INDOLE-3-CARBOXAMIDE

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/211,383, filed August 28, 2015, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] Provided herein are crystalline forms of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2- dihydropyridin- 3 - yl) methy 1) -2- methy

indole-3-carboxamide, processes for preparing the crystalline forms, pharmaceutical compositions comprising the crystalline forms, and uses of the crystalline forms and compositions thereof in modulating the activity of histone methyl modifying enzymes.

BACKGROUND OF THE INVENTION

[0003] Eukaryotic chromatin is composed of macromolecular complexes called nucleosomes. A nucleosome has 147 base pairs of DNA wrapped around a protein octamer having two subunits of each of histone protein H2A, H2B, H3, and H4. Histone proteins are subject to post-translational modifications which in turn affect chromatin structure and gene expression. One type of post-translational modification found on histones is methylation of lysine and arginine residues. Histone methylation plays a critical role in the regulation of gene expression in eukaryotes. Methylation affects chromatin structure and has been linked to both activation and repression of transcription (Zhang and Reinberg, Genes Dev. 15:2343- 2360, 2001). Enzymes that catalyze attachment and removal of methyl groups from histones are implicated in gene silencing, embryonic development, cell proliferation, and other processes.

[0004] One class of histone methylases is characterized by the presence of a Suppressor of Variegation Enhancer of Zeste Trithorax (SET) domain, comprising about 130 amino acids. Enhancer of Zeste Homo log 2 (EZH2) is an example of a human SET-domain containing methylase. EZH2 associates with EED (Embryonic Ectoderm Development) and SUZ12 (suppressor of zeste 12 homolog) to form a complex known as PRC2 (Polycomb GroupRepressive Complex 2) having the ability to tri-methylate histone H3 at lysine 27 (Cao and Zhang, Mol. Cell 15:57-67, 2004). PRC2 complexes can also include RBAP46 and RBAP48 subunits. Another example is the related methylase EZH1.

[0005] oncogenic activities of EZH2 have been shown by a number of studies. In cell line experiments, over-expression of EZH2 induces cell invasion, growth in soft agar, and motility while knockdown of EZH2 inhibits cell proliferation and cell invasion (Kleer et al., 2003, Proc. Nat. Acad. Sci. USA 100: 11606-11611; Varambally et al., (2002), "The polycomb group protein EZH2 is involved in progression of prostate cancer," Nature 419, 624-629). It has been shown that EZH2 represses the expression of several tumor

suppressors, including E-cadherin, DAB2IP and RUNX3 among others. In xenograft models, EZH2 knockdown inhibits tumor growth and metastasis. Recently, it has been shown that down modulation of EZH2 in murine models blocks prostate cancer metastasis (Min et al., "An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor- kappaB," Nat Med. 2010 Mar; 16(3):286-94). EZH2 overexpression is associated with aggressiveness of certain cancers such as breast cancer (Kleer et al., Proc. Nat. Acad. Sci. USA 100: 11606-11611, 2003). Recent studies also suggest that prostate cancer specific oncogenic fusion gene TMPRSS2-ERG induces repressive epigenetic programs via direct activation of EZH2 (Yu et al., "An Integrated Network of Androgen Receptor, Polycomb, and TMPRSS2-ERG Gene Fusions in Prostate Cancer Progression," Cancer Cell. 2010 May 18;17(5):443-454).

[0006] (R)-N-((4-methoxy-6-methyl-2-oxo- l,2-dihydropyridin-3-yl)methyl)-2-methyl- 1- (l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole- 3-carboxamide is an inhibitor of EZH2 and is useful in treating a variety of diseases, disorders or conditions, associated with a methyl modifying enzyme, such as, e.g., in treating proliferative disorders such as cancer.

[0007] The amorphous form of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin- 3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2-trifluoroethyl)piperidi n-4-yl)ethyl)-lH-indole-3- carboxamide is exemplified in U.S. Patent Publication No. 2015/0011546 as Compound 365, and is incorporated herein by reference. (R)-N-((4-methoxy-6-methyl-2-oxo-l,2- dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2-trifluor oethyl)piperidin-4-yl)ethyl)-lH- indole-3-carboxamide is represented by the following structural formula:

[0008] Given the therapeutic benefits associated with the amorphous form, there is a need for the identification, characterization, manufacturing and development of crystalline forms of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide. Crystalline forms represent an attractive alternative to amorphous solids in that they facilitate isolation, manufacturing, formulation and enhance storage stability

SUMMARY OF THE INVENTION

[0009] Provided herein are novel crystalline Forms A, B, and C of (R)-N-((4-methoxy-6- methyl-2-oxo-l,2-diliydropyridin-3-yl)methyl)-2-methyl-l-(l- (l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide .

