein interaction. These crystalline forms and pharmaceutical compositions comprising the crystalline forms may be useful in treating diseases such as cancer.

Description

[Technical field]

[0001] The present invention relates to crystalline forms of inhibitors of the menin / mixed lineage leukemia (MLL) protein-protein interaction. The present invention also relates to pharmaceutical compositions comprising crystalline forms of inhibitors of the menin / mixed lineage leukemia (MLL) protein-protein interaction. These crystalline forms and pharmaceutical compositions comprising the crystalline forms may be useful in treating diseases such as cancer. [Background technology]

[0002] Chromosomal rearrangements affecting the mixed lineage leukemia genes (MLL; MLL1; KMT2A) cause aggressive acute leukemia across all age groups and remain largely incurable, highlighting the urgent need for novel therapeutic approaches. Acute leukemias with these chromosomal translocations of MLL can be lymphocytic, myeloid, or biphenotypic and constitute 5-10% of acute leukemias in adults and approximately 70% in infants.

[0003] MLL is a histone methyltransferase that methylates histone H3 at lysine 4 (H3K4) and functions in a multiprotein complex. The use of inducible loss-of-function alleles of MLL1 demonstrated that MLL1 plays a crucial role in hematopoietic stem cell (HSC) maintenance and B cell development, but that its histone methyltransferase activity is dispensable for hematopoiesis.

[0004] Fusions of MLL with over 60 different partners have been reported to date and have been associated with leukemia formation / progression. 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. Recruitment of chromatin-modifying enzymes such as Dot1L and / or pTEFb complex by the fusion partner enhances 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.

[0005] Menin, encoded by the Multiple Endocrine Neoplasia type 1 (MEN1) gene, is ubiquitously expressed and mainly localized in the nucleus. It has been shown to interact with numerous proteins and therefore participate in various cellular processes. The best understood function of menin is its role as an oncogenic cofactor of MLL fusion proteins. Menin interacts with two motifs within the N-terminal fragment of MLL that are conserved in all fusion proteins, menin-binding motif 1 (MBM1) and MBM2. The menin / MLL interaction creates a new interaction surface for lens epithelium-derived growth factor (LEDGF). Although MLL directly binds to LEDGF, menin is essential for stable interaction between MLL and LEDGF and for gene-specific chromatin recruitment of the MLL complex via the PWWP domain of LEDGF. Furthermore, numerous genetic studies have demonstrated that menin is strictly required for oncogenic transformation by MLL fusion proteins, suggesting that the menin / MLL interaction is an attractive therapeutic target. For example, conditional deletion of MEN1 blocks leukocyte formation in myeloid progenitors that ectopically express MLL fusions. Similarly, genetic disruption of the menin / MLL fusion interaction by loss-of-function mutations abrogates the oncogenic properties of MLL fusion proteins, blocks leukemia development in vivo, and relieves the differentiation block of MLL-transformed leukemic blasts. These studies also demonstrated that menin is required for the maintenance of HOX gene expression by MLL fusion proteins. In addition, small molecule inhibitors of the menin / MLL interaction have been developed, suggesting the druggability of this protein / protein interaction and demonstrating efficacy in preclinical models of AML. Together with the observation that menin is not an essential cofactor for MLL1 during normal hematopoiesis, these data establish that disruption of the menin / MLL interaction represents a promising new therapeutic approach for treating MLL-rearranged leukemias and other cancers with an active HOX / MEIS1 gene signature.For example, intragenic partial tandem duplication (PTD) within the 5' region of the MLL gene represents another major abnormality found primarily in de novo and secondary AML and myelodysplastic syndromes. Although the molecular mechanisms and biological functions of MLL-PTD are not fully understood, new therapeutic targeting strategies affecting menin / MLL interaction may also prove effective in the treatment of MLL-PTD-associated leukemia. Furthermore, castration-resistant prostate cancer has been shown to be dependent on menin / MLL interaction.

[0006] The MLL protein is also known in the scientific community as the histone-lysine N-methyltransferase 2A (KMT2A) protein (UniProt accession number Q03164).

[0007] WO 2022 / 253167 relates to menin / MLL protein / protein interaction inhibitors. Summary of the Invention

[0008] The present invention is directed to a crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide having the following structure:

[0009] [ka]

[0010] In one embodiment, the crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide is a crystalline free base form.

[0011] In one embodiment, the crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide is a crystalline HCl salt form, particularly a crystalline mono-HCl salt variable hydrate, more particularly a crystalline mono-HCl salt trihydrate.

[0012] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide.

[0013] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide and a pharma- ceutically acceptable carrier or excipient.

[0014] Furthermore, the present invention relates to crystalline forms of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide for use as a medicament and to crystalline forms of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide for use in the treatment or prevention of cancer, including but not limited to leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0015] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide for use in the treatment or prevention of cancer, including but not limited to leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0016] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide and a pharma- ceutically acceptable carrier or excipient for use in the treatment or prevention of cancer, including but not limited to leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes. In a particular embodiment, the present invention relates to a crystalline form of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide for use in the treatment or prevention of cancer.

[0017] In certain embodiments, the cancer is selected from leukemia, lymphoma, myeloma, or solid tumor cancer (such as, for example, prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma, and glioblastoma). In some embodiments, the leukemia is selected from the group consisting of acute leukemia, chronic leukemia, myeloid leukemia, myelogenous leukemia, lymphoblastic leukemia, lymphocytic leukemia, acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML), acute lymphoblastic leukemias (ALL), chronic lymphocytic leukemias (CLL), T cell prolymphocytic leukemias (T-PLL), large granular lymphocytic leukemia, hairy cell leukemia, and / or idiopathic leukemia. These include MLL-rearranged leukemia, MLL-PTD leukemia, MLL-amplified leukemia, MLL-positive leukemia, and leukemia with HOX / MEIS1 gene expression signature.

[0018] In particular, the compounds according to the invention and pharmaceutical compositions thereof may be useful in the treatment or prevention of leukemia, in particular nucleophosmin (NPM1) mutant leukemia, such as NPM1c.

[0019] In one embodiment, compounds according to the invention may have improved metabolic stability properties.

[0020] In one embodiment, compounds according to the invention may have extended in vivo half-lives (T1 / 2).

[0021] In one embodiment, compounds according to the invention may have improved oral bioavailability.

[0022] 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.

[0023] 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.

[0024] In one embodiment, the compounds of formula (I) and their pharma- ceutically acceptable salts and solvates may have an improved safety profile (e.g., reduced hERG inhibition, improved cardiovascular safety).

[0025] In one embodiment, compounds according to the invention may be suitable for QD dosing (once per day).

[0026] 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 syndrome (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0027] 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.

