Salt and crystalline forms of activin receptor-like kinase inhibitors

Abstract

Disclosed are various salt forms of Compound (I), represented by the following structural formula, and their corresponding pharmaceutical compositions. [Formula 1] TIFF2022544272000026.tif55651:1.5 Specific single crystalline forms of Compound (I) succinate, 1:1 Compound (I) hydrochloride, and 1:1 Compound (I) fumarate are characterized by various properties and physical measurements. Methods for preparing specific crystalline forms are also disclosed. The present disclosure also provides methods for treating or ameliorating fibrodysplasia ossificans progressiva in a subject.

Description

Related Applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 885,977, filed August 13, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Activin receptor-like kinase-2 (ALK2) is encoded by the activin A receptor type I gene (ACVR1). ALK2 is a serine / threonine kinase in the bone morphogenetic protein (BMP) pathway (Shore et al., Nature Genetics 2006, 38:525-27). Inhibitors of ALK2 and mutant ALK2 have the potential to treat several diseases, including fibrodysplasia ossificans progressiva (FOP); heterotopic ossification (HO) induced by, for example, major surgical intervention, trauma (such as head injury or blast injury), prolonged immobilization, or severe burns; diffuse intrinsic pontine glioma (DIPG), a rare form of brain cancer; and anemia associated with chronic inflammatory, infectious, or neoplastic diseases.

[0003] U.S. Patent No. 10,233,186 (the entire teachings of which are incorporated herein by reference) discloses potent and highly selective inhibitors of ALK2 and mutant forms of ALK2. The structure of one of the inhibitors disclosed in U.S. Patent No. 10,233,186 (referred to herein as "Compound (I)") is shown below.

[0004] [ka] The successful development of a pharmaceutically active agent, such as Compound (I), typically requires the identification of a solid form that has properties that allow for easy isolation and purification after synthesis, are amenable to large-scale manufacturing, can be stored for extended periods with minimal water absorption, degradation, or conversion to other solid forms, is suitable for formulation, and is readily absorbed (e.g., is soluble in water and gastric fluids) after administration to a subject. Summary of the Invention

[0005] It has now been found that the free base of Compound (I) is physically unstable in moist environments and tends to become gummy when exposed to water. As a result, Compound (I) has proven difficult to isolate when prepared on a production scale.

[0006] It has also now been found that the 1.5:1 succinate salt (i.e., sesquisuccinate salt), 1:1 hydrochloride salt (1:1 hydrochloride salt), and 1:1 fumarate salt (1:1 fumarate salt) can be crystallized under well-defined conditions to provide non-hygroscopic crystalline forms (see Examples 2-7). These three salts also have good solubility in water and simulated gastric fluid (see Table 2) and high melting onsets, making them suitable for large-scale synthesis. The 1.5:1 succinate salt has the additional advantage of exhibiting a high degree of morphological stability because it exists as a single polymorph and does not undergo thermal transitions below its melting point (see Example 2.4). The designation "1:1" refers to the molar ratio between the acid (hydrochloric acid or fumaric acid) and Compound (I), and the designation "1.5:1" refers to the molar ratio between the acid (succinic acid) and Compound (I). Due to the two carboxylic acid groups on succinic acid and the three basic nitrogen atoms in Compound (I), multiple possible stoichiometries are possible. For example, Compound (I) forms both a 1:1 hydrochloride and a 2:1 hydrochloride. The 1:1 hydrochloride of Compound (I) is referred to herein as "1:1 Compound (I) HCl," and the 1.5:1 succinate is referred to herein as "1.5:1 Compound (I) sesquisuccinate."

[0007] Compound (I) HCl, Compound (I) fumarate, and Compound (I) sesquisuccinate were identified from a salt screen with 13 different acids (see Example 1). Only eight crystalline forms were identified from this salt screen. Crystalline salts were formed with benzenesulfonic acid, benzoic acid, fumaric acid, HCl (1 molar equivalent and 2 molar equivalents), maleic acid, salicylic acid, and succinic acid. Of these eight salts, the besylate, maleate, and 2:1 HCl were found to be unsuitable due to low crystallinity and instability in humid environments (deliquescence). The benzoate was found to be unsuitable due to its low solubility in water and high mass loss upon melting. The salicylate was found to be unsuitable due to its low solubility in water, high mass loss upon melting, and potential for polymorphism.

[0008] In one aspect, the present disclosure provides a succinate salt of Compound (I), wherein the molar ratio between Compound (I) and succinic acid is 1:1.5. As mentioned above, this salt is also referred to herein as "1.5:1 Compound (I) Sesqui-Succinate."

[0009] In another aspect, the present disclosure provides an HCl salt of Compound (I), wherein the molar ratio between Compound (I) and HCl acid is 1:1. As mentioned above, this salt is also referred to herein as "1:1 Compound (I) HCl salt."

[0010] In yet another aspect, the present disclosure provides a fumarate salt of Compound (I), wherein the molar ratio between Compound (I) and fumaric acid is 1:1. This salt is also referred to herein as "1:1 Compound (I) fumarate salt."

[0011] In another aspect, the present disclosure provides a pharmaceutical composition comprising 1.5:1 Compound (I) sesquisuccinate salt (or 1:1 Compound (I) HCl salt or 1:1 Compound (I) fumarate salt) and a pharmaceutically acceptable carrier or diluent.

[0012] The present disclosure provides a method of treating or ameliorating fibrodysplasia ossificans progressiva in a subject, comprising administering to a subject in need thereof a pharmaceutically effective amount of a salt disclosed herein or a corresponding pharmaceutical composition.