[0010] Also provided herein are pharmaceutical compositions comprising the crystalline Forms A, B, and C of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)- 2-methyl-l-(l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl) -lH-indole-3-carboxamide, methods for their manufacture, and uses thereof for treating a variety of diseases, disorders or conditions associated with a methyl modifying enzyme, such as, e.g., in treating proliferative disorders such as cancer.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 depicts the polarized light microscopy for mostly amorphous (R)-N-((4- methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-met hyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide , with some birefringent particles, generated from US Publication No. 2015/0011546.

[0012] Figure 2 illustrates exemplary DSC plots for Form A of (R)-N-((4-methoxy-6- methyl-2-oxo-l,2-diliydropyridin-3-yl)methyl)-2-methyl-l-(l- (l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide heated at 10 °C/minute.

[0013] Figure 3 depicts an X-ray powder diffraction pattern (XRPD) for crystalline Form A of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide, isolated from

EtOH/H ₂ 0.

[0014] Figure 4 depicts an X-ray powder diffraction pattern (XRPD) for crystalline Form A of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide, isolated from iPrOH/H ₂ 0. [0015] Figure 5 illustrates subtle peak shifts represented by an overlay of X-ray powder diffraction patterns (XRPDs) for various samples of crystalline Form A of (R)-N-((4- methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-met hyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide .

[0016] Figure 6 illustrates exemplary DSC plots for Form B of (R)-N-((4-methoxy-6- methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-methyl-l-(l-( l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide heated at 10 °C/minute.

[0017] Figure 7 depicts an X-ray powder diffraction pattern (XRPD) for crystalline Form B of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide, isolated from acetonitrile.

[0018] Figure 8 depicts an X-ray powder diffraction pattern (XRPD) for crystalline Form B of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide, isolated from acetonitrile/HiO .

[0019] Figure 9 depicts an X-ray powder diffraction pattern (XRPD) for crystalline Form B of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide, isolated from

MeOH/H ₂ 0.

[0020] Figure 10 illustrates exemplary DSC plots for Form C of (R)-N-((4-methoxy-6- methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-methyl-l-(l-( l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide heated at 10 °C/minute.

[0021] Figure 11 depicts an X-ray powder diffraction pattern (XRPD) for crystalline Form C of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l- (l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole- 3-carboxamide, isolated from isopropyl acetate.

[0022] Figure 12 depicts an overlay X-ray powder diffraction pattern (XRPD) between crystalline Form B of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)- 2-methyl-l-(l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl) -lH-indole-3-carboxamide and mostly amorphous (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2- methyl-l-(l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-l H-indole-3-carboxamide generated from US Publication No. 2015/0011546. DETAILED DESCRIPTION

Definitions

[0023] When used alone, the terms "Form A", "Form B", and "Form C" refer to the crystalline polymorphic Forms A, B, and C of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2- dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2-trifluor oethyl)piperidin-4-yl)ethyl)-lH- indole-3-carboxamide, respectively. The terms "Form A", "Form A of (R)-N-((4-methoxy-6- methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-methyl-l-(l-( l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide ", and "crystalline Form A of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide" are used

interchangeably. Similary, "Form B", "Form B of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2- dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2-trifluor oethyl)piperidin-4-yl)ethyl)-lH- indole-3-carboxamide", and "crystalline Form B of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2- dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2-trifluor oethyl)piperidin-4-yl)ethyl)-lH- indole-3-carboxamide" are used interchangeably. Similary, "Form C", "Form C of (R)-N-((4- methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-met hyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide ", and "crystalline Form C of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide" are used

interchangeably.

[0024] It will be understood, that "Form A", "Form B", and "Form C" may include some water or solvent trapped within the lattice. Such forms are included herein provided they are substantially the same as the XRPD patterns/peaks described herein. Such forms are characterized e.g., by XRPD. As used herein, "anhydrous" means that the crystalline form comprises substantially no water in the crystal lattice e.g., less than 1% by weight as determined by Karl Fisher analysis.

