Crystalline forms of histone demethylase inhibitor 3-({[(4r)-7-{methyl [4-(propan-2-yl) phenyl] amino}-3, 4-dihydro-2h-1-benzopyran-4-yl] methyl} amino) pyridine-4-carboxylic acid l-lysine salt

Abstract

Provided herein are crystalline forms of 3-({[(4R)-7-{methyl [4-(propan-2-yl) phenyl] amino}-3, 4-dihydro-2H-1-benzopyran-4-yl] methyl} amino) pyridin-4-carboxylic acid L-lysine salt as inhibitors of histone demethylase. Also provided herein are pharmaceutical compositions comprising such crystalline forms.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Patent Application 63 / 408,691, filed on September 21, 2022, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to crystalline forms of L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid, which is a histone demethylase inhibitor. Background Art

[0004] The compound 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid (Compound 1) is a selective inhibitor of the histone demethylase KDM4 family (see, for example, U.S. Patent No. 9,242,968), and its structure is shown below.

[0005]

[0006] This first-in-class epigenetic modification compound shows promise in the treatment of cancers, including gastric cancer and colon cancer.

[0007] Compound 1 has poor solubility in water and the most common organic solvents and tends to precipitate as an amorphous paste, making it difficult to filter on a large scale. Therefore, for ease of handling and further formulation, it is also desirable to provide the compound in an alternative form, such as a salt.

[0008] Accordingly, in one aspect, the present disclosure provides crystalline forms (i.e., polymorphs) of L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid (Compound 1L-lysine). The present disclosure also provides pharmaceutical compositions comprising the crystalline forms of Compound 1L-lysine. Summary of the Invention

[0009] In certain embodiments, the present disclosure describes crystalline forms of L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid, which is a histone demethylase inhibitor. The present disclosure also provides pharmaceutical compositions comprising such crystalline forms.

[0010] The present embodiment can be more fully understood by referring to the detailed description and examples that are intended to illustrate non-limiting embodiments.

[0011] Embodiment 1. A crystalline form of L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid, wherein the crystalline form is Form 1.

[0012] Embodiment 2. The crystalline form according to Embodiment 1, which has an X-ray powder diffraction showing characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°.

[0013] Embodiment 3. The crystalline form according to Embodiment 1 or 2, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.0° ± 0.2° and 7.6° ± 0.2°.

[0014] Embodiment 4. The crystalline form according to any one of Embodiments 1-3, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2° and 23.5° ± 0.2°.

[0015] Embodiment 5. The crystalline form according to any one of Embodiments 1-4, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2°, 23.5° ± 0.2° and 14.5° ± 0.2°.

[0016] Embodiment 6. The crystalline form according to any one of Embodiments 1-5, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2°, 23.5° ± 0.2°, 14.5° ± 0.2°, 4.9° ± 0.2°, 6.9° ± 0.2°, 8.5° ± 0.2°, 9.4° ± 0.2°, 10.4° ± 0.2°, 11.6° ± 0.2°, 13.2° ± 0.2°, 13.8° ± 0.2°, 16.1° ± 0.2°, 17.2° ± 0.2°, 17.8° ± 0.2° and 19.0° ± 0.2°.

[0017] Embodiment 7. The crystalline form according to any one of Embodiments 1-6, which has an X-ray powder diffraction substantially as Figure 9 shown.

[0018] Embodiment 8. The crystalline form according to any one of Embodiments 1-7, which has a differential scanning calorimetry thermogram including an endotherm at about 239.4° ± 5°.

[0019] Embodiment 9. The crystalline form according to any one of Embodiments 1-8, which has a differential scanning calorimetry thermogram substantially as Figure 11 shown.

[0020] Embodiment 10. The crystalline form according to any one of Embodiments 1-9, as determined by thermogravimetric analysis, which has no significant weight loss up to about 200 °C.

[0021] Embodiment 11. The crystalline form according to any one of Embodiments 1-10, which has a thermogravimetric analysis thermogram substantially as Figure 10 shown.

[0022] Embodiment 12. The crystalline form according to any one of Embodiments 1-11, as determined by dynamic vapor sorption, which has a reversible sorption of about 1.4% at up to 90% relative humidity.

[0023] Embodiment 13. The crystalline form according to any one of Embodiments 1-12, which has a dynamic vapor sorption plot substantially as Figure 14 shown.

[0024] Embodiment 14. A crystalline form of L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid, wherein the crystalline form is Form 2.

[0025] Embodiment 15. The crystalline form according to Embodiment 14, which has an X-ray powder diffraction showing characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°.

[0026] Embodiment 16. The crystalline form according to Embodiment 14 or 15, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.5° ± 0.2° and 18.2° ± 0.2°.

[0027] Embodiment 17. The crystalline form according to any one of Embodiments 14-16, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2° and 21.5° ± 0.2°.

[0028] Embodiment 18. The crystalline form according to any one of Embodiments 14-17, wherein the X-ray powder diffraction exhibits characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2°, 21.5° ± 0.2°, and 25.6° ± 0.2°.

[0029] Embodiment 19. The crystalline form according to any one of Embodiments 14-18, wherein the X-ray powder diffraction exhibits characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2°, 21.5° ± 0.2°, 25.6° ± 0.2°, 8.6° ± 0.2°, 13.8° ± 0.2°, and 19.3° ± 0.2°.

[0030] Embodiment 20. The crystalline form according to any one of Embodiments 14-19, which has an X-ray powder diffraction substantially as Figure 5B shown.

[0031] Embodiment 21. The crystalline form according to any one of Embodiments 14-20, which has a differential scanning calorimetry thermogram including an endotherm at about 231.7° ± 5°.

[0032] Embodiment 22. The crystalline form according to any one of Embodiments 14-21, which has no significant weight loss up to about 240 °C as determined by thermogravimetric analysis.

[0033] Embodiment 23. The crystalline form according to any one of Embodiments 14-22, which has a reversible adsorption of about 1.6% at up to 90% relative humidity as determined by gravimetric vapor sorption.

[0034] Embodiment 24. A method for preparing the crystalline form according to any one of Embodiments 1-23, which comprises:

[0035] Combining 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and methanol at about 50 °C to obtain a suspension;

[0036] Adding L-lysine to the suspension at about 50 °C to obtain a solution;

[0037] Cooling the solution; and

[0038] Isolating the crystalline form from the solution.

[0039] Embodiment 25. A solid pharmaceutical composition comprising a crystalline form as described in any one of Embodiments 1-23 and a pharmaceutically acceptable excipient.

[0040] Embodiment 26. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the solid pharmaceutical composition as described in Embodiment 25.

[0041] Embodiment 27. The method as described in Embodiment 26, wherein the cancer is selected from colorectal cancer, esophageal cancer, gastric cancer, breast cancer, and lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Aspects of the present disclosure are specifically set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and the drawings that illustrate exemplary embodiments in which the principles of the present disclosure are utilized, wherein:

[0043] Figure 1 Showing the X-ray powder diffraction (XRPD) pattern of Compound 1.

[0044] Figure 2 Showing the thermogravimetric analysis (TGA) thermogram and differential scanning calorimetry (DSC) thermogram of Compound 1.

[0045] Figure 3 Showing the predicted and measured pKa of Compound 1.

[0046] Figure 4 Showing an overlay of the X-ray powder diffraction (XRPD) patterns of the crystalline forms of Compound 1 sodium salts (Na1-Na6).

[0047] Figure 5A Showing an overlay of the X-ray powder diffraction (XRPD) patterns of the crystalline forms of Compound 1 L-lysine (LYS1 (Form 1) and LYS2 (Form 2)).

[0048] Figure 5B Showing the X-ray powder diffraction (XRPD) pattern of Compound 1 L-lysine Form 2.

[0049] Figure 6 Showing the X-ray powder diffraction (XRPD) pattern of the crystalline form of Compound 1 ethanolamine salt (EA1).

[0050] Figure 7 Showing the X-ray powder diffraction (XRPD) pattern of the crystalline form of Compound 1 N-ethylglucamine salt (Neg1).

[0051] Figure 8Show the polarized light microscopy (PLM) micrograph of Compound 1 L-lysine form 1.

[0052] Figure 9 Show the X-ray powder diffraction (XRPD) pattern of Compound 1 L-lysine form 1.

[0053] Figure 10 Show the thermogravimetric analysis (TGA) thermogram of Compound 1 L-lysine form 1.

[0054] Figure 11 Show the differential scanning calorimetry (DSC) thermogram of Compound 1 L-lysine form 1.

[0055] Figure 12 Show the high performance liquid chromatography (HPLC) chromatogram of Compound 1 L-lysine form 1.

[0056] Figure 13 Show Compound 1 L-lysine form 1 1 1H NMR chromatogram.

[0057] Figure 14 Show the dynamic vapor sorption (DVS) adsorption-desorption plot of Compound 1 L-lysine form 1.

