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, a histone demethylase inhibitor

Abstract

Provided herein is a crystalline form of the 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 as a histone demethylase inhibitor. Also provided herein is a pharmaceutical composition comprising such a crystalline form.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 408,691, filed September 21, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure generally relates to a crystalline form of the 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, a histone demethylase inhibitor. [Background technology]

[0003] 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), the structure of which is shown below, is a selective inhibitor of the KDM4 family of histone demethylases (see, e.g., U.S. Pat. No. 9,242,968).

[0004] [ka] This first-in-class epigenetic modifying compound shows promise for the treatment of cancers, including gastric and colon cancer.

[0005] Compound 1 has poor solubility in water and most common organic solvents and tends to precipitate as an amorphous paste, making large-scale filtration difficult. Therefore, there is also a need to provide alternative forms of the compound, such as salts, for ease of handling and subsequent formulation.

[0006] Thus, in one aspect, provided herein is a crystalline form (e.g., a polymorph) of the 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 1·L-lysine). Also provided herein is a pharmaceutical composition comprising a crystalline form of Compound 1·L-lysine. Summary of the Invention

[0007] In certain embodiments, the 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 is described herein as a histone demethylase inhibitor. Also provided herein are pharmaceutical compositions comprising such crystalline forms.

[0008] Embodiments of the present invention can be more fully understood by reference to the detailed description and examples, which are intended to exemplify non-limiting embodiments.

[0009] Embodiment 1. A crystalline form of the 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.

[0010] Embodiment 2. The crystalline form of embodiment 1, having an X-ray powder diffraction exhibiting a characteristic scattering angle (2θ) of at least 20.0°±0.2°.

[0011] Embodiment 3. The crystalline form of embodiment 1 or 2, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 20.0°±0.2° and 7.6°±0.2°.

[0012] Embodiment 4. The crystalline form of any one of embodiments 1-3, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 20.0°±0.2°, 7.6°±0.2°, and 23.5°±0.2°.

[0013] Embodiment 5. The crystalline form of any one of embodiments 1-4, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 20.0°±0.2°, 7.6°±0.2°, 23.5°±0.2°, and 14.5°±0.2°.

[0014] Embodiment 6. The crystalline form of any one of embodiments 1-5, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 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°.

[0015] Embodiment 7. The crystalline form of any one of embodiments 1-6, having an X-ray powder diffraction pattern substantially as shown in FIG.

[0016] Embodiment 8. The crystalline form of any one of embodiments 1-7, having a differential scanning calorimetry thermogram comprising an endotherm at about 239.4°±5°.

[0017] Embodiment 9. The crystalline form of any one of embodiments 1-8, having a differential scanning calorimetry thermogram substantially as shown in Figure 11.

[0018] Embodiment 10. The crystalline form of any one of embodiments 1-9, having no significant weight loss up to about 200° C. as determined by thermogravimetric analysis.

[0019] Embodiment 11. The crystalline form of any one of embodiments 1-10, having a thermogravimetric analysis thermogram substantially as shown in FIG.

[0020] Embodiment 12. The crystalline form of any one of embodiments 1-11, having a reversible sorption of from about 1.4% to 90% relative humidity as determined by dynamic vapor sorption.

[0021] Embodiment 13. The crystalline form of any one of embodiments 1-12, having a dynamic vapor sorption profile substantially as shown in FIG. 14.

[0022] Embodiment 14. A crystalline form of the 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.

[0023] Embodiment 15. The crystalline form of embodiment 14, having an X-ray powder diffraction exhibiting a characteristic scattering angle (2θ) of at least 20.5°±0.2°.

[0024] Embodiment 16. The crystalline form of embodiment 14 or 15, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 20.5°±0.2° and 18.2°±0.2°.

[0025] Embodiment 17. The crystalline form of any one of embodiments 14-16, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 20.5°±0.2°, 18.2°±0.2°, and 21.5°±0.2°.

[0026] Embodiment 18. The crystalline form of any one of embodiments 14 to 17, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 20.5°±0.2°, 18.2°±0.2°, 21.5°±0.2°, and 25.6°±0.2°.

[0027] Embodiment 19. The crystalline form of any one of embodiments 14 to 18, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 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°.

[0028] Embodiment 20. The crystalline form of any one of embodiments 14-19, having an X-ray powder diffraction pattern substantially as shown in Figure 5B.

[0029] Embodiment 21. The crystalline form of any one of embodiments 14-20, having a differential scanning calorimetry thermogram comprising an endotherm at about 231.7°±5°.

[0030] Embodiment 22. The crystalline form of any one of embodiments 14-21, having no significant weight loss up to about 240°C as determined by thermogravimetric analysis.

[0031] Embodiment 23. The crystalline form of any one of embodiments 14-22, having a reversible sorption of from about 1.6% to 90% relative humidity as determined by gravimetric vapor sorption.

[0032] Embodiment 24. 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 with 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; separating the crystalline form from the solution; 24. A method for preparing the crystalline form of any one of embodiments 1 to 23, comprising:

[0033] Embodiment 25. A solid pharmaceutical composition comprising the crystalline form of any one of Embodiments 1-23 and a pharmaceutically acceptable excipient.

[0034] 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 of embodiment 25.

[0035] Embodiment 27. The method of embodiment 26, wherein the cancer is selected from colorectal cancer, esophageal cancer, gastric cancer, breast cancer, and lymphoma. [Brief explanation of the drawings]

[0036] Various aspects of the present disclosure are set forth with particularity 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 that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0037] [Figure 1] FIG. 1 shows the X-ray powder diffraction (XRPD) pattern of Compound 1. [Figure 2] FIG. 2 shows both the thermogravimetric analysis (TGA) thermogram and the differential scanning calorimetry (DSC) thermogram of Compound 1. [Figure 3] FIG. 3 shows the predicted and measured pKa for Compound 1. [Figure 4] Figure 4 shows an overlay of X-ray powder diffraction (XRPD) patterns of the crystalline forms of Compound 1·sodium salt (Na1–Na6). [Figure 5A] Figure 5A shows an overlay of X-ray powder diffraction (XRPD) patterns of crystalline forms of compound 1·L-lysine (LYS1 (form 1) and LYS2 (form 2)). [Figure 5B] FIG. 5B shows the X-ray powder diffraction (XRPD) pattern of compound 1·L-lysine form 2. [Figure 6] FIG. 6 shows the X-ray powder diffraction (XRPD) pattern of the crystalline form of the ethanolamine salt of Compound 1 (EA1). [Figure 7] FIG. 7 shows the X-ray powder diffraction (XRPD) pattern of the crystalline form of the N-ethylglucamine salt of Compound 1 (Neg1). [Figure 8] FIG. 8 shows a polarized light microscopy (PLM) micrograph of Compound 1·L-lysine Form 1. [Figure 9] FIG. 9 shows the X-ray powder diffraction (XRPD) pattern of Compound 1·L-lysine Form 1. [Figure 10] FIG. 10 shows a thermogravimetric analysis (TGA) thermogram of Compound 1·L-lysine Form 1. [Figure 11] FIG. 11 shows a differential scanning calorimetry (DSC) thermogram of Compound 1·L-lysine Form 1. [Figure 12] FIG. 12 shows a high-performance liquid chromatography (HPLC) chromatogram of Compound 1·L-lysine Form 1. [Figure 13] FIG. 13 shows the H NMR chromatogram of Compound 1·L-lysine Form 1. [Figure 14] FIG. 14 shows the dynamic vapor sorption (DVS) sorption-desorption plot of Compound 1·L-lysine Form 1. [Figure 15] FIG. 15 shows an 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 OF THE INVENTION