[0025] The term "amorphous" means a solid that is present in a non-crystalline state or form. Amorphous solids are disordered arrangements of molecules and therefore possess no distinguishable crystal lattice or unit cell and consequently have no definable long range ordering. Solid state ordering of solids may be determined by standard techniques known in the art, e.g., by X-ray powder diffraction (XRPD) or differential scanning calorimetry (DSC). Amorphous solids can also be differentiated from crystalline solids e.g., by birefringence using polarized light microscopy. [0026] As used herein, "(R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-2-methyl-l-(l-(l-(2,2,2 rifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3- carboxamide" is intended to mean the following structure:

[0027] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not adversely affect the pharmacological activity of the compound with which it is formulated, and which is also safe for human use. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyvinylpyrrolidone- vinyl acetate, cellulose-based substances (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose Phthalate), starch, lactose monohydrate, mannitol, sodium lauryl sulfate, and crosscarmellose sodium, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polymethacrylate, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Description of Exemplary Compounds

[0028] In one aspect, the present disclosure provides crystalline Form A of (R)-N-((4- methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-met hyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide , represented by the following structure:

[0029] In one aspect, crystalline Form A is characterized by at least three, at least four, at least five, or by at least six X-ray powder diffraction peaks at 2Θ angles selected from 8.52°, 16.82°, 19.10°, 19.48°, 20.62°, 23.78°, and 24.26°. Alternatively, crystalline Form A is characterized by major X-ray powder diffraction peaks at 2Θ angles at 8.52°, 16.82°, 19.10°, 19.48°, 20.62°, and 24.26°. In another alternative, crystalline Form A is characterized by X- ray powder diffraction peaks at 2Θ angles 8.52°, 16.82°, 19.10°, 19.48°, 20.62°, 23.78°, and 24.26°. In another alternative, crystalline Form A is characterized by X-ray powder diffraction peaks at 2Θ angles 8.52°, 12.38°, 14.18°, 14.42°, 16.82°, 19.10°, 19.48°, 20.62°, 22.14°, 23.78°, and 24.26°.

[0030] In one aspect, crystalline Form A is characterized by a DSC pattern that is substantially the same as Figure 2.

[0031] In another aspect, crystalline Form A is characterized by an XRPD pattern that is substantially the same as Figure 3.

[0032] In another aspect, crystalline Form A is characterized by an XRPD pattern that is substantially the same as Figure 4.

[0033] In another aspect, crystalline Form A is characterized by the peaks in Table 3.

[0034] In one aspect, the present disclosure provides crystalline Form B of (R)-N-((4- methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-met hyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide , represented by the following structure:

wherein crystalline Form B is at least 50% pure by weight, at least 75% pure by weight, at least 80% pure by weight, at least 90% pure by weight, at least 95% pure by weight, or at least 98% pure by weight.

[0035] In one aspect, crystalline Form B is characterized by at least three, at least four, at least five, at least six, or by at least seven X-ray powder diffraction peaks at 2Θ angles selected from 4.54°, 17.12°, 17.52°, 19.38°, 20.08°, 21.10°, 23.92°, and 25.2°. Alternatively, crystalline Form B is characterized by major X-ray powder diffraction peaks at 2Θ angles at 4.54°, 23.92°, and 25.2°. In another alternative, crystalline Form B is characterized by X-ray powder diffraction peaks at 2Θ angles 4.54°, 17.12°, 17.52°, 19.38°, 20.08°, 21.10°, 23.92°, and 25.2°. In another alternative, crystalline Form B is characterized by X-ray powder diffraction peaks at 2Θ angles 4.54°, 10.14°, 17.12°, 17.52°, 19.38°, 21.10°, 23.18°, 23.92°, 25.20°, 29.48°, and 35.18°.

[0036] In one aspect, crystalline Form B is characterized by a DSC pattern that is substantially the same as Figure 6.

[0037] In another aspect, crystalline Form B is characterized by an XRPD pattern that is substantially the same as Figure 7.

[0038] In another aspect, crystalline Form B is characterized by an XRPD pattern that is substantially the same as Figure 8.

[0039] In another aspect, crystalline Form B is characterized by an XRPD pattern that is substantially the same as Figure 9.