[0058] Figure 15 Show the overlay of the X-ray powder diffraction (XRPD) patterns of Compound 1 L-lysine form 1 before and after analysis by dynamic vapor sorption (DVS). Detailed Description

[0059] Definitions

[0060] As used herein, the terms "comprising" and "including" are used interchangeably. The terms "comprising" and "including" shall be construed to specify the presence of the stated features or components mentioned, but do not preclude the presence or addition of one or more features or components or groups thereof. Additionally, the terms "comprising" and "including" are intended to encompass the examples covered by the term "consisting of". Thus, the term "consisting of" can be used in place of the terms "comprising" and "including" to provide a more specific embodiment of the present invention.

[0061] The term "consisting of" means that the subject matter has at least 90%, 95%, 97%, 98% or 99% of the stated features or components that make it up. In another embodiment, the term "consisting of" excludes any other features or components from the scope of any subsequent statement, except those features or components that are not essential for the technical effect to be achieved.

[0062] As used herein, the term "or" shall be construed as the inclusive "or" meaning any one or any combination thereof. Thus, "A, B, or C" means any of the following: "A; B; C; A and B; A and C; B and C; A, B, and C". An exception to this definition will occur only when the combination of elements, functions, steps, or acts is inherently mutually exclusive in some manner.

[0063] In this specification, unless otherwise indicated, any concentration range, error range, percentage range, ratio range, or integer range shall be understood to include any integral value within the stated range and, where appropriate, their fractions (such as one-tenth and one-hundredth of an integer). Additionally, unless otherwise indicated, any numerical range recited herein with respect to any physical feature shall be understood to include any integer within the stated range. As used herein, unless otherwise indicated, the terms "about" and "approximately" mean ±20%, ±10%, ±5%, or ±1% of the stated range, value, or structure.

[0064] As used herein, "treatment" means the partial or complete alleviation of a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition, or the slowing or halting of the further progression or worsening of these symptoms, or the attenuation or eradication of one or more causative factors of the disorder, disease, or condition itself. In one embodiment, the disorder is cancer or a symptom thereof as described herein.

[0065] As used herein, "prevention" means a method of delaying and / or preventing the onset, recurrence, or spread of all or part of a disorder, disease, or condition; deterring a subject from developing a disorder, disease, or condition; or reducing the risk that a subject will develop a disorder, disease, or condition. In one embodiment, the disorder is cancer or a symptom thereof as described herein.

[0066] The term "effective amount" in relation to a compound disclosed herein means an amount capable of treating or preventing a disorder, disease, or condition disclosed herein, or a symptom thereof.

[0067] The term "subject" as used herein includes animals, including but not limited to cows, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a human having cancer or at risk of developing cancer.

[0068] References to "an embodiment" or "embodiments" throughout this specification mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the phrases "in an embodiment" or "in embodiments" that appear throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0069] Although various features of the invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment.

[0070] Crystalline forms of Compound 1·L-lysine salt

[0071] The crystalline forms of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid L-lysine salt (hereinafter referred to as "Compound 1 L-lysine") described herein have properties superior to those of the free base (hereinafter referred to as "Compound 1"). For example, the crystalline forms described herein can provide improved stability, solubility, filtration characteristics, hygroscopicity, ease of handling, and / or ease of formulation into solid pharmaceutical compositions such as tablets or capsules.

[0072] In one aspect, crystalline forms of Compound 1 L-lysine are provided herein. In some embodiments, the crystalline form of Compound 1 L-lysine is Form 1. In some embodiments, the crystalline form of Compound 1 L-lysine is Form 2.

[0073] The crystalline forms of Compound 1 L-lysine described herein can be identified by their unique solid-state properties, which are characterized by, for example, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), dynamic vapor sorption (DVS), gravimetric vapor sorption (GVS), and other techniques.

[0074] Crystalline Form 1

[0075] In one aspect, crystalline forms of Compound 1 L-lysine are provided herein, wherein the crystalline form is Form 1.

[0076] In some embodiments, Compound 1 L-lysine Form 1 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 1 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.0° ± 0.2° and 7.6° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 1 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2°, and 23.5° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 1 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2°, 23.5° ± 0.2°, and 14.5° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 1 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2°, 23.5° ± 0.2°, 14.5° ± 0.2°, 4.9° ± 0.2°, 6.9° ± 0.2°, 8.5° ± 0.2°, 9.4° ± 0.2°, 10.4° ± 0.2°, 11.6° ± 0.2°, 13.2° ± 0.2°, 13.8° ± 0.2°, 16.1° ± 0.2°, 17.2° ± 0.2°, 17.8° ± 0.2°, and 19.0° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 1 has an X-ray powder diffraction substantially as Figure 9 shown.

[0077] In some embodiments, Compound 1 L-lysine Form 1 has a differential scanning calorimetry thermogram including an endotherm at about 239.4° ± 5°. In some embodiments, Compound 1 L-lysine Form 1 has a differential scanning calorimetry thermogram including an endotherm at about 239.4° ± 4°, 239.4° ± 3°, or 239.4° ± 2°. In some embodiments, Compound 1 L-lysine Form 1 has a differential scanning calorimetry thermogram including an endotherm at about 239.4° ± 10° (such as at about 239.4° ± 9°, 239.4° ± 8°, 239.4° ± 7°, or 239.4° ± 6°). In some embodiments, Compound 1 L-lysine Form 1 has a differential scanning calorimetry thermogram substantially as Figure 11 shown.

[0078] In some embodiments, as determined by thermogravimetric analysis, Compound 1 L-lysine Form 1 has no significant weight loss up to about 200°C. In some embodiments, Compound 1 L-lysine Form 1 has a substantially asFigure 10 The thermogravimetric analysis thermogram shown.

[0079] In some embodiments, as determined by dynamic vapor sorption, Compound 1 L-lysine Form 1 has a reversible sorption of about 1.4% at up to 90% relative humidity. In some embodiments, Compound 1 L-lysine Form 1 has a dynamic vapor sorption profile substantially as Figure 14 shown.

[0080] Crystalline Form 2

[0081] In another aspect, provided herein is a crystalline form of Compound 1 L-lysine, wherein the crystalline form is Form 2.

[0082] In some embodiments, Compound 1 L-lysine Form 2 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 2 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.5° ± 0.2° and 18.2° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 2 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2°, and 21.5° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 2 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2°, 21.5° ± 0.2°, and 25.6° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 2 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2°, 21.5° ± 0.2°, 25.6° ± 0.2°, 8.6° ± 0.2°, 13.8° ± 0.2°, and 19.3° ± 0.2°. In some embodiments, Compound 1 L-lysine Form 2 has an X-ray powder diffraction substantially as Figure 5B shown.

[0083] In some embodiments, Compound 1 L-lysine Form 2 has a differential scanning calorimetry thermogram with an endotherm at about 231.7° ± 5°. In some embodiments, Compound 1 L-lysine Form 2 has a differential scanning calorimetry thermogram with an endotherm at about 231.7° ± 4°, 231.7° ± 3°, or 231.7° ± 2°. In some embodiments, Compound 1 L-lysine Form 2 has a differential scanning calorimetry thermogram with an endotherm at about 231.7° ± 10°, such as about 231.7° ± 9°, 231.7° ± 8°, 231.7° ± 7°, or 231.7° ± 6°.

[0084] In some embodiments, as determined by thermogravimetric analysis, Compound 1 L-lysine Form 2 has no significant weight loss up to about 240 °C.

[0085] In some embodiments, as determined by gravimetric vapor sorption, Compound 1 L-lysine Form 2 has a reversible sorption of about 1.6% at up to 90% relative humidity.

[0086] Preparation Method

[0087] Compound 1 can be synthesized as described in U.S. Patent No. 9,242,968. An overview of the synthesis is provided in Scheme 1.

[0088] Scheme 1.

[0089]

[0090] In one aspect, provided herein is a method for preparing a crystalline form of Compound 1 (such as a crystalline form of a salt of Compound 1). In some embodiments, the method for preparing a crystalline form of Compound 1 comprises: combining Compound 1 and a solvent (such as methanol) at an elevated temperature (e.g., about 50 °C) to obtain a suspension; adding a base or an acid to the suspension at the elevated temperature to obtain a solution; cooling the solution; and isolating the crystalline form from the solution.

[0091] In some embodiments, provided herein is a method for preparing a crystalline form of Compound 1 L-lysine, which comprises: combining Compound 1 and methanol at about 50 °C to obtain a suspension; adding L-lysine to the suspension at about 50 °C to obtain a solution; cooling the solution; and isolating the crystalline form from the solution. In some embodiments, the crystalline form of Compound 1 L-lysine is Form 1. In some embodiments, the crystalline form of Compound 1 L-lysine is Form 2.

[0092] Pharmaceutical Compositions

[0093] The crystalline forms provided herein can be administered to a subject in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a crystalline form of Compound 1 (such as Compound 1 L-lysine Form 1 or Form 2) and one or more pharmaceutically acceptable excipients. The pharmaceutical composition can be administered orally, topically, or parenterally to the subject in conventional dosage forms such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions. In some embodiments, the pharmaceutical composition comprises a solid dosage form such as a capsule, microcapsule, tablet, granule, powder, pill, or suppository.