[0028] The present invention relates to products comprising compounds according to the invention and pharmaceutical agents as combined preparations for simultaneous, separate or sequential use for use in the treatment or prevention of cancer, including but not limited to leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0029] Furthermore, the present invention relates to a method for treating or preventing a cell proliferative disorder in a warm-blooded animal, the method comprising administering to the animal an effective amount of a compound according to the invention as defined herein, or a pharmaceutical composition or combination as defined herein. [Brief description of the drawings]

[0030] 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. In the drawings: [Figure 1] Figure 2 is an X-ray powder diffraction (XRPD) pattern of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide as the crystalline free base form. [Diagram 2] Figure 13 is the X-ray powder diffraction (XRPD) pattern of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide as the crystalline HCl salt form. [Diagram 3] Dynamic vapor sorption (DVS) isotherm plot of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide as the crystalline HCl salt form. [Figure 4] FIG. 1 is a dynamic vapor sorption (DVS) mass change plot of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide as the crystalline HCl salt form. Detailed Description of the Invention

[0031] As used herein, the term "compound of the invention" or "compound according to the invention" is meant to include crystalline forms of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide, such as the crystalline HCl salt form and the crystalline free base form.

[0032] As used herein, the term "subject" refers to an animal, preferably a mammal (e.g., a cat, dog, primate, or human), more preferably a human, who is or has been the object of treatment, observation, or experiment.

[0033] The term "therapeutically effective amount," as used herein, means an amount of an active compound or pharmaceutical agent that elicits the biological or medical 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.

[0034] 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.

[0035] As used herein, the term "treatment" is intended to refer to any process that can slow, hinder, inhibit, or halt the progression of a disease, although it does not necessarily indicate the complete disappearance of all symptoms.

[0036] The present invention also encompasses isotopically labeled compounds of the present invention that are identical to those enumerated herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature (or the most abundant atom found in nature), as will be apparent to one of ordinary skill in the art.

[0037] All isotopes and isotopic mixtures of any particular atom or element identified herein, whether naturally occurring or synthetically produced, either at natural abundance or in isotopically enriched form, are contemplated within the scope of the compounds of the invention.

[0038] In one embodiment, the compound of the invention is 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide as the crystalline free base form.

[0039] In one embodiment, the compound of the invention is 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide as a crystalline HCl salt form, in particular 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N The crystalline mono-HCl salt of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide is a variable hydrate, more specifically a crystalline mono-HCl salt trihydrate of 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidin-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridin-1-yl]-5-fluoro-N-isopropyl-benzamide.

[0040] The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention.

[0041] The present invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present invention and a pharma- ceutically acceptable carrier or excipient.

[0042] Experimental Department Several methods for preparing the compounds of the present invention are illustrated in the following examples. Unless otherwise specified, all starting materials were obtained from commercial suppliers and used without further purification, or could be synthesized by one of ordinary skill in the art by using well-known methods.

[0043] [Table 1-1]

[0044] [Table 1-2]

[0045] [Table 1-3]

[0046] Compounds or intermediates isolated as salt forms may be of integer stoichiometry, i.e., mono- or di-salt, or of intermediate stoichiometry. When an intermediate or compound in the experimental section below is designated as "HCl salt" without specifying the number of equivalents of HCl, this means that the number of equivalents of HCl was not determined.

[0047] The stereochemical configuration about a center in some compounds / intermediates may be designated as "R" or "S" if the mixture has been isolated and the absolute stereochemistry is known, or if only one enantiomer has been obtained and the absolute stereochemistry is known; for some intermediates, if the intermediate itself has been isolated as a single stereoisomer and is enantiomerically pure, but the absolute stereochemistry has not been determined (even if the bonds are drawn stereospecifically), the stereochemical configuration about the named center may be designated as "R" or "S" * R" or " * It is written as "S". * When a compound marked "R" is converted into another compound, the resulting compound's " * The "R" designation comes from the starting material.

[0048] For example, intermediate 18

[0049] [ka] but,

[0050] [ka] It is clear that this is the case.

[0051] 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.

[0052] If stereochemistry is not specified, this means a mixture of stereoisomers or the stereochemistry is not determined, unless otherwise specified or clear from the context.

[0053] When a stereocenter is designated "RS," this means that a racemic mixture was obtained at the indicated center, unless otherwise indicated.

[0054] A double bond designated EZ means that the compound / intermediate was obtained as a mixture of E and Z isomers.

[0055] Preparation of intermediates and compounds For intermediates that were used in the next reaction step either as crude intermediates or as partially purified intermediates, in some cases no molar amount is mentioned for such intermediate in the next reaction step or an estimated or theoretical molar amount is indicated for such intermediate in the next reaction step in the reaction protocols described below.

[0056] Preparation of intermediate 1:

[0057] [ka]

[0058] To a solution of 4-bromo-1H-pyrrolo[2,3-c]pyridine (2 g, 95% purity, 9.64 mmol) in 1,4-dioxane (30 mL) and water (4 mL) was added 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (7.26 g, 50% in THF, 28.9 mmol) and potassium carbonate (4.0 g, 28.9 mmol). The suspension was degassed and replaced with N2 twice. [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (706 mg, 0.964 mmol) was added to the reaction mixture. The reaction mixture was heated to 100 °C and stirred at this temperature overnight. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated. The resulting residue was purified by silica gel column chromatography eluting with 0%-80% ethyl acetate in petroleum ether to give intermediate 1 (1.01 g, purity 95%, yield 75.3%).

[0059] Alternatively, intermediate 1 can be prepared by the following procedure. In a 20 L 4-neck round bottom flask, 4-bromo-1H-pyrrolo[2,3-c]pyridine (1330 g, 6750 mmol, 1.00 equiv.), Pd(dppf)Cl2 (493.9 g, 675 mmol, 0.10 equiv.), K2CO3 (2798.69 g, 20250.21 mmol, 3.00 equiv.), 1,4-dioxane (13 L), HO (2 L), and 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (2542.01 g, 20250.21 mmol, 3.00 equiv.) were added at room temperature. The resulting mixture was stirred at 100° C. overnight. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (15 L). The aqueous layer was extracted with EtOAc (3×10 L) and the organic layer was washed with water (2×5 L). The resulting liquid was dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography eluting with 10% methanol in dichloromethane to give Intermediate 1 (640 g, yield: 72%) as a grey solid.

[0060] Preparation of intermediate 2:

[0061] [ka]

[0062] To a solution of intermediate 1 (918 mg, 95% purity, 6.6 mmol) in DMF (60 mL) at 0° C. was added dropwise a solution of N-bromosuccinimide (1.17 g, 6.6 mmol) in DMF (10 mL). The reaction mixture was stirred at this temperature for 30 min. The reaction mixture was quenched with water and extracted twice with ethyl acetate (50 mL). The organic layer was washed with brine (25 mL), dried over sodium sulfate, filtered and concentrated to give the crude product, which was purified by silica gel column chromatography eluting with 0% to 60% ethyl acetate in petroleum to give intermediate 2 (1.14 g, 97.1% purity, 79.5% yield) as a white solid.