[0040] In another aspect, crystalline Form B is characterized by the XRPD peaks in

Table 4.

[0041] In one aspect, the present disclosure provides crystalline Form C of (R)-N-((4- methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-met hyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide , represented by the following structure:

[0042] In one aspect, crystalline Form C is characterized by at least three, at least four, at least five, at least six, or by at least seven X-ray powder diffraction peaks at 2Θ angles selected from 7.56°, 10.24°, 14.18°, 14.38°, 16.26°, 18.04°, 19.60°, and 21.52°. Alternatively, crystalline Form C is characterized by major X-ray powder diffraction peaks at 2Θ angles selected from 7.56°, 9.94°, 10.24°, 14.18°, 14.38°, 18.04°, 18.54°, 19.60°, 21.52°, 24.16°, and 24.46°. In another alternative, crystalline Form C is characterized by X-ray powder diffraction peaks at 2Θ angles 7.56°, 10.24°, 14.18°, 14.38°, 16.26°, 18.04°, 19.60°, and 21.52°. In another alternative, crystalline Form C is characterized by X-ray powder diffraction peaks at 2Θ angles 7.56°, 9.94°, 10.24°, 14.18°, 14.38°, 16.26°, 17.14°, 17.63°, 18.04°, 18.54°, 19.60°, 20.28°, 21.52°, 22.32°, 24.16°, 24.46°, 25.00°, and 27.88°.

[0043] In one aspect, crystalline Form C is characterized by a DSC pattern that is substantially the same as Figure 10. [0044] In another aspect, crystalline Form C is characterized by an XRPD pattern that is substantially the same as Figure 11.

[0045] In another aspect, crystalline Form C is characterized by the XRPD peaks in

Table 5.

[0046] It will be understood that the 2-theta values of the X-ray powder diffraction patterns for Form A, Form B, and Form C may vary slightly from one instrument to another and also depending on variations in sample preparation and batch to batch variation.

Therefore, unless otherwise indicated, the XRPD patterns / assignments for the crystalline forms defined herein are not to be construed as absolute and can vary + 0.2 degrees. In the case of crystalline Form A, unless otherwise indicated, the XRPD patterns / assignments ranging from 13.5° to 14.5° and from 17.0° to 25.0° can vary by + 0.3 degrees. See e.g., Figure 5. Without wishing to be bound by theory, it is believed that this additional variation may be indicative of some level of solvent trapped within the lattice.

[0047] As intended herein, "substantially the same XRPD pattern as shown in Figure 3" means that for comparison purposes, the XRPD has at least 90% of the peaks shown in

Figure 3. This applies similarly to Figures 3-5, 7-9, and 11. It is to be further understood that for comparison purposes, some variability in 2Θ angles from those shown in Figures 3- 5, 7-9, and 11 are allowed, such as + 0.2 degrees, except for the 2Θ angles ranging from 13.5° to 14.5° and from 17.0° to 25.0° in the case of Form A (Figures 3-5, which can vary by + 0.3 degrees.

[0048] In one aspect, the present disclosure provides a process for preparing crystalline Form A of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l- (l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole- 3-carboxamide. Such a process includes, e.g., preparing a slurry of amorphous (R)-N-((4-methoxy-6-methyl-2-oxo-l,2- dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2-trifluor oethyl)piperidin-4-yl)ethyl)-lH- indole-3-carboxamide in e.g., neat ethanol (200 proof); mixture of ethanol in water, heptane, or methyl tert-butyl ether; dichloro methane; and isopropanol / water (50:50). Form A can also be prepared by seeding with Form A, a suspension (slurry) of Form B or Form C in ethanol / water (50:50).

[0049] In one aspect, the present disclosure provides a process for preparing crystalline Form B of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l- (l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole- 3-carboxamide. Such a process includes, e.g., preparing a slurry of amorphous (R)-N-((4-methoxy-6-methyl-2-oxo-l,2- dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2-trifluor oethyl)piperidin-4-yl)ethyl)-lH- indole-3-carboxamide in e.g., methanol, acetonitrile, methanol / water (50:50), acetonitrile / water (50:50), and methanol / methyl tert-buthyl ether. Form B can also be prepared by seeding with Form B, a suspension (slurry) of Form A or Form C in acetonitrile.