[0094] The crystalline forms disclosed herein can be administered orally, topically, or parenterally to a subject in conventional dosage forms such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions. Suitable dosage forms can be prepared by commonly employed methods using conventional, organic or inorganic additives such as excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, or calcium carbonate), binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, or starch), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropyl cellulose, sodium bicarbonate, calcium phosphate, or calcium citrate), lubricants (e.g., magnesium stearate, light anhydrous silicic acid, talc, or sodium lauryl sulfate), flavoring agents (e.g., citric acid, menthol, glycine, or orange powder), preservatives (e.g., sodium benzoate, sodium bisulfite, methyl paraben, or propyl paraben), stabilizers (e.g., citric acid, sodium citrate, or acetic acid), suspending agents (e.g., methylcellulose, polyvinylpyrrolidone, or aluminum stearate), dispersing agents (e.g., hydroxypropyl methylcellulose), diluents (e.g., water), and base waxes (e.g., cocoa butter, white petrolatum, or polyethylene glycol). The effective amount of the crystalline form of Compound 1 in the pharmaceutical composition can be at a level that will achieve the desired effect; for example, for oral and parenteral administration, the unit dose is from about 0.005 mg / kg of subject body weight to about 10 mg / kg of subject body weight.

[0095] The dosage of the crystalline forms described herein to be administered to a subject can vary quite widely and can be subject to the judgment of a healthcare practitioner. Generally, the compounds disclosed herein can be administered one to four times per day at a dosage of from about 0.001 mg / kg of subject body weight to about 10 mg / kg of subject body weight, but the above dosages can be appropriately varied depending on the age, weight, and medical condition of the subject, as well as the type of administration. In one embodiment, the dosage is from about 0.001 mg / kg of subject body weight to about 5 mg / kg of subject body weight, from about 0.01 mg / kg of subject body weight to about 5 mg / kg of subject body weight, from about 0.05 mg / kg of subject body weight to about 1 mg / kg of subject body weight, from about 0.1 mg / kg of subject body weight to about 0.75 mg / kg of subject body weight, or from about 0.25 mg / kg of subject body weight to about 0.5 mg / kg of subject body weight. In one embodiment, one dosage is given per day. In any given case, the amount of the crystalline form administered will depend on factors such as the solubility of the active ingredient, the formulation used, and the route of administration.

[0096] In some embodiments, the crystalline forms described herein are administered to a subject at a dosage of from about 0.01 mg / day to about 750 mg / day, from about 0.1 mg / day to about 375 mg / day, from about 0.1 mg / day to about 150 mg / day, from about 0.1 mg / day to about 75 mg / day, from about 0.1 mg / day to about 50 mg / day, from about 0.1 mg / day to about 25 mg / day, or from about 0.1 mg / day to about 10 mg / day.

[0097] In another embodiment, the present invention provides unit dosage formulations that comprise a crystalline form of Compound 1 between about 0.1 mg and 500 mg, between about 1 mg and 250 mg, between about 1 mg and about 100 mg, between about 1 mg and about 50 mg, between about 1 mg and about 25 mg, or between about 1 mg and about 10 mg, such as Compound 1 L-lysine Form 1 or Form 2.

[0098] In a particular embodiment, the present invention provides unit dosage formulations that comprise about 0.1 mg or 100 mg of a crystalline form of Compound 1, such as Compound 1 L-lysine Form 1 or Form 2.

[0099] In another embodiment, the present invention provides unit dosage formulations that comprise 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 35 mg, 50 mg, 70 mg, 100 mg, 125 mg, 140 mg, 175 mg, 200 mg, 250 mg, 280 mg, 350 mg, 500 mg, 560 mg, 700 mg, 750 mg, 1000 mg, or 1400 mg of a crystalline form of Compound 1, such as Compound 1 L-lysine Form 1 or Form 2.

[0100] The crystalline form of Compound 1, such as Compound 1 L-lysine Form 1 or Form 2, can be administered once, twice, three times, four times, or more times per day. In certain embodiments, a dose of 100 mg or less is administered as a once-daily dose, and a dose greater than 100 mg is administered twice daily in an amount equal to half of the total daily dose.

[0101] For convenience, the crystalline form of Compound 1, such as Compound 1 L-lysine Form 1 or Form 2, can be administered orally. In one embodiment, when administered orally, the crystalline form is administered with food and water. In another embodiment, the crystalline form is dispersed in water or fruit juice (e.g., apple juice or orange juice) or any other liquid and administered orally as a solution or suspension.

[0102] The crystalline forms of Compound 1 disclosed herein can also be administered intradermally, intramuscularly, intraperitoneally, transdermally, intravenously, subcutaneously, intranasally, epidurally, sublingually, intracerebrally, intravaginally, transdermally, rectally, mucosally, by inhalation, or topically to the ear, nose, eye, or skin. The mode of administration is at the discretion of the healthcare practitioner and can depend in part on the site of the medical condition.

[0103] In one embodiment, capsules are provided herein that contain a crystalline form of Compound 1 (such as Compound 1 L-lysine Form 1 or Form 2) without additional carriers, excipients, or vehicles.

[0104] In another embodiment, pharmaceutical compositions are provided that comprise an effective amount of a crystalline form of Compound 1 (such as Compound 1 L-lysine Form 1 or Form 2), and a pharmaceutically acceptable carrier or vehicle, wherein the pharmaceutically acceptable carrier or vehicle can include excipients, diluents, or mixtures thereof.

[0105] The pharmaceutical compositions can be in the form of tablets, chewable tablets, capsules, solutions, parenteral solutions, lozenges, suppositories, and suspensions, among others. The pharmaceutical compositions can be formulated to contain the daily dose or a convenient fraction of the daily dose in dosage units, which can be a single tablet or capsule or a convenient volume of liquid. Generally, all pharmaceutical compositions are prepared according to known methods in pharmaceutical chemistry. Capsules can be prepared by mixing the crystalline form of Compound 1 (such as Compound 1 L-lysine Form 1 or Form 2) with a suitable carrier or diluent and filling an appropriate amount of the mixture into the capsules. Commonly used carriers and diluents include, but are not limited to, inert powdered substances such as many different kinds of starches, powdered celluloses (especially crystalline and microcrystalline celluloses), sugars (such as fructose, mannitol, and sucrose), cereal flours, and similar edible powders.

[0106] Tablets can be prepared by direct compression, by wet granulation or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrants as well as the compound. Typical diluents include, for example, different types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts (such as sodium chloride) and powdered sugar. Powdered cellulose derivatives are also available. Typical tablet binders are substances such as starch, gelatin and sugars (such as lactose, fructose, glucose, etc.). Natural and synthetic gums are also convenient, which include gum arabic, alginates, methylcellulose, polyvinylpyrrolidone, etc. Polyethylene glycol, ethylcellulose and waxes can also be used as binders.

[0107] Lubricants may be necessary in tablet formulations to prevent adhesion of the tablets and the punch to the die in the mold. Lubricants can be selected from smooth solids such as talc, magnesium stearate and calcium stearate, stearic acid and hydrogenated vegetable oils. Tablet disintegrants are substances that swell upon wetting to break down the tablet and release the compound. They include starch, clay, cellulose, algin and gums. More particularly, for example, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resin, alginic acid, guar gum, citrus pulp and carboxymethyl cellulose as well as sodium lauryl sulfate can be used. Tablets can be sugar-coated as flavorants and sealants, or coated with film protectants to alter the dissolution properties of the tablets. For example, by using substances such as mannitol in the formulation, the composition can also be formulated as a chewable tablet.

[0108] When it is desired to administer a crystalline form of Compound 1 (such as Compound 1 L-lysine Form 1 or Form 2) as a suppository, typical bases can be used. Cocoa butter is a traditional suppository base and can be modified by adding wax to slightly increase its melting point. Water-miscible suppository bases, particularly those containing polyethylene glycols of various molecular weights, are widely used.

[0109] The action of a crystalline form of Compound 1 (such as Compound 1 L-lysine Form 1 or Form 2) can be delayed or prolonged by appropriate formulation. For example, slowly dissolving pellets of the crystalline form can be prepared and incorporated into tablets or capsules, or as a sustained-release implantable device. The techniques also include preparing pellets of several different dissolution rates and filling capsules with a mixture of the pellets. Tablets or capsules can be film-coated to resist dissolution over a predictable period of time. Even parenteral formulations can be made into long-acting formulations by dissolving or suspending the crystalline form of Compound 1 in an oily or emulsified vehicle that causes it to disperse slowly in the serum.

[0110] Method of Use

[0111] Compound 1 and its salts (including the crystalline forms described herein (Compound 1 L-lysine Form 1 or Form 2)) can be used to selectively inhibit the KDM4 family of histone demethylases and for the treatment of cancers associated with KDM4 activity.

[0112] Accordingly, in one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a crystalline form of Compound 1 L-lysine as described herein, such as Compound 1 L-lysine Form 1 or Form 2. In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a solid pharmaceutical composition comprising Compound 1 L-lysine Form 1. In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a solid pharmaceutical composition comprising Compound 1 L-lysine Form 2. In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound 1 L-lysine Form 1. In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound 1 L-lysine Form 2.