[0038] definition As used herein, the terms "comprising" and "including" may be used interchangeably. The terms "comprising" and "including" should be interpreted as specifying the presence of the stated features or components as referenced, but do not exclude the presence or addition of one or more features or components, or groups thereof. Furthermore, the terms "comprising" and "including" are intended to include examples encompassed by the term "consisting of." Thus, the term "consisting of" can be used in place of the terms "comprising" and "including" to provide more specific embodiments of the present invention.

[0039] The term "consisting of" means that the subject matter has at least 90%, 95%, 97%, 98%, or 99% of the recited features or components that make up the subject matter. In another embodiment, the term "consisting of" excludes from the scope of any succeeding recitation any other features or components, excepting those that are not essential to the technical effect to be achieved.

[0040] As used herein, the term "or" should be interpreted as an inclusive "or," meaning any one or any combination. 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." Exceptions to this definition occur only where combinations of elements, features, steps, or acts are inherently mutually exclusive in some way.

[0041] Any concentration range, error range, percentage range, ratio range, or integer range herein should be understood to include every integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated. Also, any numerical range recited herein for any physical characteristic should be understood to include every integer within the recited range, unless otherwise indicated. As used herein, the terms "about" and "approximately" mean ±20%, ±10%, ±5%, or ±1% of the indicated range, value, or structure, unless otherwise indicated.

[0042] "Treating," as used herein, means alleviating, in whole or in part, a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition, or slowing or arresting further progression or worsening of those symptoms, or alleviating or eradicating the cause of the disorder, disease, or condition itself. In one embodiment, the disorder is a cancer or symptom thereof as described herein.

[0043] "Preventing," as used herein, refers to a method of wholly or partially delaying and / or eliminating the onset, recurrence, or spread of a disorder, disease, or condition, a method of preventing a subject from acquiring a disorder, disease, or condition, or a method of reducing a subject's risk of acquiring a disease or condition. In one embodiment, the disorder is a cancer or symptom thereof as described herein.

[0044] The term "effective amount" in reference to a compound disclosed herein means an amount disclosed herein that is capable of treating or preventing a disorder, disease or condition, or a symptom thereof.

[0045] As used herein, the term "subject" includes animals, including, but not limited to, cows, monkeys, horses, sheep, pigs, chickens, turkeys, quail, 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 who has or is at risk of having cancer.

[0046] Throughout this specification, a reference to "one embodiment" or "one embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrase "in one embodiment" or "in one embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0047] While various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein for clarity in the context of separate embodiments, the invention may also be implemented in a single embodiment.

[0048] Crystalline form of compound 1·L-lysine salt The 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 (hereinafter referred to as "Compound 1·L-lysine") described herein has advantageous properties over the free base. For example, the crystalline forms described herein may provide improved stability, solubility, filterability, hygroscopicity, ease of handling, and / or ease of formulation into solid pharmaceutical compositions (such as tablets or capsules).

[0049] In one aspect, provided herein are crystalline forms of Compound 1·L-lysine. 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.

[0050] The crystalline forms of compound l·L-lysine described herein can be identified by their unique solid-state properties characterized by, for example, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), dynamic vapor sorption (DSV), gravimetric vapor sorption (GVS), and other techniques.

[0051] Crystalline form 1 In one aspect, provided herein is a crystalline form of Compound 1·L-lysine, wherein the crystalline form is Form 1.

[0052] In some embodiments, Compound 1·L-lysine Form 1 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) of at least 20.0°±0.2°. In some embodiments, Compound 1·L-lysine Form 1 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) of at least 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) of at least 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) of at least 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 pattern exhibiting characteristic scattering angles (2θ) of at least 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 pattern substantially as shown in FIG.

[0053] In some embodiments, Compound 1·L-lysine Form 1 has a differential scanning calorimetry thermogram comprising an endotherm at about 239.4°±5°. In some embodiments, Compound 1·L-lysine Form 1 has a differential scanning calorimetry thermogram comprising 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 comprising an endotherm at about 239.4°±10°, such as 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 shown in FIG. 11.

[0054] In some embodiments, Compound 1·L-lysine Form 1 has no significant weight loss up to about 200° C. as determined by thermogravimetric analysis. In some embodiments, Compound 1·L-lysine Form 1 has a thermogravimetric analysis thermogram substantially as shown in FIG.

[0055] In some embodiments, Compound 1·L-lysine Form 1 has a reversible sorption from about 1.4% to 90% relative humidity as determined by dynamic vapor sorption. In some embodiments, Compound 1·L-lysine Form 1 has a dynamic vapor sorption profile substantially as shown in FIG.

[0056] Crystalline form 2 In another aspect, provided herein is a crystalline form of Compound 1·L-lysine, wherein the crystalline form is Form 2.

[0057] In some embodiments, Compound 1·L-lysine Form 2 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) of at least 20.5°±0.2°. In some embodiments, Compound 1·L-lysine Form 2 has an X-ray powder diffraction exhibiting characteristic scattering angles (2θ) of at least 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) of at least 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) of at least 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 pattern exhibiting characteristic scattering angles (2θ) of at least 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 pattern substantially as shown in FIG. 5B.

[0058] In some embodiments, Compound 1·L-lysine Form 2 has a differential scanning calorimetry thermogram comprising an endotherm at about 231.7°±5°. In some embodiments, Compound 1·L-lysine Form 2 has a differential scanning calorimetry thermogram comprising 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 comprising an endotherm at about 231.7°±10°, such as about 231.7°±9°, 231.7°±8°, 231.7°±7°, or 231.7°±6°.

[0059] In some embodiments, Compound 1·L-lysine Form 2 has no significant weight loss up to about 240°C as determined by thermogravimetric analysis.

[0060] In some embodiments, Compound 1·L-lysine Form 2 has reversible sorption from about 1.6% to 90% relative humidity as determined by gravimetric vapor sorption.

[0061] Method of preparation 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.