[0050] In one aspect, the present disclosure provides a process for preparing crystalline Form C of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l- (l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole- 3-carboxamide. Such a process includes, e.g., agitating a mixture comprising amorphous (R)-N-((4-methoxy-6-methyl-2- oxo-l,2-dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4- yl)ethyl)-lH-indole-3-carboxamide and isopropyl acetate (iPrOAc) at e.g., a temperature of about 65 °C.

[0051] Crystalline Form C can also be obtained by preparing a concentrated solution of amorphous (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l- (l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole- 3-carboxamide at or near the reflux temperature of an organic solvent or solvent system; and allowing the solution to cool to ambient temperature or preparing a slurry of amorphous (R)-N-((4-methoxy-6-methyl-2- oxo-l,2-dihydropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2- trifluoroethyl)piperidin-4- yl)ethyl)-lH-indole-3-carboxamide. Exemplary organic solvents and solvent systems for this processes include e.g., ethanol (200 proof), 2-methoxyethanol, 2-propanol, n-propanol, butanol, methyl ethyl glycol, methyl acetate, ethyl acetate, isopropyl acetate, 1,2- dichloromethane, chloroform, dimethoxyethane, dimethyl sulfoxide, N-methyl 2-pyrrolidone, acetone, methyl ethyl ketone, methyl isobutyl ketone, tetrahydrofuran, 1,4-dioxane vapor diffusion, methyl t-butyl ether, toluene, benzene vapor diffusion, and xylenes. Form C can also be prepared by seeding with Form C a slurry of Form A or Form B in isopropyl acetate.

[0052] Processess for preparing each of the crystalline Forms A, B, and C also include cooling down saturated solutions with or without seeding solutions of (R)-N-((4-methoxy-6- methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2-methyl-l-(l-( l-(2,2,2- trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carboxamide . Solvent systems that are appropriate in the above slurries are also compatible with cooling down saturated solutions with or without seeding.

Uses, Formulation and Administration

Pharmaceutically acceptable compositions

[0053] According to other aspects, the present disclosure relates to a method of modulating a histone modifying enzyme using a composition comprising Form A, Form B, or Form C and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of crystalline form is a provided composition is such that is effective to measurably modulate a histone methyl modifying enzyme, or a mutant thereof, in a biological sample or in a patient. In certain embodiments, the amount of crystalline form in a provided composition is such that it is effective to measurably modulate a histone methyl modifying enzyme, or a mutant thereof, in a biological sample or in a patient.

[0054] In one aspect, pharmaceutical compositions described herein comprise least 50% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, at least 95% by weight, or at least 99% by weight of crystalline Form A, crystalline Form B, or crystalline Form C; and a pharmaceutically acceptable carrier or diluent.

[0055] According to other aspects, the present disclosure relates to a method of inhibiting EZH2 using a composition comprising the crystalline forms described herein and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of the crystalline form in a provided composition is such that is effective to measurably inhibit EZH2, or a mutant thereof, in a biological sample or in a patient. In certain aspects, a provided composition is formulated for administration to a patient in need of such composition. In some aspects, a provided composition is formulated for oral administration to a patient.

[0056] Compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0057] Other forms of administration are as described in U.S. Patent Publication No. 2012/0157428. Dosage forms for oral administration are also as described in U.S. Patent Publication No. 2015/0011546, the contents of which are incorporated herein by reference.

[0058] The amount of provided crystalline form that may be combined with carrier materials to produce a composition in a single dosage form will vary depending upon the patient to be treated and the particular mode of administration. Provided compositions may be formulated such that a dosage of between 0.001 - 100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.

[0059] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a provided crystalline form in the composition will also depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

[0060] The crystalline forms described herein and compositions thereof are generally useful for modulating the activity of one or more enzymes involved in epigenetic regulation, such as EZH2 and others described in e.g., U.S. Patent Publication No. 2015/0011546. Thus, in some aspects, the present disclosure provides a method of inhibiting one or more enzymes involved in epigenetic regulation, such as EZH2, by administering a provided crystalline form or composition.