[0113] In some embodiments, the present disclosure provides the use of a crystalline form of Compound 1 L-lysine as described herein (such as Compound 1 L-lysine Form 1 or Form 2) in the manufacture of a medicament for the treatment of cancer.

[0114] In some embodiments, the present disclosure provides the use of a crystalline form of Compound 1 L-lysine as described herein (such as Compound 1 L-lysine Form 1 or Form 2) for the treatment of cancer in a subject in need thereof.

[0115] Embodiments of the present disclosure provide a method for inhibiting the KDM4 family of histone demethylases in a subject in need thereof, the method comprising administering to the subject an effective amount of a crystalline form of Compound 1, such as Compound 1 L-lysine Form 1 or Form 2. Inhibition of the KDM4 family of histone demethylases can be evaluated and demonstrated by a variety of methods known in the art. Kits and commercially available assays can be used to determine whether the KDM4 family of histone demethylases is inhibited and the extent of inhibition.

[0116] In one aspect, the present disclosure provides a method of inhibiting the KDM4 family of histone demethylases, comprising contacting the KDM4 family of histone demethylases with an effective amount of a crystalline form of Compound 1. In some embodiments, the crystalline form is Compound 1 L-lysine Form 1. In some embodiments, the crystalline form is Compound 1 L-lysine Form 2.

[0117] In some embodiments, the crystalline forms described herein inhibit the KDM4 family of histone demethylases by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the crystalline forms described herein inhibit the KDM4 family of histone demethylases by about 1%-100%, 5%-100%, 10%-100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 5%-95%, 5%-90%, 5%-85%, 5%-80%, 5%-75%, 5%-70%, 5%-65%, 5%-60%, 5%-55%, 5%-50%, 5%-45%, 5%-40%, 5%-35%, 5%-30%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-90%, 20%-80%, 30%-70%, or 40%-60%.

[0118] In another aspect, the present disclosure provides methods for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a crystalline form described herein, such as Compound 1 L-lysine Form 1 or Form 2. In some embodiments, the present disclosure provides methods for preventing cancer (such as cancer associated with KDM4 activity) in a subject in need thereof, comprising administering to the subject an effective amount of a crystalline form described herein, such as Compound 1 L-lysine Form 1 or Form 2. Non-limiting examples of cancers for treatment include gastric cancer or colorectal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is colorectal carcinoma. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lymphoma.

[0119] In some embodiments, administering a crystalline form disclosed herein to a cancer - prone subject prevents any symptoms of cancer in the subject. In some embodiments, administering a crystalline form disclosed herein to a subject who has not yet manifested symptoms of cancer prevents any symptoms of cancer in the subject. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof reduces the degree of cancer in the subject. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof stabilizes the cancer (prevents or delays the progression of cancer). In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof delays the onset or recurrence of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof slows the progression of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof provides partial remission of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof provides complete remission of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof reduces the dosage of one or more other drugs required to treat cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof enhances the effect of another drug used to treat cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof delays the progression of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof improves the quality of life of a subject with cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof prolongs the survival of a subject with cancer.

[0120] In one aspect, the present disclosure provides a method of preventing any symptoms of cancer in a cancer - prone subject, the method comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the present disclosure provides a method of preventing any symptoms of cancer in a subject who has not yet manifested symptoms of cancer, the method comprising administering to the subject a crystalline form disclosed herein.

[0121] In some aspects, the present disclosure provides a method of reducing the degree of cancer in a subject, the method comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the present disclosure provides a method of stabilizing cancer in a subject, the method comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the method prevents cancer progression. In some embodiments, the method delays cancer progression.

[0122] In another aspect, the present disclosure provides a method of delaying the onset or recurrence of cancer in a subject, the method comprising administering to the subject a crystalline form disclosed herein.

[0123] In some embodiments, provided herein are methods of slowing the progression of cancer in a subject, the methods comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the methods provide a partial remission of cancer. In some embodiments, the methods provide a complete remission of cancer.

[0124] In a further aspect, provided herein are methods of reducing the dosage of one or more other drugs required to treat cancer in a subject, the methods comprising administering to the subject a crystalline form disclosed herein. In some embodiments, provided herein are methods of enhancing the effect of another drug used to treat cancer in a subject, the methods comprising administering to the subject a crystalline form disclosed herein.

[0125] Also provided herein are methods of delaying the progression of cancer in a subject, the methods comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the methods improve the quality of life of a subject with cancer. In some embodiments, the methods prolong the survival of a subject with cancer.

[0126] Examples

[0127] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0128] Abbreviations used:

[0129]

[0130]

[0131] Example 1. Salt screening study of Compound 1 and identification of Compound 1 L-lysine Form 1.

[0132] Compound 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid (Compound 1) was synthesized as described in U.S. Patent No. 9,242,968. Compound 1 has poor solubility in water and the most common organic solvents and tends to precipitate as an amorphous paste. To identify crystalline non-hygroscopic forms suitable for further development, a salt screening assay was performed.

[0133] 1. Instrument and method details.

[0134] X - ray powder diffraction (XRPD) was performed using a Bruker AXS C2 GADDS.

[0135] X-ray powder diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), an automated XYZ stage, a laser video microscope for automated sample positioning, and a HiStar two-dimensional area detector. The X-ray optics consisted of a single multilayer mirror and a 0.3 mm pinhole collimator connected in series. Weekly performance checks were performed using the certified standard NIST 1976 Corundum (flat plate).

[0136] The beam divergence, i.e., the effective size of the X-ray beam on the sample, was approximately 4 mm. A θ-θ continuous scan mode was used, where the sample-detector distance was 20 cm, which gave an effective 2θ range of 3.2° - 29.7°. Typically, the sample was exposed to the X-ray beam for 120 s. The software used for data collection was GADDS for XP / 2000 4.1.43, and the data was analyzed and presented using Diffrac Plus EVAv15.0.0.0.

[0137] Environmental conditions. Samples run under environmental conditions were prepared as flat specimens using as-received powder without grinding. Approximately 1 - 2 mg of the sample was gently pressed onto a glass slide to obtain a flat surface.

[0138] Non-environmental conditions. Samples run under non-environmental conditions were mounted on a silicon wafer with a thermal conducting compound. The sample was then heated to the appropriate temperature at 10 °C / min and subsequently held isothermally for 1 minute before starting data collection.

[0139] Nuclear magnetic resonance (NMR)

[0140] 1H NMR spectra were collected on a Bruker 400 MHz instrument equipped with an autosampler and controlled by a DRX400 console. 1 Standard Bruker loading experiments were used, and automated experiments were obtained using ICON-NMR v4.0.7 running on Topspin v1.3. For non-conventional spectroscopy, data was obtained only by using Topspin. Samples were prepared in DMSO-d6 unless otherwise stated. Offline analysis was performed using ACD Spectrus Processor 2012.

[0141] X - ray powder diffraction (XRPD) was performed using a Bruker AXSD8 Advance

[0142] X-ray powder diffraction patterns were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), a θ-2θ goniometer, and V4 divergence and receiving slits, a Ge monochromator, and a Lynxeye detector. The performance of the instrument was checked using a certified Corundum standard (NIST 1976). The software used for data collection was Diffrac Plus XRD Commander v2.6.1, and the data was analyzed and presented using Diffrac Plus EVA v15.0.0.0.

[0143] The as-received powder was used as a flat sample, and the sample was run under ambient conditions. The sample was gently loaded into a cavity cut into a polished zero-background (510) silicon wafer. During analysis, the sample was rotated in its own plane. The details of data collection were: angular range: 2° to 42° 2θ; step size: 0.05° 2θ; collection time: 0.5 s / step.

[0144] Differential scanning calorimetry (DSC)

[0145] DSC data was collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Sapphire was used for the calibration of heat capacity, and certified indium was used for the calibration of energy and temperature. Typically, 0.5 - 3 mg of each sample in a pierced aluminum pan was heated from 25 °C to 300 °C at 10 °C / min. A dry nitrogen purge of 50 ml / min was maintained above the sample. Temperature-modulated DSC was performed using a base heating rate of 2 °C / min and temperature modulation parameters of ±0.636 °C (amplitude) every 60 seconds (cycle). The instrument control software was Advantage for Q Series v2.8.0.394 and Thermal Advantage v5.5.3, and the data was analyzed using Universal Analysis v4.5A.

[0146] Thermogravimetric analysis (TGA)

[0147] TGA data was collected on a TA Instruments Q500 TGA equipped with a 16-position autosampler. The temperature of the instrument was calibrated using certified Alumel and nickel. Typically, 5 - 10 mg of each sample was loaded onto a pre-tared DSC aluminum pan and heated from ambient temperature to 350 °C at 10 °C / min. A nitrogen purge of 60 ml / min was maintained above the sample.

[0148] The instrument control software is Advantage and Thermal Advantage v5.5.3 for Q Series v2.5.0.256, and data is analyzed using Universal Analysis v4.5A.