[0062] [ka]

[0063] In one aspect, provided herein are methods 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 includes 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 acid to the suspension at an elevated temperature to obtain a solution, cooling the solution, and separating the crystalline form from the solution.

[0064] In some embodiments, provided herein are methods for preparing a crystalline form of Compound 1·L-lysine, the methods comprising 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 separating 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.

[0065] Pharmaceutical Composition 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 to a subject orally, topically, or parenterally 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 formulation such as a capsule, microcapsule, tablet, granule, powder, pill, or suppository.

[0066] The crystalline forms disclosed herein can be administered to a subject orally, topically, or parenterally in conventional formulations such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions. Suitable formulations may contain 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, calcium carbonate ... The pharmaceutical composition can be prepared by conventional methods using conventional organic or inorganic additives such as cereals such as cereals containing ...

[0067] The dose of the crystalline forms described herein administered to a subject can vary widely and be subject to the judgment of a medical professional. Generally, the compounds disclosed herein can be administered at a dose of about 0.001 mg / kg to about 10 mg / kg of subject body weight, one to four times daily, although this dosage can vary appropriately depending on the subject's age, weight, and medical condition, as well as the type of administration. In one embodiment, the dose is about 0.001 mg / kg to about 5 mg / kg of subject body weight, about 0.01 mg / kg to about 5 mg / kg of subject body weight, about 0.05 mg / kg to about 1 mg / kg of subject body weight, about 0.1 mg / kg to about 0.75 mg / kg of subject body weight, or about 0.25 mg / kg to about 0.5 mg / kg of subject body weight. In one embodiment, one dose is given per day. In any given case, the amount of crystalline form administered will depend on factors such as the solubility of the active ingredient, the formulation used, and the route of administration.

[0068] In some embodiments, the crystalline forms described herein are administered to a subject at a dose of about 0.01 mg / day to about 750 mg / day, about 0.1 mg / day to about 375 mg / day, about 0.1 mg / day to about 150 mg / day, about 0.1 mg / day to about 75 mg / day, about 0.1 mg / day to about 50 mg / day, about 0.1 mg / day to about 25 mg / day, or about 0.1 mg / day to about 10 mg / day.

[0069] In another embodiment, provided herein is a unit dosage formulation comprising about 0.1 mg to 500 mg, about 1 mg to 250 mg, about 1 mg to about 100 mg, about 1 mg to about 50 mg, about 1 mg to about 25 mg, or about 1 mg to about 10 mg of a crystalline form of Compound 1, e.g., Compound 1·L-lysine Form 1 or Form 2.

[0070] In certain embodiments, provided herein are unit dosage formulations comprising 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.

[0071] In another embodiment, provided herein is a unit dose formulation comprising 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.

[0072] A 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 daily. In certain embodiments, doses of 100 mg or less are administered as a single daily dose, and doses greater than 100 mg are administered twice daily in an amount equal to half of the total daily dose.

[0073] Crystalline forms of Compound 1, such as Compound 1·L-lysine Form 1 or Form 2, can be administered orally for convenience. 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 juice (e.g., apple juice or orange juice) or any other liquid and administered orally as a solution or suspension.

[0074] The crystalline forms of Compound 1 disclosed herein may 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 left to the discretion of the healthcare professional and may depend, in part, on the site of the disease.

[0075] In one embodiment, provided herein is a capsule containing a crystalline form of Compound 1, such as Compound 1·L-lysine Form 1 or Form 2, without an additional carrier, excipient, or vehicle.

[0076] In another embodiment, provided herein is a pharmaceutical composition comprising 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 an excipient, a diluent, or a mixture thereof.

[0077] Pharmaceutical compositions can be in the form of tablets, chewable tablets, capsules, liquids, parenteral solutions, lozenges, suppositories, suspensions, and the like. Pharmaceutical compositions can be formulated to contain a daily dose, or a convenient fraction of a daily dose, in a dosage unit that 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 a crystalline form of Compound 1, such as Compound 1·L-lysine Form 1 or Form 2, with a suitable carrier or diluent and filling the appropriate amount of the mixture into capsules. Typical carriers and diluents include, but are not limited to, inert powdered substances such as many different types of starch, powdered cellulose, particularly crystalline and microcrystalline cellulose, sugars such as fructose, mannitol, and sucrose, grain flours, and similar edible powders.

[0078] Tablets can be prepared by direct compression, wet granulation, or dry granulation. These formulations usually incorporate diluents, binders, lubricants, and disintegrants together with the compound. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride, and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are substances such as starch, gelatin, and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginate, methylcellulose, polyvinylpyrrolidine, and the like, are also convenient. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.

[0079] Lubricants may be necessary in tablet formulations to prevent the tablet and punch from sticking in the dye. Lubricants can be selected from slippery solids such as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils. Tablet disintegrants are substances that swell when wet, causing the tablet to disintegrate and release the compound. These include starch, clay, cellulose, algin, and gum. More specifically, for example, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resin, alginic acid, guar gum, citrus pulp, and carboxymethylcellulose, and sodium lauryl sulfate can be used. Tablets can be coated with sugar as a flavoring and sealant, or with a film-forming protective agent to modify the dissolution properties of the tablet. The composition can also be formulated as a chewable tablet, for example, by using substances such as mannitol in the formulation.

[0080] 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 it can be modified by the addition of waxes to slightly raise its melting point. Water-miscible suppository bases, particularly those containing polyethylene glycols of various molecular weights, are widely used.

[0081] The effects of crystalline forms 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 sustained-release implantable devices. This technique also involves creating pellets with several different dissolution rates and filling capsules with a mixture of the pellets. Tablets or capsules can be coated with a film that resists dissolution for a predictable period of time. Even parenteral preparations can be made long-acting by dissolving or suspending the crystalline form of Compound 1 in an oily or emulsifying vehicle that allows it to disperse slowly in serum.

[0082] How to use Compound 1 and its salts, including the crystalline forms described herein, Compound 1·L-lysine Form 1 or Form 2, are useful for selectively inhibiting the KDM4 family of histone demethylases and are useful for treating cancers associated with KDM4 activity.

[0083] Thus, in one aspect, provided herein are methods 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, Compound 1·L-lysine Form 1 or Form 2, as described herein. In some embodiments, provided herein are methods 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, provided herein are methods 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, provided herein are methods 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, provided herein are methods 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.

[0084] In some embodiments, provided herein is the use of a crystalline form of Compound 1·L-lysine described herein, such as Compound 1·L-lysine Form 1 or Form 2, in the manufacture of a medicament for treating cancer.

[0085] In some embodiments, provided herein is the use of a crystalline form of Compound 1·L-lysine described herein, such as Compound 1·L-lysine Form 1 or Form 2, to treat cancer in a subject in need thereof.