[0061] The present disclosure also relates to treating diseases and/or disorders associated with overexpression of EZH1 or EZH2 and/or expression of a mutant form of EZH2, particularly those mutant forms that alter EZH2 substrate activity (e.g., as those described in U.S. Patent Publication No. 2015/0011546), with a crystalline form or composition thereof described herein. The study of EZH2 deletions, missense and frameshift mutations suggest that EZH2 functions as a tumor suppressor in blood disorders such as myelodysplastic syndromes (MDS) and myeloid malignancies (Ernst et al., Nat Genet. 2010 Aug;

42(8):722-6; Nikoloski et al., Nat Genet. 2010 Aug; 42(8):665-7). In some embodiments, crystalline forms and compositons thereof are useful in treating diseases and/or disorders associated with the presence of EZH2 having a Y641N, Y641C, Y641F, Y641H, Y641S, A677G, or A687 mutation. In one aspsect, the EZH2 has a Y641N mutation.

[0062] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.

[0063] Diseases and conditions treatable according to the methods described herein include, but are not limited to, diseases and/or disorders associated with cellular proliferation. In some embodiments, the crystalline forms and compositions thereof described herein are useful in treating diseases and/or disorders associated with misregulation of cell cycle or DNA repair. In some embodiments, the crystalline forms and compositions thereof described herein are useful in treating cancer. Exemplary types of cancer include e.g., adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma,

angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney,

craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma,

dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin' s lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non- small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor

T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

[0064] In one aspect, the cancer treated by the crystalline forms and compositions thereof described herein is selected from breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme cancer, bladder cancer, melanoma, bronchial cancer, lymphoma, liver cancer, multiple myeloma, lymphoma, ovarian cancer, NSCL, pancreatic cancers, malignant rhabdoid tumor, synovial sarcoma, and glioma.

[0065] Another aspect of the present disclosure is the use of one or more of the crystalline forms as described herein in the manufacture of a medicament for use in the treatment of a disorder or disease herein. Another object of the present disclosure is one or more of the crystalline forms or composition described herein for use in the treatment of a disorder or disease herein.

EXEMPLIFICATION

[0066] As depicted in the Examples below, in certain exemplary embodiments, crystalline Forms A, B, and C are prepared according to the following general procedures.

[0067] XRPD analyses were conducted using a Rigaku Ultima III X-Ray diffractometer operating with a Cu radiation source at 45 kV, 40 niA through a Ni filter with a divergence slit of 2/3°, Divergence H.L. slit of 10 mm, scatter slit set to "Auto", and receiving slit of 0.3 mm. Samples were placed on Si zero-return sample holders and analysis was performed using continuous scan from 3° to 40° 2Θ at 2 min with a step size of 0.02 second and a step time of 0.6 point/second. Samples were rotated plane parallel to sample surface at 60 rpm. Peak assignments were performed using Jade 7 software.

[0068] Differential Scanning Calorimetry was performed on a TA Instruments DSC Q1000 on the sample "as is." Sample were weighed into an aluminum pan, covered with a pierced lid, and then crimped and analyzed from -25 to 280 °C ramped at 10 °C/min.

[0069] For TGA, weight loss was monitored as a function of temperature using a TA Instruments TGA Q50. Samples were run from 25 °C to 300 °C or > 20% decomposition at a heating rate of 10 °C/min in open platinum TGA pans under an inert nitrogen atmosphere. Weight losses were calculated from the highest point in the baseline to the point at which the onset of melt occurred or to the visible plateau before the onset of decomposition.

[0070] Moisture sorption profiles were generated isothermally at 25 °C using a Hiden IGAsorp moisture sorption analyzer. Samples were dried on the instrument under inert nitrogen at 40 °C for 60 min. Following drying the samples moisture sorption was determined by performing one full cycle of weight measurements at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 95 %RH, with exposure time at each humidity set point dependent upon a minimum of 99% confidence in the Fl fit model or 15 min, with a maximum timeout of 120 min. The moisture sorption isotherms were plotted as a function of weight change (relative weight % of the dried starting weight) and % RH.

[0071] The material obtained for (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin- 3-yl)methyl)-2-methyl-l-(l-(l-(2,2,2 rifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3- carboxamide, following the methods described in Example 28, compound 365 of U.S. Patent Publication No. 2015/0011546 produces mostly amorphous material with very weak XRPD signal having some similarity to Form B. The microscopy results of this material is shown in Figure 1 and the XRPD overlay with Form B is shown in Figure 12.