[0063] Alternatively, intermediate 2 can be prepared by the following procedure. In a 10 L 4-neck round bottom flask, intermediate 1 (640 g, 4842.39 mmol, 1.00 equiv) and DMF (5.00 L) were added at room temperature. To the above mixture, NBS (861.87 g, 4842.40 mmol, 1.00 equiv) was added portionwise at room temperature over 1 h. The resulting mixture was stirred at room temperature for another 30 min. The reaction was quenched by adding an aqueous solution of Na2S2O3 (10 L, 10% (w / v)) at room temperature. The aqueous layer was extracted with EtOAc (3×5 L) and the organic layer was washed with brine (1×5 L). The resulting liquid was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography eluting with 20% ethyl acetate in petroleum ether to give intermediate 2 (800 g, yield: 78%) as a grey solid.

[0064] Preparation of intermediate 4:

[0065] [ka]

[0066] To a solution of intermediate 2 (1.14 g, 97.1% purity, 5.24 mmol) in DMF (80 mL) was added 5-fluoro-2-iodobenzoic acid (1.40 mg, 5.24 mmol), copper powder (333 mg, 5.24 mmol), and potassium carbonate (2.18 g, 15.7 mmol). The reaction mixture was heated to 100° C. and stirred at this temperature overnight. After the mixture was cooled to room temperature, the reaction mixture was concentrated and the resulting residue was acidified to pH=about 3 with HCl (1N). The resulting mixture was filtered and the filter cake was washed twice with water. The filter cake was dried under vacuum to give crude intermediate 4 (1.8 g, 91% purity, 89.4% yield) as a yellow solid.

[0067] Alternatively, intermediate 4 can be prepared by the following procedure: In a 10 L 4-neck round bottom flask, intermediate 2 (560 g, 2653.24 mmol, 1.00 equiv), Cu (252.91 g, 3979.87 mmol, 1.50 equiv), K2CO3 (1100.08 g, 7959.74 mmol, 3.00 equiv), and 5-fluoro-2-iodobenzoic acid (705.79 g, 2653.24 mmol, 1.00 equiv) in DMF (6.00 L) were added at room temperature. The resulting mixture was stirred at 100 °C under nitrogen atmosphere for another 2 h. The resulting mixture was filtered, the filter cake was washed with DMF (1 x 5 L), and the filtrate was concentrated under reduced pressure. The resulting mixture was diluted with water (8 L). The mixture was acidified to pH = 3 with aqueous HCl (conc.). The precipitated solid was collected by filtration and washed with water (3×3 L). The resulting solid was dried under vacuum to give Intermediate 4 (1300 g, crude) as a grey solid.

[0068] Intermediate 110 was synthesized in a similar manner starting from intermediate 1.

[0069] [Table 2]

[0070] Preparation of intermediate 6:

[0071] [ka]

[0072] To a solution of intermediate 4 (1.8 g, 91% purity, 4.69 mmol) in DMF (50 mL) at 0° C., HATU (4.46 g, 11.7 mmol), N,N-diisopropylethylamine (3.03 g, 23.5 mmol), and N-methylpropan-2-amine (858 mg, 11.7 mmol) were added. After addition, the mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the resulting residue was purified by silica gel column chromatography eluting with 0% to 5% methanol in dichloromethane to give intermediate 6 (2.0 g, 93% purity, 98.1% yield) as a yellow oil.

[0073] Alternatively, intermediate 6 can be prepared by the following procedure. In a 20 L 4-neck round bottom flask, intermediate 4 (920 g, 2634.90 mmol, 1.00 equiv., same as crude 1300 g), DMF (7.5 L), HATU (1102.06 g, 2898.39 mmol, 1.10 equiv.), and DIEA (1021.63 g, 7904.70 mmol, 3.00 equiv.) were added at room temperature. The resulting mixture was stirred at room temperature for another 30 min. To the above mixture, N-methylpropan-2-amine (211.99 g, 2898.39 mmol, 1.10 equiv.) was added dropwise over 10 min at 0° C. The resulting solution was stirred overnight at room temperature. The reaction was quenched by adding water (20 L) at room temperature. The aqueous layer was extracted with EtOAc (3×7 L) and the organic layer was washed with water (3×5 L). The resulting liquid was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 50% ethyl acetate in petroleum ether (1:1) to give intermediate 6 (700 g, yield: 66%) as a light yellow solid.

[0074] Intermediate 111 was synthesized by a similar method starting from intermediate 110.

[0075] [Table 3]

[0076] Alternative Approach to Prepare Intermediate 6 Intermediate 111 (1.3 g, 4.0 mmol) was dissolved in MeCN (40 mL). CuBr2 (2.7 g, 12 mmol) was then added and the mixture was stirred at room temperature for 5 h. 7N NH3 / MeOH (20 mL) was then added. The reaction mixture was stirred vigorously for about 30 min. Water (40 mL) and isopropyl acetate were then added. The layers were separated and the aqueous layer was extracted twice with isopropyl acetate. The organic layers were combined, washed with brine, dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by silica gel column chromatography eluting with 0%-3% methanol in dichloromethane to give intermediate 6 (1.2 g, 72% yield) as an orange oil.

[0077] Preparation of intermediate 9:

[0078] [ka]

[0079] To a mixture of intermediate 6 (4 g, 4.312 mmol), tert-butyl 3-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)azetidine-1-carboxylate (2.92 g, 9.9 mmol), and potassium carbonate (2.7 g, 19.7 mmol) in 1,4-dioxane (70 mL) and water (23 mL) was added Pd(dppf)Cl2 (724 mg, 0.99 mmol). The mixture was degassed under nitrogen atmosphere three times and the reaction was stirred under nitrogen atmosphere at 100 °C for 16 h. After the mixture was cooled to room temperature, the reaction mixture was diluted with H2O and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with 90% ethyl acetate in petroleum ether to give intermediate 9 (1.8 g, 45.7% purity, 38.7% yield) as a yellow solid.

[0080] Preparation of intermediate 10:

[0081] [ka]

[0082] A mixture of intermediate 6 (12.0 g, 29.8 mmol), tert-butyl 3-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene)pyrrolidine-1-carboxylate (9.2 g, 29.8 mmol), and potassium carbonate (12.3 g, 89.1 mmol) in 1,4-dioxane (120 mL) and water (20 mL) was degassed and replaced with N2 twice. Pd(dppf)Cl2 (2.16 g, 2.95 mmol) was added and the reaction mixture was heated to 100 °C and stirred at this temperature overnight. After cooling the reaction mixture to room temperature, the resulting mixture was concentrated and the residue was purified by silica gel column chromatography eluting with 0% to 80% ethyl acetate in petroleum ether to give intermediate 10 (12.0 g, 79.4% yield) as a yellow oil.