[0086] Embodiments of the present disclosure provide methods for inhibiting the KDM4 family of histone demethylases in a subject in need thereof, 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 assessed and demonstrated by a variety of methods known in the art. Kits and commercially available assays can be used to determine whether and to what extent the KDM4 family of histone demethylases are inhibited.

[0087] In one aspect, provided herein are methods 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 of Compound 1·L-lysine is Form 1. In some embodiments, the crystalline form of Compound 1·L-lysine is Form 2.

[0088] 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 lyse 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%, Inhibits 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%.

[0089] In another aspect, provided herein are 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, provided herein are methods for preventing cancer in a subject in need thereof, such as a cancer associated with KDM4 activity, 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 to be treated include gastric cancer or colon cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lymphoma.

[0090] In some embodiments, administering a crystalline form disclosed herein to a subject susceptible to cancer prevents the subject from developing any symptoms of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject who does not yet exhibit symptoms of cancer prevents the subject from developing any symptoms of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof reduces the extent 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 slows the progression of the 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 results in a partial remission of the cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need thereof results in a complete remission of the cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need reduces the dose of one or more other medications required to treat cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need enhances the effectiveness of another medication used to treat cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need slows the progression of cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need improves the quality of life of a subject with cancer. In some embodiments, administering a crystalline form disclosed herein to a subject in need prolongs the survival of a subject with cancer.

[0091] In one aspect, provided herein is a method of preventing a subject susceptible to cancer from developing any symptoms of cancer, comprising administering to the subject a crystalline form disclosed herein. In some embodiments, provided herein is a method of preventing a subject who does not yet exhibit symptoms of cancer from developing any symptoms of cancer, comprising administering to the subject a crystalline form disclosed herein.

[0092] In some aspects, provided herein are methods for reducing the extent of cancer in a subject, comprising administering to the subject a crystalline form disclosed herein. In some embodiments, provided herein are methods for stabilizing cancer in a subject, comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the method prevents the cancer from worsening. In some embodiments, the method delays the cancer from worsening.

[0093] In another aspect, provided herein is 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.

[0094] In some embodiments, provided herein are methods for slowing the progression of cancer in a subject, comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the method results in a partial remission of the cancer. In some embodiments, the method results in a complete remission of the cancer.

[0095] In a further aspect, provided herein is a method of reducing the dose of one or more other medications required to treat cancer in a subject, comprising administering to the subject a crystalline form disclosed herein. In some embodiments, provided herein is a method of enhancing the effect of another medication used to treat cancer in a subject, comprising administering to the subject a crystalline form disclosed herein.

[0096] Also provided herein is a method for delaying the progression of cancer in a subject, comprising administering to the subject a crystalline form disclosed herein. In some embodiments, the method improves the quality of life of a subject with cancer. In some embodiments, the method prolongs the survival of a subject with cancer. [Example]

[0097] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0098] The following abbreviations have been used:

[0099] [Table 1]

[0100] Example 1. Compound 1 Salt Screening Study and Identification of Compound 1·L-Lysine Form 1. 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) was synthesized as described in U.S. Pat. No. 9,242,968. Compound 1 has poor solubility in water and most common organic solvents and tends to precipitate as an amorphous paste. A salt screening assay was performed to identify a crystalline, non-hygroscopic form suitable for further development.

[0101] 1. Details of equipment and methods. X-ray powder diffraction (XRPD) using a Bruker AXS C2 GADDS. X-ray powder diffraction patterns were collected on a Bruker AXS C2 GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), an automated XYZ stage, a laser video microscope for automated sample positioning, and a HiStar 2D area detector. The X-ray optics consisted of a single Göbel multilayer mirror coupled with a 0.3 mm pinhole-type collimator. Weekly performance checks were performed using certified standard NIST 1976 Corundum plates.

[0102] The beam divergence, i.e., the effective diameter of the X-ray beam on the sample, was approximately 4 mm. A θ-θ continuous scan mode was used with a sample-to-detector distance of 20 cm, giving an effective 2θ range of 3.2° to 29.7°. Typically, the sample would be exposed to the X-ray beam for 120 seconds. The software used for data collection was GADDS for XP / 2000 4.1.43, and the data were analyzed and presented using Diffrac Plus EVA v15.0.0.0.

[0103] Ambient conditions. Samples run under ambient conditions were prepared using the powder as received, without grinding, as flat specimens. Approximately 1-2 mg of sample was gently pressed onto a glass slide to obtain a flat surface.

[0104] Non-ambient conditions. Samples run under non-ambient conditions were mounted on a silicon wafer with thermal compound. The sample was then heated to the appropriate temperature at 10°C / min, followed by an isothermal hold for 1 min, at which point data collection began.

[0105] nuclear magnetic resonance (NMR) 1H NMR spectra were collected on a Bruker 400 MHz instrument equipped with an autosampler and controlled by a DRX400 console. ICON-NMR v4.0.7 running with Topspin v1.3 allowed for automated experiments using standard Bruker loaded experiments. For non-routine spectroscopy, data were obtained exclusively through the use of Topspin. Unless otherwise specified, samples were prepared in DMSO-d6. Offline analysis was performed using an ACD Spectrus Processor 2012.

[0106] X-ray powder diffraction using a Bruker AXS D8 Advance X-ray powder diffraction patterns were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), a theta-2-theta goniometer, V4 divergence and receiving slits, a Ge monochromator, and a Lynxeye detector. Certified Corundum standards (NIST 1976) were used to check instrument performance. The software used for data collection was Diffrac Plus XRD Commander v2.6.1, and Diffrac Plus EVA v15.0.0.0 was used to analyze and present the data.

[0107] The powder was used as received as a flat plate specimen and the sample was run under ambient conditions. The sample was gently packed into a cavity cut into a polished, zero-background (510) silicon wafer. The sample was rotated in its own plane during analysis. Data collection details were as follows: angular range: 2–42° 2θ, step size: 0.05° 2θ, collection time: 0.5 s / step.

[0108] Differential scanning calorimetry (DSC) DSC data were collected on a TA Instruments Q2000 with a 50-position autosampler. Heat capacity calibration was performed using sapphire, and energy and temperature calibrations were performed using certified indium. Typically, 0.5–3 mg samples were heated from 25°C to 300°C at 10°C / min in pinhole-equipped aluminum pans. A dry nitrogen purge of 50 ml / min was maintained over the sample. Modulated temperature DSC was performed using a base heating rate of 2°C / min and temperature modulation parameters of ±0.636°C (amplitude) every 60 seconds (duration). The instrument control software was Advantage for Q Series v2.8.0.394 and Thermal Advantage v5.5.3, and data were analyzed using Universal Analysis v4.5A.

[0109] Thermogravimetric analysis (TGA) TGA data were collected on a TA Instruments Q500 TGA equipped with a 16-position autosampler. The instrument was temperature calibrated using certified Alumel and Nickel. Typically, 5-10 mg of each sample was placed on a pre-tared aluminum DSC pan and heated from ambient to 350 °C at 10 °C / min. A nitrogen purge at 60 ml / min was maintained over the sample.