[0072] Example 1: Preparation of monohydrate crystalline Form A, B, and C of (R)-N- ((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-2 -methy 1-1 -(1-(1 -(2,2,2- trifluoroethyl)piperid -4-yl)ethyl)-lH-indole-3-carboxamide

[0073] Polymorph screen samples were prepared by initially in 33 solvent systems with 29 neat solvents and 4 combinations of 1: 1 solvent:water. Polymorph screen samples were prepared with amorphous (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-2-methyl-l-(l-(l-(2,2,2 rifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3- carboxamide by aliquoting from a stock solution of amorphous (R)-N-((4-methoxy-6-methyl- 2-oxo- 1 ,2-dihydropyridin-3-yl)methyl)-2-methyl- 1 -( l-( l-(2,2,2-trifluoroethyl)piperidin-4- yl)ethyl)-lH-indole-3-carboxamide in dichloromethane and evaporating to dryness. Samples that did not generate solids as a saturated solution at ambient conditions were slowly evaporated to dryness. Dry samples that did not yield crystalline solids were reconstituted in the original solvent and subject to vapor diffusion at ambient temperature with MBTE as an antisolvent. Dry sample that yield trace crystalline solids were re-slurried in a few drops of the original solvent to break up the solids and promote additional crystal growth. Vapor diffusion samples yielding mostly amorphous solids were also re-slurried in the original solvent to generate crystalline material.

[0074] Resulting solids were isolated by spatula or centrifugation and screened for birefringence using polarized light microscopy. The resulting form was determined by XRPD analysis of the wet cake for all birefringent solids. DSC and TGA analysis after vacuum drying at 40 °C for at least 4 hours was performed in isolated instances to spot check and build data for each form. Table 1 details the results.

Table 1

Solvent Abbreviation Solvent Name Resulting Form

CHCI3 Chloroform C

DME Dimethoxyethane C

DMSO Dimethyl sulfoxide _C

NMP N-methyl 2-pyrrolidone _c kS

ACE Acetone _c kS

MEK Methyl ethyl ketone

MIBK Methyl isobutyl ketone C

THF Tetrahydro furan

DIOX/MTBE 1,4-Dioxane vapor diffusion

MTBE Methyl t-butyl ether C

HEP Heptane N/A ^EV

TOL Toluene

Benz/MTBE Benzene vapor diffusion

Xyl Xylenes C

TFE Trifluoroethanol N/A

MeOH/H ₂ 0 50:50 Methanol/water 50:50 B

EtOH/H ₂ 0 50:50 Ethanol/water 50:50 A

ACN/H ₂ 0 50:50 Acetonitrile/water 50:50 B

IPA/H ₂ 0 50:50 Isopropanol/water 50:50 A

EV = slow evaporation

VD = vapor diffusion

RS = reslurry

N/A = data not available

[0075] Table 2 compares certain physical characteristics of Form A, Form B, and Form C of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carb oxamide.

Table 2

[0076] A representative listing of XRPD peaks for each of Form A, Form B, and Form C of (R)-N-((4-methoxy-6-methyl-2-oxo-l,2-dihydropyridin-3-yl)met hyl)-2-methyl-l-(l-(l- (2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-lH-indole-3-carb oxamide are provided in Tables 3-5.

Table 3 - Form A

Table 4 - Form B

Rel. Peak

2-Theta Height Height A%

[%]

17.141 302 36 22.7

17.627 245 29.1 20.3

18.042 737 87.8 68.9

18.539 391 46.6 45.3

19.599 674 80.3 63.1

20.279 312 37.2 32

21.519 683 81.3 70.3

22.318 209 24.9 24.7

22.962 143 17 12.4

24.161 270 32.2 32.5

24.461 256 30.5 41.5

25.001 241 28.7 17.9

26.284 111 13.2 9.6

27.881 285 34 23.8

28.701 135 16.1 15.8

29.02 140 16.7 15.3

[0077] A scale up of crystalline Form C was performed as follows.