[0083] Preparation of intermediate 15:

[0084] [ka]

[0085] A mixture of intermediate 9 (6.0 g, 12.2 mmol) in methanol (100 mL) was degassed under nitrogen atmosphere three times. 10 w / w% palladium on carbon (3 g) was added and the mixture was degassed under hydrogen atmosphere three times. The mixture was stirred under hydrogen atmosphere (balloon) at room temperature for 16 h. The mixture was filtered and the filtrate was concentrated and purified by silica gel column chromatography eluting with 50% ethyl acetate in petroleum ether to give intermediate 15 (5.2 g, 97% purity, 83.7% yield) as a yellow solid.

[0086] Preparation of intermediates 16, 17, and 18:

[0087] [ka]

[0088] To a solution of intermediate 10 (2.5 g, 93% purity, 4.59 mmol) in methanol (40 mL) was added 10 w / w% palladium on charcoal (1 g) under N2. The suspension was degassed under vacuum and purged with H2 several times. The reaction mixture was heated to 30 °C and stirred at this temperature overnight. After the reaction was cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated and purified by silica gel column chromatography eluting with 0% to 5% methanol in dichloromethane to give intermediate 16 (2.5 g, 93% purity, 99.6% yield) as a yellow oil.

[0089] Intermediate 16 (8 g, purity 95%, 14.9 mmol) was purified by chiral IG-SFC (separation conditions: column: IG; mobile phase: CO2-IPA: 65:35, 60 mL / min; temperature: 40 °C; wavelength: 214 nm) to give intermediate 17 (1st fraction, 3.29 g, purity 98%, yield 42.4%) as a yellow oil and intermediate 18 (2nd fraction, 3.36 g, purity 98%, yield 43.3%) as a yellow solid.

[0090] Chiral SFC method 2 was used to match the stereochemistry of intermediate 18 and intermediate 201 (retention time = 5.97-6.10 min).

[0091] Preparation of intermediate 25:

[0092] [ka]

[0093] To a cooled (ice bath) solution of intermediate 15 (1.1 g, 2.2 mmol) in dichloromethane (14 mL) was added TFA (7 mL) dropwise. The mixture was then stirred at room temperature for 2 h. The solvent was removed by evaporation and the residue was dissolved in DCM, the pH was adjusted to 8-9 with saturated aqueous sodium carbonate solution and extracted with DCM. The organic phase was dried over Na2SO4 and concentrated under vacuum to give intermediate 25 (680 mg, 72% yield) as a white solid.

[0094] Intermediate 27 was synthesized by a similar method as described for intermediate 25.

[0095] [Table 4]

[0096] Preparation of Intermediate 201 - Method A:

[0097] [ka]

[0098] To a 2 L 4-neck round bottom flask, THF (345 mL) and Zn (120.87 g, 1847.90 mmol, 5.00 equiv.) were added under nitrogen atmosphere at 30° C. A solution of TMSCl (8.03 g, 73.91 mmol, 0.2 equiv.) and 1-bromo-2-chloroethane (10.60 g, 73.91 mmol, 0.20 equiv.) in THF (230 mL) was added to the round bottom flask equipped with a Lead Fluid-BT100F peristaltic pump (speed: 10 mL / min) under nitrogen atmosphere. The resulting mixture was stirred at 30° C. for another 40 min. Next, the solvent in the RBF was quickly removed using a Lead Fluid-BT100F peristaltic pump, and then fresh THF (575 mL) was recharged under nitrogen atmosphere. The mixture was heated to 60° C. Next, a solution of tert-butyl (3R)-3-(iodomethyl)pyrrolidine-1-carboxylate (115 g, 369.58 mmol, 1.00 equiv) in THF (575 mL) was added to the above RBF under nitrogen atmosphere using a Lead Fluid-BT100F peristaltic pump (rate: 15.0 mL / min) (temperature rises to 60-65 °C). The solution was stirred at 60 °C for an additional 1 h. The mixture was then cooled to 30 °C and allowed to stand for 1 h. The solution of {[(3R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl]methyl}(iodo)zinc was used as is in the next step. The concentration of the product was about 0.37 mol / L in THF.

[0099] To a 2 L 4-neck round bottom flask, intermediate 6 (105 g, 259.71 mmol, 1.00 equiv) and THF (500 mL) were added under nitrogen atmosphere at 30° C. To the stirred solution, 4th generation RuPhos Pd precatalyst (5.65 g, 6.49 mmol, 0.025 equiv) was added under nitrogen atmosphere. Then, a solution of {[(3R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl]methyl}(iodo)zinc was added rapidly (excess zinc dust was not transferred) to a 2 L 4-need RBF under nitrogen atmosphere using a Lead Fluid-BT100F peristaltic pump. The resulting mixture was stirred at 50° C. for an additional 16 h. The reaction was repeated six times in parallel. The reaction was quenched by adding saturated aqueous NH4Cl solution (12 L). The aqueous layer was extracted with EtOAc (3×6 L) and the organic layer was washed with water (2×3 L) and brine (1×3 L). The resulting mixture was dried over Na2SO4 and concentrated under reduced pressure. The crude product (1100 g, crude) as a black oil was used directly in the next step (preparation of intermediate 202).

[0100] Alternatively, the following procedure can be used to prepare intermediate 201-Method B. A column (1.5 cm x 15 cm) was plugged with absorbent cotton and packed with 22 g of granular zinc (20-30 mesh). The column volume of the packed column was determined by measuring the time it took for THF to fill the column at a flow rate of 1 mL / min. Column volume = 4.3 mL. The zinc was activated by running a strong activation solution through the column at 0.5 mL / min for 10 min. The strong activation solution consisted of 1 mL of TMSCl (0.67 M) and 0.75 mL of chlorobromoethane (0.71 M) in 10 mL of THF. After activation, the column was washed with dry THF: 10 mL, 1 mL / min. tert-Butyl (R)-3-(iodomethyl)pyrrolidine-1-carboxylate (10 g, 37 mmol) was dissolved in THF (60 mL). The iodide solution was run through the activated zinc column at 50 °C and a flow rate of 0.45 mL / min. After reaction: Titration with iodine indicates a concentration of 0.30M.

[0101] Intermediate 6 (1.2 g, 2.4 mmol) was added to RuPhos Pd G4 (0.051 g, 0.06 mmol) in a sealed vial equipped with a stir bar in a glove box. A solution of freshly made R-((1-(tert-butoxycarbonyl)-3-yl)methyl)zinc(II) iodide (12 mL, 0.3 M, 3.6 mmol), prepared by the procedure described above, was then added. The solution was then heated to 50° C. under a nitrogen atmosphere for 16 h. The solution was concentrated in vacuo and the residue was redissolved in DCM. Water was then added followed by aqueous Na4EDTA (pH>10). The layers were separated and the aqueous layer was extracted once more with DCM. The organic layers were combined, dried over Na2SO4 and evaporated to dryness. The residue was purified by silica gel column chromatography eluting with 0% to 10% methanol in dichloromethane to give intermediate 201 (1.4 g, 1.5 mmol (55% purity), 63% yield).