[0149] Polarized light microscopy (PLM)

[0150] Samples were studied on a Leica LM / DM polarized light microscope with a digital camera for image capture. A small amount of each sample was placed on a glass slide, mounted in immersion oil and covered with glass putty to isolate individual particles as much as possible. Samples were observed at appropriate magnifications and partial polarized light, coupled with a λ false color filter.

[0151] Scanning electron microscopy (SEM)

[0152] Data was collected on a Phenom Pro scanning electron microscope. A small amount of sample was mounted on an aluminum stub using conductive double-sided tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s).

[0153] Moisture determination was carried out by Karl Fischer titration (KF)

[0154] The water content of each sample was measured at 150 °C using a Metrohm 874 oven sample processor with Hydranal Coulomat AG oven reagent and nitrogen purge, and an 851 Titrano Coulometer. The weighed solid sample was introduced into a sealed sample bottle. Approximately 10 mg of sample was used for each titration, and duplicate determinations were made. Data collection and analysis were performed using Tiamo v2.2.

[0155] Gravimetric vapor sorption (GVS)

[0156] Adsorption isotherms were obtained using an SMS DVS Intrinsic moisture sorption analyzer controlled by DVS Intrinsic control software v1.0.1.2 (or v1.0.1.3). The sample temperature was maintained at 25 °C by instrument control. Humidity was controlled by mixing dry and wet nitrogen gas flows with a total flow rate of 200 ml / min. Relative humidity was measured by a calibrated Rotronic probe (dynamic range 1.0 - 100% RH) located near the sample. The weight change (mass relaxation) of the sample as a function of %RH was continuously monitored by a microbalance (accuracy ±0.005 mg).

[0157] Typically, 5 - 20 mg of the sample is placed in a tared stainless steel wire basket under ambient conditions. The sample is loaded and unloaded at 40% RH and 25 °C (typical indoor conditions). The moisture sorption isotherm is performed as outlined in Table 1 below (2 scans to obtain 1 complete cycle). The standard isotherm is performed at 25 °C in the range of 0 - 90% RH at 10% RH intervals. Using Microsoft Excel, data analysis is carried out using the DVS analysis package v6.2 (or 6.1 or 6.0). After the isotherm is completed, the sample is recovered and re - analyzed by XRPD.

[0158] Table 1. Methods for SMS DVS Intrinsic experiments.

[0159] Parameter Value Adsorption - scan 1 40-90 Desorption / Adsorption - scan 2 90-0，0-40 Interval (%RH) 10 Number of scans 2 Flow rate (mL / min) 200 Temperature (°C) 25 Stability (°C / min) 0.2 Adsorption time (hours) 6 hours, overtime

[0160] Chemical purity determination was carried out by HPLC

[0161] Purity analysis is carried out on an Agilent HP1100 series system equipped with a diode array detector and using ChemStation software vB.04.03 using the methods detailed in Table 2 below.

[0162] Table 2. HPLC methods for chemical purity determination.

[0163]

[0164]

[0165] Ion chromatography (IC)

[0166] Data is collected using IC Net software v2.3 on a Metrohm 761 Compact IC (for cations) and a Metrohm 861 Advanced Compact IC (for anions) and summarized as in Tables 3 and 4. The accurately weighed sample is prepared as a stock solution in an appropriate dissolution solution and diluted appropriately before testing. Quantification is achieved by comparison with a standard solution of known concentration of the ion being analyzed.

[0167] Table 3. IC methods for cation chromatography.

[0168]

[0169]

[0170] Table 4. IC methods for anion chromatography.

[0171]

[0172] pKa determination and prediction.

[0173] Data were collected on a Sirius T3 instrument. Measurements were carried out by UV photometric titration with a cosolvent at 25 °C. The ionic strength adjustment (ISA) of the titration medium was carried out with 0.15 M KCl (aqueous solution). The data were refined using Sirius T3 Refine version 1.1.3.0. The pKa values were predicted using ACD / Labs Percepta 2012. The Log P values were predicted using ACD / Labs Percepta 2012.

[0174] 2. Experimental procedure (salt screening).

[0175] Preliminary solubility assessment. Compound 1 (15 mg) was treated with increasing volumes of solvent at 50 °C until the material was completely dissolved or until a maximum of 20 volumes of solvent was used. Sodium hydroxide (1.1 equivalents) was added, which in most cases produced a clear solution. The temperature was ramped up to 5 °C at 0.1 °C / min and stirred overnight at this temperature. Any solids were filtered, air-dried, and analyzed by XRPD. Any solutions were evaporated to dryness and the solid residues were analyzed by XRPD.

[0176] Materials. Commercially available chemicals and solvents were purchased from Aldrich or Fluka. The following chemicals were used to prepare stock solutions: sodium hydroxide, potassium hydroxide, L-lysine, L-arginine, N-methylglucosamine, ammonium hydroxide, choline, calcium chloride, ethanolamine, dimethylaminoethanol, N-ethylglucosamine, and tromethamine. The basic stock solutions were prepared at a concentration of 1.0 M, except for L-arginine and calcium chloride which were prepared at a concentration of 0.5 M. Water was used as the solvent to prepare the basic stock solutions, except for calcium chloride which was prepared using ethanol.

[0177] Salt screening - general procedure (cooling). Compound 1 (20 mg) was suspended in a solvent system (10 or 15 volumes) at 50 °C. The suspension was treated with the selected counterion. Then at this stage, the solution or suspension was cooled to 5 °C at 0.1 °C / min and stirred overnight at this temperature. The resulting solid was filtered, air-dried, and analyzed by XRPD. The solution was subjected to the procedure detailed in the following paragraph.

[0178] Salt screening - evaporating the solution. Any solution obtained from the paragraph titled "Salt screening - general procedure (cooling)" above was concentrated by evaporation under ambient conditions and the solid residue was initially analyzed by XRPD. The gel was subjected to the procedure detailed in the following paragraph.

[0179] Salt screening - addition of antisolvent. Any gel, oil or amorphous solid obtained from the paragraph above titled "Salt screening - general procedure (cooling)" was stirred overnight at 30 °C with TBME (10 volumes). Any solids were analyzed by XRPD.

[0180] Preparation of calcium salt via ion exchange. Compound 1 (20 mg) was suspended in a solvent system at 50 °C. The suspension was treated with sodium hydroxide (1.1 eq), showing a clear solution. Calcium hydroxide (0.5 eq) was added to the solution, yielding a precipitate. These experiments were then cooled to 5 °C at 0.1 °C / min and stirred overnight at this temperature. The solid was filtered and air-dried and initially analyzed by XRPD. Any gel, oil or amorphous solid obtained at this stage was subjected to the above procedure.

[0181] Water solubility determination. Compound 1 or its corresponding salt was accurately weighed into a vial. Water was added in increments with stirring at 25 °C until complete dissolution was observed or up to a maximum of 200 - 240 volumes of water (depending on the sample). After stirring for a few minutes, a visual assessment of dissolution was made.

[0182] 3. Characterization of Compound 1.

[0183] The characterization data of Compound 1 are summarized in Table 5 below.

[0184] Table 5. Characterization data of Compound 1.

[0185]

[0186]

[0187] Compound 1 was characterized as a dark yellow amorphous solid. A representative XRPD pattern of Compound 1 is shown in Figure 1 . When the material was stored at 40 °C / 75% RH and 25 °C / 97% RH for 13 days, partial deliquescence was observed at the edges of the sample. Thermal analysis showed a 4.8% loss of moisture, which corresponded to a broad and unresolved endothermic event obtained by DSC ( Figure 2 ). The material showed low solubility in aqueous solvents (< 5 mg / ml in water at 25 °C) and organic solvents (difficulty in preparing NMR samples with deuterated methanol and DMSO, and difficulty in preparing pKa / ion chromatography samples in acetonitrile).

[0188] Determination of pKa using the sodium salt of Compound 1. The low solubility of Compound 1 made it unsuitable for these determinations. The predicted and measured pKa values are shown in Figure 3In the case of the compound, due to its insolubility, LogP measurement could not be performed. Two LogP experiments were attempted, but precipitation was observed on both occasions. The compound exists in cationic, zwitterionic, and anionic forms at different pH values. The neutral form is the minor species.

[0189] 4. Preliminary studies.

[0190] The solubility of Compound 1 was evaluated in a series of solvent systems, followed by preliminary salt formation experiments using sodium hydroxide. The results are summarized in Table 6.

[0191] At 50 °C, Compound 1 was insoluble in any of the selected solvent systems. However, upon addition of the corresponding base (1.1 eq), complete dissolution was observed in most vials. Three crystalline solids and one amorphous solid were obtained. Although slight differences were observed by XRPD, after storage at 40 °C / 75% RH for 7 days, all solids were converted to the same crystalline form, except for the sample from acetone:water, which showed a gel (amorphous).

[0192] Table 6. Solubility evaluation of Compound 1.

[0193]

[0194] * An oil was initially observed after evaporation; a solid was observed after standing in a fume hood at room conditions for 48 h.

[0195] Based on the above results and their diversity, methanol, acetone / 10% water, and THF were selected for primary screening.

[0196] 5. Salt screening - Results.