[0110] The instrument control software was Advantage for Q Series v2.5.0.256 and Thermal Advantage v5.5.3, and data were analyzed using Universal Analysis v4.5A.

[0111] Polarized Light Microscopy (PLM) Samples were analyzed with a Leica LM / DM polarized light microscope equipped with a digital video camera for image capture. A small amount of each sample was placed on a glass slide, mounted in immersion oil, and covered with a glass slip to separate individual particles as much as possible. Samples were viewed under appropriate magnification and partially polarized light coupled with a λ false color filter.

[0112] Scanning Electron Microscopy (SEM) Data were collected on a Phenom Pro Scanning Electron Microscope. A small sample was mounted on an aluminum stub using conductive double-sided adhesive tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 seconds).

[0113] Water Determination by Karl Fischer Titration (KF) The water content of each sample was measured on a Metrohm 874 Oven Sample Processor at 150°C with an 851 Titrano Coulometer using Hydranal Coulomat AG oven reagent and nitrogen purge. Weighed solid samples were introduced into sealed sample vials. Approximately 10 mg of sample was used per titration, and replicate measurements were performed. Information was collected and analyzed using Tiamo v2.2.

[0114] Gravimetric Vapor Sorption (GVS) Sorption 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). Sample temperature was maintained at 25°C by the instrument controls. Humidity was controlled by mixing dry and humid nitrogen streams at a total flow rate of 200 ml / min. Relative humidity was measured by a calibrated Rotronic probe (dynamic range of 1.0-100% RH) located near the sample. Sample weight change (mass relaxation) as a function of % RH was constantly monitored with a microbalance (accuracy ±0.005 mg).

[0115] Typically, 5-20 mg of sample was placed in a tared mesh stainless steel basket under ambient conditions. The sample was loaded and unloaded at 40% RH and 25°C (typical room conditions). Moisture sorption isotherms were performed (two scans give one full cycle) as outlined in Table 1 below. Standard isotherms were run at 25°C over the range of 0-90% RH at 10% RH intervals. Data analysis was performed using Microsoft Excel using DVS Analysis Suite v6.2 (or 6.1 or 6.0). Samples were recovered after completion of the isotherm and reanalyzed by XRPD.

[0116] [Table 2]

[0117] Chemical Purity Measurement by HPLC Purity analysis was performed on an Agilent HP1100 series system equipped with a diode array detector using ChemStation software vB.04.03 as shown in Table 2 below.

[0118] [Table 3]

[0119] Ion Chromatography (IC) Data were 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 are summarized in Tables 3 and 4. Accurately weighed samples were prepared as stock solutions in the appropriate dissolution medium and diluted appropriately before testing. Quantification was achieved by comparison with standard solutions of known concentrations of the ion being analyzed.

[0120] [Table 4]

[0121] [Table 5]

[0122] pKa measurement and prediction Data were collected on a Sirius T3 instrument. Measurements were obtained at 25°C by UV-metric titration with a cosolvent. The ionic strength of the titration medium was adjusted to 0.15 M KCl(aq) (ISA). Data were refined using Sirius T3 Refine version 1.1.3.0. ACD / Labs Percepta 2012 was used to predict pKa values. ACD / Labs Percepta 2012 was used to predict log P values.

[0123] 2. Experimental procedure (salt screening). Preliminary solubility assessment. Compound 1 (15 mg) was treated with increasing volumes of solvent at 50°C until the material was completely dissolved or a maximum of 20 volumes had been used. Sodium hydroxide (1.1 equivalents) was added, and in most cases a clear solution was obtained. The lamp was set to 5°C at 0.1°C / min and stirred at this temperature overnight. Any solids were filtered, air-dried, and analyzed by XRPD. Any solutions were evaporated to dryness, and the solid residues were also analyzed by XRPD.

[0124] Materials. Commercial chemicals and solvents were purchased from Aldrich or Fluka. The following chemicals were used to make stock solutions: sodium hydroxide, potassium hydroxide, L-lysine, L-arginine, N-methylglucamine, ammonium hydroxide, choline, calcium chloride, ethanolamine, dimethylaminoethanol, N-ethylglucamine, and tromethamine. Base 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 for preparing the base stock solutions, except for calcium chloride, for which ethanol was used.

[0125] 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 counterion of choice. At this stage, the solution or suspension was cooled to 5°C at 0.1°C / min and stirred at this temperature overnight. The resulting solid was filtered, air-dried, and analyzed by XRPD. The solution was subjected to the procedure detailed in the following paragraphs.

[0126] Salt Screening - Evaporation of Solution. Any solution obtained from the above paragraph entitled "Salt Screening - General Procedure (Cooling)" was concentrated by evaporation at ambient conditions, and the solid residue was first analyzed by XRPD. The gum was subjected to the procedure detailed in the following paragraph.

[0127] Salt Screening - Antisolvent Addition. Any gum, oil, or amorphous solid obtained from the above paragraph entitled "Salt Screening - General Procedure (Cooled)" was stirred with TBME (10 volumes) at 30°C overnight. All solids were analyzed by XRPD.

[0128] Preparation of calcium salt by ion exchange. Compound 1 (20 mg) was suspended in the solvent system at 50°C. The suspension was treated with sodium hydroxide (1.1 equivalents), revealing a clear solution. Calcium hydroxide (0.5 equivalents) was added to the solution, resulting in a precipitate. These experiments were then cooled to 5°C at 0.1°C / min and stirred at this temperature overnight. The solid was filtered, air-dried, and initially analyzed by XRPD. Any gum, oil, or amorphous solid obtained at this stage was subjected to the procedure described above.

[0129] Determination of aqueous solubility. Compound 1 or its corresponding salt was accurately weighed into a vial. Water was gradually added with stirring at 25 °C until complete dissolution was observed or up to 200–240 volumes, depending on the sample. After stirring for several minutes, a visual assessment of dissolution was performed.

[0130] 3. Characterization of Compound 1. The characterization of Compound 1 is summarized in Table 5.

[0131] [Table 6]

[0132] Compound 1 was characterized as a dark yellow amorphous solid. A representative XRPD pattern of compound 1 is shown in Figure 1. Evidence of partial deliquescence was observed at the edge of the sample upon storage of this material at 40 °C / 75% RH and 25 °C / 97% RH for 13 days. Thermal analysis showed the loss of 4.8% water, corresponding to a broad, unresolved endothermic event by DSC (Figure 2). This material exhibited low solubility in both aqueous solvents (<5 mg / ml in water at 25 °C) and organic solvents (difficulty in preparing NMR samples using deuterated methanol and DMSO and pKa / ion chromatography samples in acetonitrile).