[0102] Preparation of intermediate 202:

[0103] [ka]

[0104] A mixture of intermediate 201 (17 g, 33.09 mmol) in dichloromethane (50 mL) was added to a solution of 24 mL of chlorine hydrogen (7 M in ethyl acetate). After stirring at room temperature for 5 h, the reaction mixture was concentrated, the residue was diluted with DCM and basified with aqueous sodium hydroxide (1 M) to pH ∼10. The layers were separated, the aqueous layer was extracted three times with DCM and the combined organic layers were washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated to give intermediate 202 (13 g, 31.1 mmol, 94.2% yield) as a yellow solid, which was used in the next step without purification.

[0105] Alternatively, intermediate 202 can be prepared as a diTFA salt by using the following procedure: Intermediate 201 (5.2 g, 6.95 mmol, 68% purity) was dissolved in DCM (44.5 mL), TFA (5.3 mL) was added and stirred at room temperature for 4 h. The solution was concentrated in vacuo and coevaporated with toluene. The mixture was then washed with 1M NaOH and extracted 4 times with 10 DCM and EtOAc and Me-THF to give the combined organics, which were then dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified via silica gel column chromatography eluting with 0%-10% methanol in dichloromethane (containing 7N NH3) to give intermediate 202 as a di-TFA salt.

[0106] Alternatively, intermediate 202 can be prepared by the following procedure. To a 10 L 4-neck round bottom flask was added 4N HCl in 1,4-dioxane (1.8 L). Then, crude intermediate 201 in THF (3 L) was added dropwise at 0° C. (735 g of intermediate 201, 1.82 mol, calculated by 1.0 equiv.). The resulting mixture was stirred at 0° C. for another 2 h. The resulting mixture was diluted with ethyl acetate (3 L) and water (3 L). The aqueous layer was washed with DCM (10×1 L). The pH of the aqueous layer was adjusted to pH 8 with saturated aqueous Na2CO3 and extracted with CHCl2 (4×2 L). The organic layer was dried over Na2SO4 and concentrated under vacuum to give intermediate 202 (389 g, 53% yield for two steps) as a light yellow solid.

[0107] Preparation of intermediate Z:

[0108] [ka]

[0109] To a solution of intermediate 27 (3.5 g, 95%, 8.14 mmol) in DCM (80 mL) was added tert-butyl 4-formylpiperidine-1-carboxylate (3.66 g, 16.3 mmol) and sodium triacetoxyborohydride (2.58 g, 12.2 mmol). After stirring at room temperature for 6 h, the reaction mixture was poured into saturated aqueous sodium bicarbonate and extracted twice with dichloromethane (80 mL). The combined organic layers were washed with brine (80 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography eluting with 0% to 6% methanol in dichloromethane to give intermediate Z (4.66 g, 95% purity, 89.8% yield) as a white oil.

[0110] Preparation of intermediate Y:

[0111] [ka]

[0112] To a solution of intermediate Z (650 mg, 95% purity, 1.02 mmol) in DCM (8 mL) at 0° C. was added hydrogen chloride in ethyl acetate (2.2 mL, 7 M). After stirring at room temperature for 2 h, the reaction mixture was concentrated and the residue was basified with aqueous sodium hydroxide (1 M) and extracted twice with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give intermediate Y, which was used in the next step without purification.

[0113] Preparation of compound 51:

[0114] [ka]

[0115] To a solution of intermediate Y (1.04 g, 95% purity, 1.95 mmol) in DCM (10 mL) at 0° C., acetyl chloride (160 mg, 2.05 mmol) and triethylamine (592 mg, 5.85 mmol) were added. After stirring at room temperature for 2 h, the resulting mixture was poured into water and extracted twice with dichloromethane (20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated to give the crude product, which was purified by preparative HPLC (column: Xbridge C18 150×19 mm×5 um, mobile phase A: water (0.1% NH4HCO3), mobile phase B: acetonitrile, flow rate: 15 mL / min, gradient condition 15% B to 60% B). The collected fractions were lyophilized to give compound 51 (1.25 g, 99.8% purity, 74.9% yield) as a white solid.

[0116] Alternatively, compound 51 can be prepared by the following procedure. Intermediate 202 (as 2TFA salt) (0.20 g, 0.49 mmol) and 1-acetylpiperidine-4-carbaldehyde (0.097 g, 0.62 mmol) were dissolved in MeOH (5.5 mL). After stirring at ambient temperature for about 5 min, solid NaCNBH3 (0.039 g, 0.62 mmol) was added. The resulting mixture was stirred at ambient temperature for about 2 h, after which saturated aqueous NaHCO3 was added. Most of the MeOH was then evaporated to dryness and DCM was added. The pH of the aqueous layer was adjusted to pH>10 with 1 M aqueous NaOH. The layers were separated and the aqueous layer was extracted three more times with DCM. The organic layers were combined, dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography eluting with 0% to 10% methanol in dichloromethane (+1% 7N NH3 in MeOH) to give compound 51 (0.060 g, 0.11 mmol, 35% yield).

[0117] Compound 51 (derived from the route via intermediate 202; 0.051 g, 99.7% purity, LC / MS Method 32) was dissolved in 2-3 drops of isopropyl acetate (IPAC) and the resulting solution was then stirred at 45° C. for approximately 5 hours. The mixture was then stirred at ambient temperature for 48 hours and then filtered to give a white solid material corresponding to the crystalline free base form of compound 51. Melting point (by DSC): T 開始 =121.6℃.

[0118] Compound 51 (derived from the route via intermediate 202; ca. 1 g, 98.7% purity, LC / MS Method 33) was dissolved in cyclopentyl methyl ether (CPME) (3 mL), then heptane (2 mL) was added slowly, followed by ca. 10 mg of seed crystals (obtained via the previous procedure). Next, 1 mL of heptane was added and the mixture was stirred for 20 h, after which the suspension was filtered to obtain a solid material, which was dried under vacuum at 40° C. to obtain compound 51 in its crystalline free base form (96% yield).

[0119] Chiral SFC method 1 was used to match the stereochemistry of compound 51 obtained via the route using intermediate Y or intermediate 202. Retention time = 4.73-4.77 min.

[0120] Preparation of compound 51a:

[0121] [ka]

[0122] Compound 51 (0.50 g, 0.91 mmol, 95.2% purity (as determined by LC / MS Method 32)) was dissolved in acetone (0.50 mL) and stirred to obtain a clear solution. A solution of 1 M HCl in acetone was then prepared as follows: 1 mL of concentrated aqueous HCl was added to 11 mL of acetone. A solution of 1 M HCl in acetone (0.92 mL, 1 eq.) was then added to maintain the solution. The solution was stirred at ambient temperature for approximately 30-60 minutes, after which heptane (5.0 mL) was added. Acetone was then added (3.0 mL). Vigorous stirring was commenced and the mixture was stirred overnight. The suspension was then filtered once a fine white suspension was obtained. The solid was rinsed with heptane and dried to obtain compound 51a as the mono HCl trihydrate salt (as determined via dynamic vapor sorption analysis of approximately 3 eq. of water) as a white solid (0.48 g, 78% yield). Melting point (by DSC): T 開始 =139℃.