[0078] A 100 L round bottom reactor was charged with CDI (1.14 kg, 7.0 mol). The reactor was equipped with mechanical stirring, and static N ₂ . Tetrahydrofuran was charged (10.8 L, 5 L/kg) and the suspension was heated to 58 °C. In a separate vessel, (R)-2- methyl- 1- (l-(l-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxylic acid (2.15 kg) was dissolved in tetrahydrofuran (8.6 L, 4 L/kg). The solution was added to the CDI mixture via addition funnel at a rate such that the C0 ₂ off-gassing was controlled and the reaction temperature was maintained at <60 °C (~1 hour). The reaction was agitated at 60 °C until conversion to the acyl- imidazole was complete by HPLC (<1 %). 2-Propanol (895 mL, 11.7 mol) was charged and the resulting mixture was stirred at 60 oC for 15 min. Charged 3- (aminomethyl)-4-methoxy-6-methylpyridin-2(lH)-one (1.252 kg, 7.0 mol) to the reactor and agitated at 60 °C overnight until >96 % conversion of the acyl-imidazole was observed by HPLC. The reaction was cooled to room temperature and transferred to a separatory funnel, where it was further diluted with z ^' PrOAc (54 L, -25 L/kg). The reaction stream was washed with water (10.8 L, 5 L/kg) two times. The resulting product-rich z ^' PrOAc layer was polish filtered and concentrated to a target volume of (~2 L/kg based on input).

[0079] A second reaction was run on 2.17 kg scale under reaction conditions identical to those described above. After the aqueous workup and polish filtration operations were completed for the second reaction, it was combined with the first reaction (see paragraph above) in a 100 L round bottom reactor for further distillation and product isolation. The combined mixture was distilled to a target volume of 2 L/kg (based on the combined input) and was then diluted with z ^' PrOAc (34.5 L, 8 L/kg) resulting in a final concentration of -10 L/kg. The mixture was then heated to 65 °C and agitated overnight to achieve conversion to Form C. Note - During the form conversion, the initial solution is pale yellow, and subsequent crystallization results in a thick slurry that gradually becomes less viscous. The slurry was subsequently cooled to 25 °C and agitated at room temperature (< 30 °C) overnight. The product was collected by vacuum filtration, and the flask and filter cake were rinsed with z ^' PrOAc (4 L/kg total in two portions). The product was dried under vacuum at 55 °C until constant mass was achieved. Product analysis by HPLC shows 97.5 area % chromatographic purity and 93.3 w/w% potency. Yield - 5.695 kg, 87% (potency corrected) Pharmacokinetic of Form C and Amorphous (R)-N-((4-methoxy-6-methyl-2-oxo-l _< 2- dihvdropyridin-3-yl)methyl)-2-methyl-l-(l-(l-(2 _< 2 _< 2-trifluoroethyl)piperidin-4-yl)ethyl)- lH-indole-3-carboxamide in Rat and Dog

[0080] After once-daily oral administration of Form C as a suspension in 0.5%

methylcellulose, 0.1% Tween 80 in purified water, the systemic exposure in rats (both genders) was measured by the AUC(o-24), increased dose-proportionally between 100 and 600 mg/kg. See Table 6. A similar proportionality was observed for the maximal plasma concentration achieved after administration (C _max ).

Table 6

[0081] After twice-daily oral administration of Form C as a suspension in 0.5% methylcellulose, 0.1% Tween 80 in purified water, the systemic exposure in dogs (both genders) was measured by the AUC(o-24), increased mostly dose-proportionally between 50 and 500 mg/kg BID. See Table 7. The maximum plasma concentration observed after administration, C _max also increased with doses. Doses levels ranging from 50 mg/kg to 500 mg/kg were permitted to achieve pharmacological relevant systemic exposures.

Table 7 These results are comparable with the material generated for Compound 365 following the procedures outlined in U.S. Patent Publication No. 2015/0011546. Compound 365 was formulated similarly to Form C, suspension in 0.5% methylcellulose, 0.1% Tween 80 in purified water. At both 100 and 300 mpk the Cmax and AUC are slightly superior to what was achieved with Form C. This was expected since the aqueous solubility of Compound 365 is greater than the aqueous solubility of Form C (Table 2). However, the differences in exposure are minimal demonstrating that Form C is mostly equivalent to Compound 365 when administered at these doses in rat. The advantages of Form C compared to the mostly amorphous solid Compound 365 are e.g., isolation and purification (crystallization is a purification step), stability (Form C being the most stable polymorph), and ease to handle / formulate.

Table 8

[0082] While have described a number of embodiments of this, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this disclosure. Therefore, it will be appreciated that the scope of this disclosure is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

[0083] The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.