[0133] The pKa was determined using the sodium salt of Compound 1. Due to the low solubility of Compound 1, Compound 1 was unsuitable for these measurements. The predicted and measured pKa are shown in Figure 3. Due to the compound's insolubility, LogP measurements were not possible. Two LogP experiments were attempted, but precipitation was observed on both occasions. The compound exists as a cation, zwitterion, and anion at different pHs. The neutral form is the minor species.

[0134] 4. Preliminary Research A solubility evaluation of compound 1 in a range of solvent systems was performed, followed by preliminary salt formation experiments with sodium hydroxide, and the results are summarized in Table 6.

[0135] Compound 1 was not soluble in any of the selected solvent systems at 50 °C. However, after adding the corresponding base (1.1 equivalents), complete dissolution was observed in most vials. Three crystalline solids and one amorphous solid were obtained. Although slight differences were observed by XRPD, all solids were converted to the same crystalline form after 7 days of storage at 40 °C / 75% RH, except for the sample from acetone:water, which showed a gum (amorphous).

[0136] [Table 7]

[0137] Based on the above results and their versatility, methanol, acetone / 10% water, and THF were selected for the primary screening.

[0138] 5. Salt screening – results. Salt screening was performed in three solvent systems using the procedure described above. Antisolvent addition was performed as necessary. Crystalline salts were obtained with sodium, L-lysine, ethanolamine, and N-ethylglucamine. Crystalline solids were obtained from experiments with tromethamine and ammonium, but 1 The absence of counterions was verified by H NMR or ion chromatography. The data are summarized in Table 7. Recoveries from these processes were low but were not quantified at this stage.

[0139] [Table 8-1] [Table 8-2] [Table 8-3]

[0140] To generate sufficient material for characterization, the salt formation experiments were repeated on a 50 mg scale. Partial characterization details for the counterions / coformers sodium, L-lysine, ethanolamine, and N-ethylglucamine are summarized in Table 8. Attempts to form salts with ammonium or tromethamine were unsuccessful and only yielded the crystalline free form of Compound 1.

[0141] [Table 9]

[0142] Representative XRPD patterns of various crystalline forms of the sodium salt of Compound 1, the L-lysine salt of Compound 1, the ethanolamine salt of Compound 1, and the N-ethylglucamine salt of Compound 1 are shown in Figures 4-7.

[0143] 6. Discussion of results. Crystalline salts were obtained with sodium, L-lysine, ethanolamine, and N-ethylglucamine, and crystalline free form material was also obtained through unsuccessful salt formation experiments with ammonium and tromethamine.

[0144] 1 Determining the stoichiometry of salts with organic counterions by 1 H NMR was difficult due to overlapping signals. 1 H NMR work was performed in deuterated DMSO, deuterated methanol, and combinations of the two to assess the presence of counterions. Solid mixtures of compound 1 with the corresponding counterions were also prepared and dissolved in deuterated solvents for comparison. Therefore, the expected spectra can be used as a reference for the experimental salts.

[0145] The monosodium salt, Na2, is crystalline and likely a monohydrate based on its water content. Water desorption was observed by TGA and corresponds to multiple endothermic events by DSC. The salt was stable at 40 °C / 75% RH for 1 week but exhibited deliquescence after a total of 36 days of storage.

[0146] Mono-L-lysine salt (Compound 1·L-lysine), LYS1 (Form 1), is crystalline, anhydrous, and exhibits a melting endotherm (associated with decomposition) at 233 °C. This material was stable upon storage at 40 °C / 75% RH for 36 days.

[0147] The N-ethylglucamine salt, NEG1, is crystalline and 1H NMR indicates a slight excess of counter ions (most likely due to incomplete salt formation). This material was stable upon storage for 36 days at 40°C / 75% RH. The N-ethylglucamine salt exhibits an endothermic event (possible melting) at 110°C. At this stage, insufficient material was available for TGA analysis. This material was stable upon storage for 36 days at 40°C / 75% RH.

[0148] Finally, the ethanolamine salt, EA1, is also crystalline. 1 H NMR confirmed monostoichiometry. The salt exhibits an endothermic event (possible melting) at 143°C. At this stage, insufficient material was available for TGA analysis. The material was stable upon storage at 40°C / 75% RH for 7 days.

[0149] Four different polymorphs were observed of the free form of Compound 1. The material FF1 obtained from the unsuccessful tromethamine salt formation was a yellow crystalline solid with a broad endotherm at 217° C. by DSC. 1 Although a small amount of unidentified impurity was observed by H NMR, the small weight loss observed by TGA likely corresponds to the desorption of water. After storage at 40 °C / 75% RH for 1 week, slight changes were observed to form FF4. Another partially crystalline material, FF3, was obtained from another unsuccessful tromethamine formation experiment. Partially crystalline FF2 was generated via a failed attempt to make the ammonium salt. This material exhibited multiple endotherms by DSC and was stable after storage at 40 °C / 75% RH for 1 week. These results indicate that the free base can crystallize under certain conditions. The solubility of the crystalline free form was significantly lower in deuterated solvents. In some cases, larger solvent volumes and filtration were required to achieve complete dissolution and obtain optimal NMR spectra.

[0150] Based on the desired solid state properties of the salts, the salts of sodium, L-lysine, and N-ethylglucamine were selected for the scale-up phase.

[0151] Example 2. Scaling up selected salts of Compound 1 and identification of Compound 1·L-lysine Form 2. Sodium and L-lysine salts. Compound 1 (~500 mg) was suspended in methanol (5 volumes) at 50 °C. Sodium hydroxide or L-lysine (1 M in water, 1.1 equivalents) was added at 50 °C to form a clear solution. After approximately 10 minutes of stirring, a white precipitate was observed in both cases. In the case of the L-lysine salt, an immobile precipitate formed; in contrast, the sodium salt formed a mobile slurry. The ramp was set to 30 °C at 0.1 °C / min, and then the vial was placed at 4 °C overnight. The bulk solid was suction filtered and dried in a vacuum oven at 25 °C overnight. The white solid was used for characterization.

[0152] N-Ethylglucamine Salt. Compound 1 (∼300 mg) was suspended in methanol (17 volumes) at 65°C. N-Ethylglucamine (1 M in water, 1.1 equivalents) was added at 65°C. After 30 minutes, complete dissolution was not observed, and the solution was filtered. The ramp was then set at 0.1°C / min to 5°C. Seeds of NEG1 (material from the screening) were added (∼10 mg) at 63°C. A clear solution was still observed at 5°C, so TBME was added (10 volumes) as an antisolvent. The solution was concentrated by uncapping the vial at room temperature. Additional seeds were added, and the vial was placed at -20°C overnight, but this did not aid crystallization. Evaporation to dryness yielded a gum, which was sonicated without improvement. Another attempt to prepare the N-ethylglucamine salt was made without filtration using a methanol / TBME mixture containing a slight excess of base (1.5 equivalents) at 50°C. Upon evaporation, a gum / oil was also observed. Both the gum / oil were dried under vacuum to yield amorphous solids. These were slurried in 10 solvent systems using a temperature cycle of 25-50°C overnight. A slurry in ethyl acetate showed a crystalline white solid after 1 hour at room temperature. Based on these results, the bulk amorphous solid was slurried overnight in ethyl acetate (cycled from 25-50°C) to yield a crystalline solid. These solids were used for characterization.