[0123] Compound 51a was obtained as a variable hydrate with equilibrium water content that varied as a function of humidity - primarily the trihydrate at ambient % relative humidity.

[0124] Pharmacology It has been found that the compounds of the present invention can block the interaction of menin with MLL proteins and oncogenic MLL fusion proteins themselves or can be metabolized in vivo to a more active form (prodrug).Accordingly, the compounds according to the present invention and pharmaceutical compositions comprising such compounds can be useful in the treatment or prevention, particularly in the treatment, of diseases such as cancer, including but not limited to leukemia, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and diabetes.

[0125] 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, myeloid leukemia, myeloid leukemia, lymphoblastic leukemia, lymphocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), T-cell 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.

[0126] 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).

[0127] In particular, the compounds according to the invention and pharmaceutical compositions thereof may be useful in the treatment or prevention of leukemia, in particular nucleophosmin (NPM1) mutant leukemia, such as NPM1c.

[0128] In particular, the compounds according to the present invention and pharmaceutical compositions thereof are useful in treating AML, particularly nucleophosmin (NPM1) mutant AML (i.e., NPM1 mut The present invention may be useful in the treatment or prevention of NPM1-mutated AML, and more specifically, in the treatment or prevention of NPM1-mutated AML.

[0129] 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.

[0130] In particular, the compounds according to the present invention and pharmaceutical compositions thereof may be useful for treating or preventing leukemia having an MLL gene alteration, particularly AML or ALL having an MLL gene alteration.

[0131] In particular, the compounds according to the invention and pharmaceutical compositions thereof may be suitable for QD dosing (once a day).

[0132] In particular, the compounds according to the invention and pharmaceutical compositions thereof may be useful for the treatment or prevention of hematological cancers in subjects exhibiting NPM1 gene mutations and / or mixed lineage leukemia gene (MLL; MLL1; KMT2A) alterations, mixed lineage leukemia (MLL), MLL-related leukemia, MLL-associated leukemia, MLL-positive leukemia, MLL-induced leukemia, rearranged mixed lineage leukemia, leukemia associated with MLL rearrangements / alterations or MLL gene rearrangements / alterations, acute leukemia, chronic leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), insulin resistance, pre-diabetes, diabetes or risk of diabetes, hyperglycemia, chromosomal rearrangements on chromosome 11q23, type 1 diabetes, type 2 diabetes; promoting the proliferation of pancreatic cells, where the pancreatic cells are islet cells, beta cells, and the proliferation of said beta cells is evidenced by increased beta cell production or insulin production; and inhibiting menin-MLL interaction, where the target gene of the MLL fusion protein is HOX or MEIS1 in humans.

[0133] The present invention therefore relates to the compounds according to the invention for use as medicaments.

[0134] The present invention also relates to the use of a compound according to the invention or a pharmaceutical composition according to the invention for the manufacture of a medicament.

[0135] The present invention also relates to a compound of the formula 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, whose treatment or prevention is affected or promoted by blocking the interaction of menin with MLL proteins and oncogenic MLL fusion proteins.

[0136] The present invention also relates to the use of a compound according to the present invention, or a pharmaceutical composition according to the present 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.

[0137] 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.

[0138] The present invention also relates to the compounds according to the invention for use in treating or preventing any one of the aforementioned diseases.

[0139] 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.

[0140] The compounds of the invention can be administered to mammals, preferably humans, to treat or prevent any one of the aforementioned diseases.

[0141] In view of the utility of the compounds according to the invention, there is provided a method for treating warm-blooded animals, including humans, suffering from any one of the aforementioned diseases.

[0142] The method involves the administration, ie, systemic or local administration, of a therapeutically effective amount of a compound according to the invention to a warm-blooded animal, including man.

[0143] Therefore, the present invention relates to a method for treating or preventing any one of the aforementioned diseases, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound according to the invention.

[0144] Those skilled in the art will recognize 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 on, among other things, the type of disease, the concentration of the compound in the therapeutic formulation, and the condition of the patient. An effective therapeutic daily dose is 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, for example, depending 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.

[0145] The present invention also provides a composition for preventing or treating the disorders mentioned herein, comprising a therapeutically effective amount of a compound according to the invention and a pharma- ceutically acceptable carrier or diluent.

[0146] While it is possible for the active ingredient (e.g., a compound of the present invention) to be administered alone, it is preferable to administer 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.

[0147] The pharmaceutical compositions may be prepared by any of the methods well known in the art of pharmacy.

[0148] 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.

[0149] Thus, an 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.

[0150] One or more other pharmaceutical agents and the compound according to the present invention can be administered simultaneously (e.g., in separate compositions or unitary compositions) 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 respective dosage amounts and regimes for each component of the combination will depend on the specific other pharmaceutical agents and compounds of the present invention administered, their administration routes, the specific pathology, particularly tumor, being treated, and the specific host being treated.

[0151] 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 in each method. Additional detectors were included if necessary (see methods table below).

[0152] The flow from the column was delivered to a 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.

[0153] 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] - 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.

[0154] 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.

[0155] [Table 5]

[0156] [Table 6]

[0157] 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 equipped with a high-pressure flow cell capable of withstanding up to 400 bar. If 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.

[0158] [Table 7]

[0159] NMR: NMR method Some NMR experiments were carried out at ambient temperature (298.6 K) using a Bruker Avance III 400 spectrometer equipped with a BBO 400 MHz S1 5 mm probehead with z-gradients, operating at 400 MHz for protons and 100 MHz for carbon, using an internal deuterium lock. Chemical shifts (d) are reported in parts per million (ppm). J values ​​are expressed in Hz.

[0160] Some NMR experiments were performed at ambient temperature (298.6 K) using a Varian 400-MR spectrometer equipped with a Varian 400 4NUC PFG probehead with z-gradients, operating at 400 MHz for protons and 100 MHz for carbon, using an internal deuterium lock. Chemical shifts (d) are reported in parts per million (ppm). J values ​​are expressed in Hz.

[0161] Some NMR experiments were performed at ambient temperature (298.6 K) using a Varian 400-VNMRS spectrometer equipped with a Varian 400 ASW PFG probehead with z-gradients using an internal deuterium lock and operating at 400 MHz for protons and 100 MHz for carbon. Chemical shifts (d) are reported in parts per million (ppm). J values ​​are in Hz.