[0197] Salt screening was performed in three solvent systems using the above procedure. Anti-solvent addition was carried out if necessary. Crystalline salts were obtained with sodium, L-lysine, ethanolamine, and N-ethylglucosamine. Crystalline solids were obtained from experiments using tromethamine and ammonium, although the absence of counterions was proven by 1 1H NMR or ion chromatography. The data are summarized in Table 7. The recoveries of these processes were low, although not quantified at this stage.

[0198] Table 7. Salt screening results.

[0199]

[0200]

[0201]

[0202] * A salt was suspected at that time; characterization confirmed that ammonium does not exist in the form of a salt former.

[0203] The salt formation experiment was repeated on a 50 mg scale to produce sufficient material for characterization. Table 8 summarizes the details of the partial characterization of the counterions / cocrystal formers sodium, L-lysine, ethanolamine, and N-ethylglucosamine. Attempts to form salts with ammonium or tromethamine were unsuccessful, and only the crystalline free form of Compound 1 was produced.

[0204] Table 8. Preliminary characterization of salts from screening.

[0205]

[0206]

[0207] Representative XRPD patterns of various crystalline forms of Compound 1 sodium salt, Compound 1 L-lysine salt, Compound 1 ethanolamine salt, and Compound 1 N-ethylglucosamine are shown in Figures 4 - 7 .

[0208] 6. Results and discussion.

[0209] Crystalline salts were obtained with sodium, L-lysine, ethanolamine, and N-ethylglucosamine. Crystalline free form material was also obtained via unsuccessful salt formation experiments with ammonium and tromethamine.

[0210] Due to signal overlap, it is challenging to determine the stoichiometry of salts with organic counterions by 1 1H NMR. 1H NMR work was carried out in deuterated DMSO, deuterated methanol, and a combination of both to assess the presence of the counterion. Solid mixtures of Compound 1 and the corresponding counterion were also prepared and dissolved in deuterated solvents for comparison. Thus, the expected spectra can be used as a reference for the experimental salts. 1 The monosodium salt Na2 is crystalline and may be a monohydrate based on the water content. Loss of water was observed by TGA, and the water loss corresponded to multiple endothermic events obtained by DSC. The salt was stable after storage at 40 °C / 75% RH for one week but showed deliquescence after a total storage of 36 days.

[0211] The monosodium salt Na2 is crystalline and may be a monohydrate based on the water content. Loss of water was observed by TGA, and the water loss corresponded to multiple endothermic events obtained by DSC. The salt was stable after storage at 40 °C / 75% RH for one week but showed deliquescence after a total storage of 36 days.

[0212] The monosodium salt Na2 is crystalline and may be a monohydrate based on the water content. Loss of water was observed by TGA, and the water loss corresponded to multiple endothermic events obtained by DSC. The salt was stable after storage at 40 °C / 75% RH for one week but showed deliquescence after a total storage of 36 days.

[0213] The N-ethylglucosamine salt NEG1 is crystalline and by 11H NMR showed a slight excess of counterion (most likely due to incomplete salification). The material was stable after storage at 40 °C / 75% RH for 36 days. The N-ethylglucosamine salt showed an endothermic event (possibly melting) at 110 °C. At this stage, there was insufficient material available for TGA analysis. The material was stable after storage at 40 °C / 75% RH for 36 days.

[0214] Finally, the ethanolamine salt EA1 was also crystalline and the monostoichiometry was confirmed by 1 1H NMR. The salt showed an endothermic event (possibly melting) at 143 °C. At this stage, there was insufficient material available for TGA analysis. The material was stable after storage at 40 °C / 75% RH for 7 days.

[0215] Four different polymorphs of the free form of compound 1 have been observed. The material FF1 obtained from the unsuccessful tromethamine salification was a yellow crystalline solid with a broad endotherm at 217 °C by DSC. The small weight loss observed by TGA may correspond to water loss, although a small amount of unidentified impurities were observed by 1 1H NMR. Slight changes were observed after storage at 40 °C / 75% RH for one week, and FF4 was formed. Another partially crystalline material FF3 was obtained from another unsuccessful tromethamine formation experiment. The unsuccessful attempt to prepare the ammonium salt resulted in the partially crystalline FF2. The material showed multiple endotherms by DSC and was stable after storage at 40 °C / 75% RH for one week. These results suggest that the free base is capable of crystallizing under specific conditions. The solubility of the crystalline free form in deuterated solvents is significantly lower. In some cases, larger solvent volumes and filtration are required to achieve complete dissolution and obtain optimal NMR spectra.

[0216] Based on the desired solid-state properties of the salts, the sodium salt, L-lysine salt, and N-ethylglucosamine salt were selected for the scale-up phase.

[0217] Example 2. Scale-up of the selected salts of compound 1 and identification of the L-lysine form 2 of compound 1.

[0218] Sodium salt and L-lysine salt. Compound 1 (ca. 500 mg) was suspended in methanol (5 volumes) at 50 °C. Sodium hydroxide or L-lysine (1 M in water, 1.1 equiv) was added at 50 °C to form a clear solution. After stirring for approximately 10 minutes, a white precipitate was observed in both cases. In the case of the L-lysine salt, a fixed precipitate was formed, as opposed to the sodium salt which formed a flowing slurry. The ramp was set at 0.1 °C / min to 30 °C and then the vial was placed at 4 °C overnight. The bulk solid was filtered by suction and dried overnight in a vacuum oven at 25 °C. The white solid was used for characterization.

[0219] N-Ethylglucosamine salt. Compound 1 (ca. 300 mg) was suspended in methanol (17 volumes) at 65 °C. N-Ethylglucosamine (1 M in water, 1.1 eq.) was added at 65 °C. Complete dissolution was not observed after 30 min, and the solution was filtered. Then the temperature was ramped up to 5 °C at 0.1 °C / min. NEG1 seed (from the screening material) (ca. 10 mg) was added at 63 °C. A clear solution was still observed at 5 °C, so TBME was added as an anti-solvent (10 volumes). The solution was concentrated by opening the vial cap at room temperature. More seeds were added, and the vial was placed at -20 °C overnight, which did not help crystallization. Evaporation to dryness gave a gum, which was sonicated with no improvement. Another attempt to prepare the N-ethylglucosamine salt was made using a methanol / TBME mixture with a slight excess of base (1.5 eq.) at 50 °C without filtration. A gum / oil was also observed after evaporation. Both the gum / oil were dried under vacuum to yield an amorphous solid. The amorphous solids were slurried overnight in ten solvent systems using temperature cycling between 25 and 50 °C. The slurry in ethyl acetate showed a crystalline white solid after 1 h at room temperature. Based on these results, the bulk amorphous solid was slurried overnight in ethyl acetate (with temperature cycling between 25 and 50 °C) to yield a crystalline solid. These solids were used for characterization.

[0220] Results and characterization of the selected salts. The sodium salt, L-lysine salt, and N-ethylglucosamine salt were scaled up and fully characterized. Two new forms of the L-lysine salt (LYS2 (form 2)) and N-ethylglucosamine salt (NEG2) were isolated compared to the screening material. The yields were generally low, 47% and 58% for the sodium salt and L-lysine salt, respectively. The yield of the N-ethylglucosamine salt was not quantified considering the multiple steps required to obtain the crystalline material. A summary of the characterization details of these three selected salts can be seen in Table 9.

[0221] Table 9. Solid-state characterization of the selected salt forms.

[0222]

[0223]

[0224]

[0225] *The sodium salt appears to exist in different hydrated states, and the changes observed throughout the characterization may be due to differences in environmental humidity on the day of analysis.

[0226] After separation and storage in a sealed vial under ambient conditions for 10 days, the conversion of Na2 to Na4 was observed.

[0227] The sodium salt crystallizes in the form of Na2. However, after 10 days of storage in a closed vial under ambient conditions, a form change to Na4 was observed. The sodium salt is hydrated, and depending on the amount of water of hydration, the diffraction pattern shows slight variations. Additionally, these slight variations may be due to differences in ambient humidity on the day of analysis. Ion chromatography shows 0.8 equivalents of sodium. DSC shows multiple endothermic events, which are related to the loss of moisture observed by TGA. VT-XRPD was performed on the material. The starting material was checked before commencing this analysis, and a change to Na4 was observed. Na4 shows two form changes (Na1 and a new form Na5) after heating, and further changes (to a new form, designated Na6, suspected to be anhydrous) after cooling at the end of the experiment. Representative XRPD patterns of different sodium salt forms are shown in Figure 4 . Some of these XRPD patterns may be mixtures of different forms. GVS analysis also confirmed multiple steps of hydration. The starting material showed 5.7% moisture (1.5 equivalents), compared to the material at the end of the experiment, which showed 4.4% moisture (1 equivalent, showing Na4 by XRPD). At 90% RH, a total of 9.7% moisture (2.5 equivalents) was absorbed in the first cycle, and a total of 16.0% moisture (4.5 equivalents) was absorbed in the second cycle. Hysteresis was observed in both cycles, indicating that the hydration and dehydration steps show different kinetics. Overall, due to the fact that it exists in different levels of hydration and in order to achieve a 1:1 stoichiometry, the sodium salt poses challenges in process chemistry.