[0153] Results and Characterization of Selected Salts. Sodium, L-lysine, and N-ethylglucamine salts were scaled up and fully characterized. Compared to the screening material, two new forms were isolated for L-lysine (LYS2 (Form 2)) and N-ethylglucamine (NEG2). Yields were generally low, 47% and 58% for sodium and L-lysine, respectively. Given the multiple steps required to achieve crystalline material, yields were not quantified for the N-ethylglucamine salt. A summary of the characterization details for the three selected salts can be found in Table 9.

[0154] [Table 10-1] [Table 10-2]

[0155] The sodium salt crystallized as form Na2. However, after 10 days of storage at room temperature in a closed vial, a change to form Na4 was observed. The sodium salt is hydrated, and the diffractogram shows slight variations depending on the amount of water of hydration. These slight variations may be due to differences in ambient humidity on the day of analysis. Ion chromatography indicates 0.8 equivalents of sodium. DSC showed multiple endothermic events associated with the desorption of water by TGA. This material was subjected to VT-XRPD. The starting material was identified prior to the start of this analysis, and a change to Na4 was observed. Na4 showed two changes in form upon heating (Na1 and a new form, Na5), and further change upon cooling at the end of the experiment (a change to a new form, designated Na6, believed to be anhydrous). Representative XRPD patterns of different sodium salt forms are shown in Figure 4. Some of these XRPD patterns may represent mixtures of different forms. GVS analysis also confirmed multiple stages of hydration. The starting material showed 5.7% water (1.5 equivalents), compared to 4.4% water (1 equivalent, Na4 by XRPD) at the end of the experiment. A total of 9.7% water was incorporated (2.5 equivalents) at 90% RH in the first cycle, and 16.0% (4.5 equivalents) in the second cycle. Hysteresis was observed in both cycles, indicating hydration and dehydration phases with different kinetics. Overall, the sodium salts present process chemistry challenges to achieve 1:1 stoichiometry, plus challenges due to the fact that they exist at different hydration levels.

[0156] The N-ethylglucamine salt, NEG2, deliquesced after 2 days at 40°C / 75% RH and 25°C / 97% RH. Reversible moisture absorption of 22.5% w / w was observed by GVS between 0 and 90% RH, after which a gummy solid with low crystallinity was recovered (designated NEG3). TGA showed slight water desorption (1.1% below 125°C), followed by an endotherm observed by DSC (106°C). In summary, both salts formed with N-ethylglucamine had poor thermal profiles and are not recommended for further development.

[0157] Also, the L-lysine salt, LYS2 (Form 2), is stable upon storage at 40°C / 75% RH and 25°C / 97% RH for 8 days, as well as during GVS experiments. Thermal analysis showed melting and decomposition at 231°C. 1 The H NMR spectrum confirmed monostoichiometry. Slight variations in variability were observed in some peaks in the aromatic region, the nature of which is unknown. Aqueous solubility was evaluated for LYS2, which showed a cloudy yellow solution at 5 mg / mL.

[0158] The solubilities of the three salts were evaluated at 25°C. The aqueous solubilities of Na2 and NEG2 are >200 mg / ml. For both substances, clear solutions were observed immediately after adding water with stirring. After approximately 20 minutes, a thick white precipitate was observed, which redissolved overnight. Dissolution attempts with LYS2 resulted in a cloudy yellow solution at 5 mg / ml (200 volumes added and stirred overnight).

[0159] Summary: Crystalline salts were obtained from sodium, L-lysine, N-ethylglucamine, and ethanolamine. Two forms of L-lysine salts (LYS1 (Form 1) and LYS2 (Form 2)) isolated during the study are anhydrous, stable at high humidity, and melt / decompose at approximately 230 °C. Based on its higher crystallinity and solubility, L-lysine salt LYS1 (Form 1) was selected for further development.

[0160] Example 3. Polymorph Screening of Compound 1·L-Lysine Salt A polymorph screening study was conducted to identify new solid-state forms of compound 1·L-lysine. Experiments consisted of solvent-mediated solid-state transformation, temperature cycling, antisolvent vapor diffusion, solvent drop milling, pH swing, cooling crystallization, and antisolvent addition to solution. The solid-state properties of compound 1·L-lysine, Form 1, were also fully characterized.

[0161] 1. Details of equipment and methods. Optical microscopy. Micrographs were obtained using an Olympus BX51 polarizing microscope fitted with a JENOPTIK ProgRes camera and operated by ProgRes Capture Pro 2.8.8 software. Samples were dispersed on microscope slides in silicone oil and examined under transmitted polarized light.

[0162] X-ray diffraction experiments. X-ray powder diffraction data were collected on a Rigaku Miniflex 600 diffractometer using Cu K alpha (1.5406 Å) radiation under ambient conditions. Powder patterns were collected on a zero-background holder with a 0.1 mm indent at 40 kV and 15 mA from 2 to 40° 2θ at a scan rate of 2° / min.

[0163] Differential Scanning Calorimetry (DSC). Differential scanning calorimetry was performed on a TA Discovery series DSC using a few milligrams of material in Tzero aluminum pans sealed with Tzero airtight lids containing two pinholes. Samples were scanned at 10 °C / min under a nitrogen flow of 50 mL / min.

[0164] Thermogravimetric Analysis (TGA). Thermogravimetric data were collected using a TA Discovery series TGA. A few milligrams of material were analyzed in an aluminum sample pan. Data were collected from room temperature to 300°C at a scan rate of 10°C / min.

[0165] Dynamic Vapor Sorption (DVS). Dynamic vapor sorption experiments were performed on a DVS Intrinsic system by Surface Measurement Systems. Samples were exposed to relative humidity cycling from 0% RH to 90% RH, and weights were equilibrated and measured at each humidity step. Temperature was set and held constant at 25°C throughout the experiment.

[0166] Nuclear magnetic resonance (NMR). 1 H NMR spectra were recorded on a Varian Inova 300 Hz spectrometer.

[0167] High Performance Liquid Chromatography (HPLC). Chromatographic separations were performed on a Thermo Fisher SpectraSystem using the conditions presented in Table 10.

[0168] [Table 11]

[0169] 2. Initial Characterization of Compound 1·L-lysine, Form 1. Initial characterization data was generated 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 shown in Figures 8-15. A summary of the physical properties is provided in Table 11.