[0162] [Table 8]

[0163] DSC Melting points (MP) were determined for many compounds using a TA Instrument (Discovery DSC 250 or DSC 2500). Melting points were measured with a temperature gradient of 10° C. / min. The maximum temperature was 300° C. Values ​​are the onset of the melting peak.

[0164] XPRD Compound 51 as a crystalline free base form Compound 51, as a crystalline free base form, can be characterized by its X-ray powder diffraction pattern.

[0165] X-ray powder diffraction (XRPD) analysis was performed on a PANalytical Empyrean diffractometer. Compounds were loaded onto a zero background silicon wafer sample holder by gently pressing the powder sample onto a flat surface.

[0166] Samples were subjected to XRPD using the following method: Radiation: Cu K-alpha (λ=1.5418Å) Tube voltage / current: 45kV / 40mA Divergence slit: 1 / 8° Geometry: Bragg-Brentano Scanning mode: Continuous scanning Scanning range: 3~40°2θ Step size: 0.013° 2θ Scanning speed: 20.4s / process Rotation: On Detector: PIXcel 1D

[0167] Those skilled in the art will recognize that the diffraction patterns and peak positions are generally substantially independent of the diffractometer used and the particular calibration method employed. Typically, peak positions may vary by no more than about ±0.2° 2θ. The intensity (and relative intensity) of each particular diffraction peak may also vary as a function of various factors, including, but not limited to, particle size, orientation, and sample purity.

[0168] The X-ray powder diffraction pattern includes peaks at 9.3, 12.6, 15.7, 21.9, and 22.5 °2θ±0.2 °2θ. The X-ray powder diffraction pattern may further include at least one peak selected from 8.1, 11.6, 13.2, 16.8, 18.5, 18.7, 19.2, 19.9, 20.5 °2θ±0.2 °2θ.

[0169] Compound 51 as a crystalline free base form can be further characterized by a powder X-ray diffraction pattern having four, five, six, seven, eight, nine or more peaks selected from the peaks identified in Table 2a.

[0170] Compound 51 as a crystalline free base form can be further characterized by a powder X-ray diffraction pattern comprising the peaks identified in Table 2a, 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 appreciate that the relative intensities of the peaks may vary between different samples and between different measurements on the same sample.

[0171] Compound 51, as a crystalline free base form, can be further characterized by an X-ray powder diffraction pattern substantially as depicted in FIG.

[0172] [Table 9]

[0173] Compound 51a crystalline HCl salt form (mono HCl trihydrate salt) Compound 51a (crystalline HCl salt form - mono HCl trihydrate salt - Compound 51a was obtained as a variable hydrate with equilibrium water content varying as a function of humidity - primarily the trihydrate at ambient relative humidity %) can be characterized by its X-ray powder diffraction pattern.

[0174] X-ray powder diffraction (XRPD) analysis was performed on a PANalytical Empyrean diffractometer. Compounds were loaded onto a zero background silicon wafer sample holder by gently pressing the powder sample onto a flat surface.

[0175] Samples were subjected to XRPD using the following method: Radiation: Cu K-alpha (λ=1.5418Å) Tube voltage / current: 45kV / 40mA Divergence slit: 1 / 8° Geometry: Bragg-Brentano Scanning mode: Continuous scanning Scanning range: 3~40°2θ Step size: 0.013° 2θ Scanning speed: 20.4s / process Rotation: On Detector: PIXcel 1D

[0176] Those skilled in the art will recognize that the diffraction patterns and peak positions are generally substantially independent of the diffractometer used and the particular calibration method employed. Typically, peak positions may vary by no more than about ±0.2° 2θ. The intensity (and relative intensity) of each particular diffraction peak may also vary as a function of various factors, including, but not limited to, particle size, orientation, and sample purity.

[0177] The X-ray powder diffraction pattern includes peaks at 5.2, 13.2, 14.1, 18.8, and 20.3 °2θ±0.2 °2θ. The X-ray powder diffraction pattern may further include at least one peak selected from 9.7, 10.0, 15.4, 15.8, 18.3, 21.3, 24.3 °2θ±0.2 °2θ.

[0178] Compound 51a may be further characterized by a powder X-ray diffraction pattern having 4, 5, 6, 7, 8, 9 or more peaks selected from those identified in Table 2b.

[0179] Compound 51a can be further characterized by a powder X-ray diffraction pattern comprising the peaks identified in Table 2b, 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 appreciate that the relative intensities of the peaks may vary between different samples and between different measurements on the same sample.

[0180] Compound 51a can be further characterized by an X-ray powder diffraction pattern substantially as depicted in FIG.

[0181] [Table 10]

[0182] Dynamic Vapor Sorption (DVS) Moisture sorption analysis (DVS) was performed using a ProUmid GmbH & Co.KG Vsorp Enhanced dynamic vapor sorption apparatus. The results are shown in Figures 3 and 4. Moisture profiles were assessed by monitoring vapor sorption / desorption over a relative humidity range of 0-90% relative humidity at 25 °C. Sample weight balance criteria were set at ≦0.01% change in 45 minutes with minimum and maximum acclimation times of 50 and 120 minutes, respectively. Moisture profiles consisted of two cycles of vapor sorption / desorption.

[0183] The DVS mass change plot of the crystalline HCl salt form (compound 51a) shows that the crystalline form is hygroscopic and the water content varies with relative humidity, dehydrating rapidly below 10% RH (relative humidity) and becoming completely dehydrated at 0% RH. In the humidity range of 20-90% RH, the crystalline form slowly and reversibly adsorbs and desorbs water, on average to 2.5% by mass. Based on the DVS, the crystalline HCl salt form can contain, at equilibrium, approximately 3 equivalents of water (8.5-9.5% total water mass) at typical ambient RH of 40%-75%. The XRPD patterns of fractions obtained after DVS testing were comparable to the starting material. No indication of solid-state morphological changes was observed.

[0184] Pharmacological part 1) Menin / MLL homogenous time-resolved fluorescence (HTRF) assay To a non-treated white 384-well microtiter plate, 40 nL of 200× test compound in DMSO and 4 μL of 2× 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 incubation of the test compound and terbium chelate-labeled menin for 30 min at ambient temperature, 4 μL of 2× FITC-MBM1 peptide (FITC-β-alanine-SARWRFPARPGT-NH2) (where "FITC" stands for fluorescein isothiocyanate) in assay buffer was added, the microtiter plate was centrifuged at 1000 rpm for 1 min, and the assay mixture was incubated at ambient temperature for 15 min. 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 at ambient temperature using an EnVision microplate reader (excitation 337 nm / terbium emission 490 nm / FITC emission 520 nm). The 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 The binding assay is expressed as a function of time (490 nm). The 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 typically performed using an 11-point 4-fold serial dilution scheme starting at 10 μM.