[0228] The N-ethylglucosamine NEG2 salt deliquesces after two days at 40 °C / 75% RH and 25 °C / 97% RH. By GVS, a reversible moisture sorption rate of 22.5% w / w was observed between 0 - 90% RH, after which a viscous solid with low crystallinity (designated NEG3) was recovered. TGA shows a small loss of moisture (1.1% below 125 °C), followed by an endotherm (106 °C) observed by DSC. In summary, any salt formed with N-ethylglucosamine has a poor thermal profile and is not recommended for further development.

[0229] The L-lysine salt LYS2 (form 2) is also anhydrous and stable after storage at 40 °C / 75% RH and 25 °C / 97% RH for 8 days and during the GVS experiment. Thermal analysis indicates melting and decomposition at 231 °C. 1 1H NMR spectroscopy confirmed the monostoichiometry. Slight variations in multiplicity were observed in several peaks (of unknown nature) in the aromatic region. The water solubility of LYS2 was evaluated, which showed a turbid yellow solution of 5 mg / mL.

[0230] The solubility of these three salts was evaluated at 25 °C. The water solubility of Na2 and NEG2 was > 200 mg / ml. For both materials, a clear solution was immediately observed upon addition of water with stirring. After about 20 minutes, a thick white precipitate was observed, but this precipitate redissolved overnight. The dissolution attempt with LYS2 resulted in a turbid yellow solution of 5 mg / ml (adding 200 volumes and stirring overnight).

[0231] Summary. Crystalline salts were obtained from sodium, L-lysine, N-ethylglucosamine, and ethanolamine. Two forms of L-lysine salt (LYS1 (form 1) and LYS2 (form 2)) isolated during the study were anhydrous, stable at elevated humidity, and melted / decomposed at about 230 °C. Based on its higher crystallinity and solubility, L-lysine salt LYS1 (form 1) was selected for further development.

[0232] Example 3. Polymorph Screening of Compound 1 L-Lysine Salt

[0233] Polymorph screening was carried out with the aim of identifying new solid forms of compound 1 L-lysine. The experiments consisted of: solvent-mediated solid form transformation, temperature cycling, anti-solvent vapor diffusion, solvent-drop grinding, pH swing (pHSwing), cooling crystallization, and addition of anti-solvent to the solution. The solid-state properties of compound 1 L-lysine form 1 were also fully characterized. 1. Instrument and method details.

[0234] Optical microscopy. Micrographs were taken using an Olympus BX51 polarized light microscope equipped with a JENOPTIK ProgRes camera and operated by ProgRes Capture Pro 2.8.8 software. The sample was dispersed on a microscope slide with silicone oil and examined in transmitted polarized light.

[0235] X-ray diffraction experiment. X-ray powder diffraction data were collected on a Rigaku Miniflex600 diffractometer using Cu Kα (1.5406 Å) radiation under ambient conditions. The powder pattern was collected on a zero-background holder with a 0.1 mm indent at 40 kV and 15 mA, with a scan rate of 2 to 40° 2θ, 2° / min.

[0236] Differential scanning calorimetry (DSC). Differential scanning calorimetry was performed using a TA Discovery series DSC with a few milligrams of material sealed in a Tzero aluminum pan sealed with a Tzero seal lid with two pinholes. The sample was scanned at 10 °C / min under a nitrogen flow of 50 mL / min.

[0237] Thermogravimetric analysis (TGA). Thermogravimetric analysis data were collected using a TA Discovery series TGA. A few milligrams of the material were analyzed in an aluminum sample pan. Data were collected from room temperature to 300 °C at a scan rate of 10 °C / minute.

[0238] Dynamic vapor sorption (DVS). Dynamic vapor sorption experiments were performed on a DVS Intrinsic system by Surface Measurement Systems. The sample was exposed to a relative humidity cycling from 0% RH to 90% RH, where the weight was equilibrated and measured at each humidity step. The temperature was set at 25 °C and kept constant throughout the experiment.

[0239] Nuclear magnetic resonance (NMR). Recorded on a Varian Inova 300 Hz spectrometer 1 1H NMR spectra.

[0240] High performance liquid chromatography (HPLC). Chromatographic separation was performed on a Thermo Fisher Spectra system using the conditions provided in Table 10.

[0241] Table 10. HPLC method.

[0242]

[0243]

[0244] 2. Preliminary characterization of compound 1 L-lysine form 1.

[0245] Generate initial characterization data for compound 1 L-lysine form 1. Optical microscopy, X-ray powder diffraction (XRPD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), solution nuclear magnetic resonance spectroscopy (NMR), dynamic vapor sorption (DVS), and post-DVS XRPD data are presented in Figures 8 - 15 . A summary of the physical properties is presented in Table 11.

[0246] Table 11. Summary of the physical properties of compound 1 L-lysine form 1.

[0247] XRPD Crystallization Microscopy Needle - like morphology TGA There is no significant weight loss up to 200 °C. Decomposition occurs above 225 °C. DSC One endotherm with an onset temperature of 239.4 °C. <![CDATA 1 H NMR]]> Consistent with the L - lysine salt. HPLC The purity is 99.1% (by area). DVS At up to 90% relative humidity, 1.4% reversible adsorption. XRPD after DVS There is no change in the solid state form.

[0248] 3. Solid form screening of compound 1 L-lysine salt.

[0249] Thermodynamic stability form screening. Solvent-mediated solid form transformation experiments were carried out within the ranges of temperature, water activity, and solvent type to explore thermodynamically more stable polymorphs, hydrates, and solvates. Approximately 75 to 85 mg of the material was weighed into a 4 mL amber glass vial. Approximately 1 mL of solvent was added, followed by a stir bar, and the vial was capped. The vial was placed on its respective temperature stir plate and stirred at 500 rpm for 3 weeks. The solid from the slurry was analyzed by XRPD for any changes in the solid form. A summary of the experiments and results is provided in Table 12. No new crystalline forms of Compound 1 L-lysine were observed.

[0250] Table 12. Thermodynamic stability form screening of Compound 1 L-lysine.

[0251] Solvent Temperature Result Ethyl acetate 50℃ Initial lysine salt form (Form 1) 2 - Propanol 50℃ Initial lysine salt form (Form 1) Acetone 25℃ Initial lysine salt form (Form 1) Dichloromethane 4℃ Initial lysine salt form (Form 1) Acetonitrile 50℃ Initial lysine salt form (Form 1) Tetrahydrofuran 25℃ Initial lysine salt form (Form 1) Water 25℃ Amorphous Water / methanol (1 / 3, by volume) 25℃ Initial lysine salt form (Form 1) Water / acetonitrile (1 / 3, by volume) 4℃ Initial lysine salt form (Form 1) Ethanol 50℃ Initial lysine salt form (Form 1) 2 - Methyl THF 25℃ Initial lysine salt form (Form 1) CPME 50℃ Initial lysine salt form (Form 1) Toluene 50℃ Initial lysine salt form (Form 1) Methanol 25℃ Initial lysine salt form (Form 1)

[0252] Temperature cycling. Approximately 20 to 30 mg of the material was weighed into a vial. Approximately 0.2 to 0.3 mL of solvent was added, and the vial was capped. The vial was placed in a temperature cycling chamber and cycled from 10 °C to 60 °C at 10 °C / hour for 3 consecutive weeks. The solid was analyzed by XRPD for any changes in the solid form. A summary of the experiments and results is provided in Table 13. No new crystalline forms of Compound 1 L-lysine were observed.

[0253] Table 13. Temperature cycling experiment of Compound 1 L-lysine.

[0254]

[0255]

[0256] Antisolvent vapor diffusion. Approximately 25 to 30 mg of the material was weighed into a 4 mL vial. Approximately 2 mL of solvent was added, and the vial was sonicated to attempt to prepare a solution. Any slurry was filtered into a clean 4 mL vial to generate a solution for all experiments. These uncapped 4 mL vials were then placed into larger 20 mL vials containing the antisolvent. The 20 mL vials were capped to allow the antisolvent vapor to slowly diffuse into the solution in the uncapped 4 mL vials. All experiments were maintained at ambient conditions. A summary of the experiments and results is provided in Table 14. No crystalline solid was obtained.

[0257] Table 14. Antisolvent vapor diffusion experiment of Compound 1 L-lysine.

[0258]

[0259]

[0260] Solvent-drop grinding. Approximately 20 to 25 mg of the material was weighed into a 2 mL amber vial. 3 to 6 alumina beads were added to approximately 20 to 40 μL of solvent. The vial was capped and placed on a temperature-controlled mixer set at 800 rpm and 20 °C. The sample was mixed overnight and then the solid was analyzed by XRPD for solid form changes. A summary of the experiments and results is provided in Table 15. No new crystalline forms of Compound 1 L-lysine were observed.

[0261] Table 15. Solvent-drop grinding experiments of Compound 1 L-lysine.