[0170] [Table 12]

[0171] 3. Screening of solid-state forms of compound 1·L-lysine salt. Thermodynamic Stability Form Screening. Solvent-mediated solid-state form conversion experiments were conducted across a range of temperatures, water activities, and solvent types to identify thermodynamically more stable polymorphs, hydrates, and solvates. Approximately 75–85 mg of 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 a temperature-dependent stir plate and stirred at 500 rpm for 3 weeks. The solid from the slurry was analyzed by XRPD for any changes in solid form. A summary of the experiments and results is shown in Table 12. No new crystalline forms were observed for compound 1·L-lysine.

[0172] [Table 13]

[0173] Temperature Cycling. Approximately 20-30 mg of material was weighed into a vial. Approximately 0.2-0.3 mL of solvent was added, and the vial was capped. The vial was placed in a temperature cycling chamber and continuously cycled from 10 to 60 °C at 10 °C / hour for 3 weeks. The solid was analyzed by XRPD for any changes in solid form. A summary of the experiment and results is shown in Table 13. No new crystalline forms were observed for compound 1·L-lysine.

[0174] [Table 14]

[0175] Antisolvent Vapor Diffusion. Approximately 25-30 mg of material was weighed into a 4 mL vial. Approximately 2 mL of solvent was added, and the vial was sonicated in an attempt to create a solution. All slurries were filtered into clean 4 mL vials, producing the solution for all experiments. These uncapped 4 mL vials were then placed inside larger 20 mL vials containing the antisolvent. The 20 mL vials were capped, and antisolvent vapor was allowed to slowly diffuse into the solution in the uncapped 4 mL vial. All experiments were maintained at ambient conditions. A summary of the experiments and results is shown in Table 14. No crystalline solids were obtained.

[0176] [Table 15]

[0177] Solvent Trituration. Approximately 20-25 mg of material was weighed into a 2 mL amber vial. Approximately 20-40 μL of solvent was added along with 3-6 alumina beads. 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-state morphology changes. A summary of the experiments and results is shown in Table 15. No new crystalline forms were observed for compound 1·L-lysine.

[0178] [Table 16]

[0179] pH Swing. Experiments to generate new solid forms of compound 1·L-lysine were performed by varying the pH of aqueous solutions. Four experiments were performed: (1) adding a base to an acidic solution of compound 1·L-lysine, (2) adding an acid to a basic solution of compound 1·L-lysine, (3) adding an acidic solution of compound 1·L-lysine to a base, or (4) adding a basic solution of compound 1·L-lysine to an acid.

[0180] 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 stir plate, 1.5 mL of 0.1 M NaOH was slowly added to obtain a final pH of 12.0. No solids were observed.

[0181] 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 stir plate, 1.5 mL of 0.1 M HCl was slowly added to obtain a pH of 9.6. An additional 0.5 mL of 0.1 M HCl was added to form a gel with a pH of 3.0.

[0182] 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 1.5 mL of 0.1 M NaOH solution, stirring on a stir plate at 25 °C. The final pH was 12.1. No solids were observed.

[0183] Experiment 4: 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 to produce a solution with a pH of 12.2. This solution was slowly added to a 4 mL amber glass vial containing 1.5 mL of 0.1 M HCl solution stirring on a 25 °C stir plate. 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.

[0184] Cooling crystallization. Solutions of compound 1·L-lysine were prepared in various solvents and heated to 50°C for 20–30 minutes. The samples were transferred to a 4°C refrigerator and monitored for solid formation. A summary of the experiments and results is shown in Table 16. No crystalline solids were obtained.

[0185] [Table 17]

[0186] Antisolvent addition to solution. To discover new polymorphs, antisolvent was added via pipette to the sample remaining as solution from the cooling crystallization experiment above. A summary of the experiment and results is shown in Table 17. No crystalline solid was obtained.

[0187] [Table 18]

[0188] 4. Solubility of Compound 1·L-lysine, Form 1. Solubility was determined gravimetrically in the solvent used in the solvent-mediated solid form transformation experiments described above. Each slurry sample was centrifuged at 2000 rpm for 5 minutes at ambient temperature, and the supernatant was then transferred to a microfuge tube and centrifuged again for 30 minutes at 16400 rpm and 21°C. A 0.500 mL aliquot of the final supernatant solution was transferred to a pre-tared vial, and the solvent was removed by evaporation. The vial was reweighed to calculate the final weight. The solubility values ​​are listed in Table 18.

[0189] [Table 19]

[0190] 5. Summary A polymorphism screen was conducted to identify new solid-state forms of compound 1·L-lysine. Experiments consisted of solvent-mediated solid-state transformation, temperature cycling, antisolvent vapor diffusion, solvent drop trituration, pH swing, cooling crystallization, and antisolvent addition to solution. Since all crystalline forms corresponded to Form 1, no new crystalline forms were discovered in this screen.

[0191] The present disclosure has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

Claims

1. A crystalline form of the 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. 2. The crystalline form of claim 1 having an X-ray powder diffraction exhibiting a characteristic scattering angle (2θ) of at least 20.0°±0.2°.

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

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

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

6. 6. The crystalline form of any one of claims 1 to 5, wherein X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 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. 7. The crystalline form of any one of claims 1 to 6, having an X-ray powder diffraction pattern substantially as shown in Figure 9.

8. 8. The crystalline form of any one of claims 1 to 7, having a differential scanning calorimetry thermogram comprising an endotherm at about 239.4°±5°.

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

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

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

12. 12. The crystalline form of any one of claims 1 to 11, having a reversible sorption of from about 1.4% to 90% relative humidity as determined by dynamic vapor sorption.

13. 13. The crystalline form of any one of claims 1 to 12, having a dynamic vapor sorption profile substantially as shown in Figure 14.

14. A crystalline form of the 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. 15. The crystalline form of claim 14, having an X-ray powder diffraction exhibiting a characteristic scattering angle (2θ) of at least 20.5°±0.2°.

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

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

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

19. 19. The crystalline form of any one of claims 14 to 18, wherein the X-ray powder diffraction exhibits characteristic scattering angles (2θ) of at least 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. 20. The crystalline form of any one of claims 14 to 19, having an X-ray powder diffraction pattern substantially as shown in Figure 5B.

21. 21. The crystalline form of any one of claims 14 to 20, having a differential scanning calorimetry thermogram comprising an endotherm at about 231.7°±5°.

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

23. 23. The crystalline form of any one of claims 14 to 22, having a reversible sorption of from about 1.6% to 90% relative humidity as determined by gravimetric vapor sorption.

24. A method for preparing the crystalline form of any one of claims 1 to 23, comprising the steps of: 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 with 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; separating the crystalline form from the solution; A method comprising:

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

26. 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 of claim 25.

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

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