[0185] Compound potency was determined by first calculating the % inhibition at each compound concentration according to Equation 1: Inhibition% = ((HC-LC)-(HTRF 化合物 -LC) / (HC-LC) × 100 (Equation 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 (where, 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 calculated by fitting these data to Equation 2. 50 Get the value: Inhibition% = bottom + (top - bottom) / (1 + 10^((logIC 50 -log[compound])×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 inhibits the signal by 50%, and h is the Hill coefficient).

[0186] 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 1x Hepes buffer. 6.67 µM 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 over 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.

[0187] MENIN protein sequence (SEQ ID NO:1): MGLKAAQKTLFPLRSIDDVVRLFAAELGREEPDLVLLSLVLGFVEHFLAVNRVIPTNVPELTFQPSPAPDPPGGLTYFPVADLSIIAALYARFTAQIRGAVDLSLYPREGGVSSRELVKKVSDVIWNSLSRSYFKDRAHIQSLFSFITGTKLDS SGVAFAVVGACQALGLRDVHLALSEDHAWVVFGPNGEQTAEVTWHGKGNEDRRGQTVNAGVAERSWLYLKGSYMRCDRKMEVAFMVCAINPSIDLHTDSLELLQLQQKLLWLLYDLGHLERYPMALGNLADLEELEPTPGRPDPLTLYHKGIAS AKTYYRDEHIYPYMYLAGYHCRNRNVREALQAWADTATVIQDYNYCREDEEIYKEFFEVANDVIPNLLKEAASLLEAGEERPGEQSQGTQSQGSALQDPECFAHLLRFYDGICKWEEGSPTPVLHVGWATFLVQSLGRFEGQVRQKVRIVSREA EAAEAEEPWGEEAREGRRRGPRRESKPEEPPPPKKPALDKGLGTGQGAVSGPPRKPPGTVAGTARGPEGGSTAQVPAPAASPPPEGPVLTFQSEKMKGMKELLVATKINSSAIKLQLTAQSQVQMKKQKVSTPSDYTLSFLKRQRKGLHHHHHH

[0188] 2a) Proliferation assay The anti-proliferative effect of menin / MLL protein / protein interaction inhibitor test compounds was evaluated in human leukemia cell lines. The cell line MOLM14 has an MLL translocation and expresses the MLL fusion protein MLL-AF9, as well as 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., MOLM14) and NPM1c mutant cell lines show stem cell-like HOXA / MEIS1 gene expression signatures. KO-52 was used as a control cell line containing two MLL (KMT2A) wild-type alleles to exclude compounds that show general cytotoxic effects.

[0189] MOLM14 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 α-MEM (Sigma Aldrich) supplemented with 20% heat-inactivated fetal bovine serum (HyClone), 2 mM L-glutamine (Sigma Aldrich), and 50 μg / ml gentamicin (Gibco). During culture, cells were maintained at 0.3–2.5 million cells / ml and did not exceed 20 passages.

[0190] To evaluate antiproliferative effects, 200 MOLM14 cells, 200 OCI-AML3 cells, or 300 KO-52 cells were seeded in 200 μL / well of medium in 96-well round-bottom ultra-low attachment plates (Costar, catalog 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 at 37°C and 5% CO2 for 8 days. Spheroid-like growth was measured in real time by live cell imaging (IncuCyteZOOM, Essenbio, 4x objective) with image acquisition on day 8. Confluence (%) as a measure of spheroid size was determined using the integrated analysis tool.

[0191] To determine the effect of test compounds over time, the confluence in each well was calculated as a measure of spheroid size. 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).

[0192] Absolute IC as the percentage change in confluence as follows: 50 The values ​​were calculated: 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) Efficacy% = 100-(100 x (Sample-LC) / (HC-LC))

[0193] IC was calculated using GraphPad Prism (version 7.00). 50 The dose-response equation for plots of % effect versus Log10 compound concentration with variable slope and max fixed at 100% and min fixed at 0% was used.

[0194] 2b) MEIS1 mRNA expression assay MEIS1 mRNA expression upon treatment with compounds was examined by Quantigene Singleplex assay (Thermo Fisher Scientific). This technology allows direct quantification of mRNA targets using probes hybridizing to predefined target sequences of interest, and the signal is detected using the Multimode plate reader Envision (PerkinElmer). The MOLM14 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 with 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 custom assay hybridization plates (Thermo Fisher Scientific) and incubated at 55 °C for 18–22 h. The plate was then washed to remove unbound material, followed by sequential addition of preamplifier, amplifier, and labeled probe. Signal (= number of genes) was measured on a Multimode plate reader Envision. IC was determined by dose-response modeling using appropriate software. 50For all non-housekeeper gene responses, equal numbers were 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.

[0195] [Table 11]

Claims

1. A pharmaceutical composition comprising 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidine-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridine-1-yl]-5-fluoro-N-isopropyl-benzamide as a therapeutically effective amount of crystalline free base, and a pharmaceutically acceptable carrier or excipient.

2. The pharmaceutical composition according to claim 1, wherein the crystalline free base morphology is characterized by an X-ray diffraction pattern including peaks at 9.3, 12.6, 15.7, 21.9, and 22.5°2θ ± 0.2°2θ.

3. A pharmaceutical composition comprising 2-[3-[[(3R)-1-[(1-acetyl-4-piperidyl)methyl]pyrrolidine-3-yl]methyl]-4-methyl-pyrrolo[2,3-c]pyridine-1-yl]-5-fluoro-N-isopropyl-benzamide as a therapeutically effective amount of its crystalline HCl salt form, and a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition according to claim 3, wherein the HCl salt form is monoHCl trihydrate.

5. The pharmaceutical composition according to claim 3, wherein the crystalline HCl salt form is characterized by an X-ray diffraction pattern including peaks at 5.2, 13.2, 14.1, 18.8, and 20.3°2θ ± 0.2°2θ.

6. A pharmaceutical composition according to any one of claims 1 to 5, for use in the prevention or treatment of cancer.

7. A pharmaceutical composition according to any one of claims 1 to 5, for use in the prevention or treatment of leukemia, myelodysplastic syndrome (MDS), and myeloproliferative neoplasm (MPN).

8. The pharmaceutical composition for use according to claim 7 in the prevention or treatment of leukemia, wherein the leukemia is nucleophosmin (NPM1) variant leukemia.

9. The pharmaceutical composition for use according to claim 6, wherein the cancer is selected from leukemia, lymphoma, myeloma, or solid tumor cancers such as prostate cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, liver cancer, melanoma, and glioblastoma.

10. The pharmaceutical composition for use in the prevention or treatment of leukemia according to claim 7, wherein the leukemia is selected from acute leukemia, chronic leukemia, myeloid leukemia, myeloid leukemia, lymphoblastic leukemia, lymphocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), T-cell prelymphoblastic leukemia (T-PLL), large granular lymphocytic leukemia, hairy cell leukemia (HCL), MLL rearrangement leukemia, MLL-PTD leukemia, MLL amplification leukemia, MLL-positive leukemia, and leukemia exhibiting a HOX / MEIS1 gene expression signature.

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