[0262] Solvent Result DMSO Initial lysine salt form (Form 1) NMP Initial lysine salt form (Form 1) Water Initial lysine salt form (Form 1) DMF Initial lysine salt form (Form 1) Ethyl lactate Initial lysine salt form (Form 1) Chloroform Initial lysine salt form (Form 1) Isopropyl acetate Initial lysine salt form (Form 1) 1 - Propanol Initial lysine salt form (Form 1) MIBK Initial lysine salt form (Form 1) p - Cymene Initial lysine salt form (Form 1)

[0263] pH swing. Experiments were conducted to generate new solid forms of Compound 1 L-lysine by varying the pH of the aqueous solution. Four experiments were performed, which consisted of: (1) adding base to an acidic solution of Compound 1 L-lysine; (2) adding acid to a basic solution of Compound 1 L-lysine; (3) adding an acidic solution of Compound 1 L-lysine to base; or (4) adding a basic solution of Compound 1 L-lysine to acid.

[0264] Experiment 1: Approximately 30 mg of Compound 1 L-lysine was weighed into a 4 mL amber glass vial and 1.5 mL of 0.1 M HCl was added. The suspension was filtered to obtain a clear solution with a pH of 1.7. While stirring on a 25 °C magnetic stirrer, 1.5 mL of 0.1 M NaOH was slowly added, resulting in a final pH of 12.0. No solid was observed.

[0265] Experiment 2: Approximately 30 mg of Compound 1 L-lysine was weighed into a 4 mL amber glass vial and 1.5 mL of 0.1 M NaOH was added. A solution with a pH of 12.2 was obtained. While stirring on a 25 °C magnetic stirrer, 1.5 mL of 0.1 M HCl was slowly added, resulting in a pH of 9.6. An additional 0.5 mL of 0.1 M HCl was added, resulting in the formation of a gel with a pH of 3.0.

[0266] Experiment 3: Approximately 30 mg of Compound 1 L-lysine was weighed into a 4 mL amber glass vial and 1.5 mL of 0.1 M HCl was added. The suspension was filtered to obtain a clear solution with a pH of 1.6. This solution was slowly added to a 4 mL amber glass vial containing a stirred solution of 1.5 mL of 0.1 M NaOH on a 25 °C magnetic stirrer. The final pH was 12.1. No solid was observed.

[0267] Experiment 4: Weigh approximately 30 mg of Compound 1 L-lysine into a 4 mL amber glass vial and add 1.5 mL of 0.1 M NaOH to produce a solution with a pH of 12.2. Slowly add this solution to a 4 mL amber glass vial containing a stirred solution of 1.5 mL of 0.1 M HCl on a stirring plate at 25 °C. The pH was 9.5, so an additional 0.5 mL of 0.1 M HCl was added, resulting in the formation of a gel with a pH of 3.3.

[0268] Cooling crystallization. Prepare solutions of Compound 1 L-lysine in various solvents and heat them up to 50 °C for 20 to 30 minutes. Transfer the samples to a 4 °C refrigerator and monitor the formation of solids. A summary of the experiments and results is provided in Table 16. No crystalline solids were obtained.

[0269] Table 16. Cooling crystallization experiments of Compound 1 L-lysine.

[0270]

[0271]

[0272] Addition of antisolvent to the solution. Pipette the antisolvent into the samples from the above cooling crystallization experiments (which were kept as solutions to discover new polymorphs). A summary of the experiments and results is provided in Table 17. No crystalline solids were obtained.

[0273] Table 17. Addition of antisolvent to the solution of Compound 1 L-lysine.

[0274] Solvent Antisolvent Result DMAc Water Amorphous DMF Water Amorphous Water / MeCN (1 / 3) Water Amorphous 1 - Propanol Water Solution Methanol Water Amorphous NMP Water Amorphous THF Heptane Solution 2 - Butanone Heptane Amorphous 1,2 - Dichloroethane Heptane Solution

[0275] 4. Solubility of Compound 1 L-lysine Form 1.

[0276] In the solvents used for the above solvent-mediated solid form transformation experiments, the solubility was determined gravimetrically. Centrifuge each slurry sample at 2000 rpm for 5 minutes under ambient conditions, then transfer the supernatant to a microcentrifuge tube and centrifuge again at 16400 rpm at 21 °C for 30 minutes. Transfer a 0.500 mL aliquot of the final supernatant to a pre-weighed vial and remove the solvent by evaporation. Reweigh the vial to calculate the final weight. The solubility values are listed in Table 18.

[0277] Table 18. Solubility of Compound 1 L-lysine Form 1. *

[0278]

[0279]

[0280] * Solubility in water could not be determined.

[0281] 5. Summary.

[0282] Polymorph screening was carried out with the aim of identifying new solid forms of compound 1L-lysine. The experiments consisted of: solvent-mediated solid form transformation, temperature cycling, anti-solvent vapor diffusion, solvent-drop grinding, pH swing, cooling crystallization, and addition of anti-solvent to the solution. No new crystalline forms were found in the screening because all crystalline forms corresponded to Form 1.

[0283] Although the present disclosure has been described in detail for purposes of clarity of understanding by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety.

Claims

1. A crystalline form of L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid, wherein the crystalline form is Form 1.

2. The crystalline form according to claim 1, which has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°.

3. The crystalline form according to claim 1 or 2, wherein the X-ray powder diffraction exhibits characteristic scattering angles (2θ) at least at: 20.0° ± 0.2° and 7.6° ± 0.2°.

4. The crystalline form according to any one of claims 1-3, wherein the X-ray powder diffraction exhibits characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2° and 23.5° ± 0.2°.

5. The crystalline form according to any one of claims 1-4, wherein the X-ray powder diffraction exhibits characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2°, 23.5° ± 0.2° and 14.5° ± 0.2°.

6. The crystalline form according to any one of claims 1-5, wherein the X-ray powder diffraction exhibits characteristic scattering angles (2θ) at least at: 20.0° ± 0.2°, 7.6° ± 0.2°, 23.5° ± 0.2°, 14.5° ± 0.2°, 4.9° ± 0.2°, 6.9° ± 0.2°, 8.5° ± 0.2°, 9.4° ± 0.2°, 10.4° ± 0.2°, 11.6° ± 0.2°, 13.2° ± 0.2°, 13.8° ± 0.2°, 16.1° ± 0.2°, 17.2° ± 0.2°, 17.8° ± 0.2° and 19.0° ± 0.2°.

7. The crystalline form according to any one of claims 1-6, which has an X-ray powder diffraction substantially as shown in Figure 9.

8. The crystalline form according to any one of claims 1-7, which has a differential scanning calorimetry thermogram including an endotherm at about 239.4° ± 5°.

9. The crystalline form according to any one of claims 1-8, which has a differential scanning calorimetry thermogram substantially as shown in Figure 11.

10. The crystalline form according to any one of claims 1-9, as determined by thermogravimetric analysis, which has no significant weight loss up to about 200 °C.

11. The crystalline form according to any one of claims 1-10, which has a thermogravimetric analysis thermogram substantially as shown in Figure 10.

12. The crystalline form according to any one of claims 1-11, as determined by dynamic vapor sorption, which has a reversible sorption of about 1.4% at up to 90% relative humidity.

13. The crystalline form according to any one of claims 1-12, which has a dynamic vapor sorption plot substantially as shown in Figure 14.

14. A crystalline form of L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid, wherein the crystalline form is Form 2.

15. The crystalline form according to claim 14, which has an X-ray powder diffraction showing characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°.

16. The crystalline form according to claim 14 or 15, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.5° ± 0.2° and 18.2° ± 0.2°.

17. The crystalline form according to any one of claims 14-16, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2° and 21.5° ± 0.2°.

18. The crystalline form according to any one of claims 14-17, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2°, 21.5° ± 0.2° and 25.6° ± 0.2°.

19. The crystalline form according to any one of claims 14-18, wherein the X-ray powder diffraction shows characteristic scattering angles (2θ) at least at: 20.5° ± 0.2°, 18.2° ± 0.2°, 21.5° ± 0.2°, 25.6° ± 0.2°, 8.6° ± 0.2°, 13.8° ± 0.2° and 19.3° ± 0.2°.

20. The crystalline form according to any one of claims 14-19, which has an X-ray powder diffraction substantially as shown in Figure 5B.

21. The crystalline form according to any one of claims 14-20, which has a differential scanning calorimetry thermogram including an endotherm at about 231.7° ± 5°.

22. The crystalline form according to any one of claims 14-21, which, as determined by thermogravimetric analysis, has no significant weight loss up to about 240 °C.

23. The crystalline form according to any one of claims 14-22, which, as determined by gravimetric vapor sorption, has a reversible sorption of about 1.6% at up to 90% relative humidity.

24. A method for preparing the crystalline form according to any one of claims 1-23, which comprises: Combining 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and methanol at about 50 °C to obtain a suspension; Adding L-lysine to the suspension at about 50 °C to obtain a solution; Cooling the solution; and Separating the crystalline form from the solution.

25. A solid pharmaceutical composition comprising the crystalline form according to any one of claims 1-23 and a pharmaceutically acceptable excipient.

26. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the solid pharmaceutical composition as described in claim 25.

27. The method according to claim 26, wherein the cancer is selected from colorectal cancer, esophageal cancer, gastric cancer, breast cancer, and lymphoma.

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