[0013] The present disclosure provides a method of treating or ameliorating diffuse intrinsic pontine glioma in a subject, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of a salt disclosed herein or a corresponding pharmaceutical composition.

[0014] The present disclosure also provides a method of inhibiting aberrant ALK2 activity in a subject, comprising administering to a subject in need thereof a pharmaceutically effective amount of a salt or corresponding pharmaceutical composition disclosed herein.

[0015] The present disclosure also provides the use of a salt of the present disclosure or a pharmaceutical composition comprising the same in any of the methods of the present disclosure described above. In one embodiment, a salt of the present disclosure or a pharmaceutical composition comprising the same is provided for use in any of the methods of the present disclosure described herein. In another embodiment, a salt of the present disclosure or a pharmaceutical composition comprising the same is provided for use in the manufacture of a medicament for any of the methods of the present disclosure described. [Brief explanation of the drawings]

[0016] [Figure 1] 1 shows the X-ray powder diffraction (XRPD) pattern of 1.5:1 Compound (I) sesquisuccinate salt. [Figure 2] 1 shows thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) thermograms of 1.5:1 Compound (I) sesquisuccinate salt. [Figure 3] 1.5:1 H-nuclear magnetic resonance spectroscopy (H-NMR) of Compound (I) sesquisuccinate. [Figure 4] 1 shows the DVS isotherm of 1.5:1 Compound (I) sesquisuccinate salt. [Figure 5]1 shows the XRPD patterns of 1.5:1 Compound (I) sesquisuccinate salt (Form A) before (bottom) and after (top) DVS measurement. [Figure 6] 1 shows the XRPD patterns of 1.5:1 Compound (I) sesquisuccinate salt (Form A) at varying humidity. From bottom to top, XRPD diffractograms were acquired in situ for varying humidity steps of 40% RH, 60% RH, 90% RH, 40% RH, 0% RH, and again 40% RH. [Figure 7] 1 shows the variable temperature XRPD patterns of 1.5:1 Compound (I) sesquisuccinate salt (Form A). From bottom to top, XRPD diffractograms were acquired in situ for variable temperature steps of ambient conditions, 40° C., 60° C., 80° C., 100° C., 120° C., 140° C., 160° C., and again at 25° C. [Figure 8] FIG. 1 shows the XRPD pattern of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A). [Figure 9] 1 shows TGA and DSC thermograms of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A). [Figure 10] 1 shows the H-NMR of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A). [Figure 11] 1 shows the DVS isotherm of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A). [Figure 12] 1 shows the XRPD patterns of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A) before (bottom) and after (top) DVS measurement. The extra peak observed after DVS is indicated by the arrow. [Figure 13] 1 shows the XRPD patterns of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A) at varying humidity. From bottom to top, each XRPD diffractogram was acquired in situ for varying humidity steps of ambient conditions, 40% RH, 90% RH, 0% RH, and again 40% RH. [Figure 14]1 shows the variable temperature XRPD patterns of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A). From bottom to top, XRPD diffractograms were acquired in situ for variable temperature steps of ambient conditions, 50° C., 100° C., 160° C., and again at 25° C. [Figure 15] 1 shows the XRPD patterns of anhydrous 1:1 Compound (I) crystalline HCl salt (Form D) observed during initial screening (bottom) and scale-up (top). [Figure 16] FIG. 1 shows TGA and DSC thermograms of anhydrous 1:1 Compound (I) crystalline HCl salt (Form D). [Figure 17] 1 shows the H-NMR of anhydrous 1:1 Compound (I) crystalline HCl salt (Form D). [Figure 18] 1 shows XRPD patterns of anhydrous 1:1 Compound (I) crystalline HCl salt (Form G) observed during screening (bottom), scale-up (wet) (middle), and drying (top). [Figure 19] 1 shows TGA and DSC thermograms of anhydrous 1:1 Compound (I) crystalline HCl salt (Form G). [Figure 20] 1 shows the H-NMR of anhydrous 1:1 Compound (I) crystalline HCl salt (Form G). [Figure 21] 1 shows the XRPD patterns of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form I) observed during initial screening (bottom) and scale-up (top). [Figure 22] 1 shows TGA and DSC thermograms of anhydrous 1:1 Compound (I) crystalline HCl salt (Form I). [Figure 23] FIG. 1 shows the H-NMR of anhydrous 1:1 Compound (I) crystalline HCl salt (Form I). [Figure 24] 1 shows the DVS isotherm of the free base of Compound (I). [Figure 25] FIG. 1 shows the XRPD pattern of 2:1 Compound (I) crystalline HCl salt (Form B). [Figure 26] 1 shows the XRPD patterns of the anhydrous 1:1 Compound (I) crystalline fumarate salt (Form A) observed during initial screening (bottom) and scale-up (top). [Figure 27] 1 shows TGA and DSC thermograms of anhydrous 1:1 Compound (I) crystalline fumarate salt (Form A). [Figure 28] 1 shows the H-NMR of anhydrous 1:1 Compound (I) crystalline fumarate salt (Form A). [Figure 29] FIG. 1 shows the XRPD pattern of 1:1 Compound (I) crystalline fumarate salt (Form C). [Figure 30] FIG. 1 shows the XRPD pattern of 1:1 Compound (I) crystalline fumarate salt (Form D). DETAILED DESCRIPTION OF THE INVENTION

[0017] The present disclosure is directed to a novel succinate salt (i.e., 1:1.5 sesquisuccinate salt), a novel hydrochloride salt (i.e., 1:1 hydrochloride salt), and a novel fumarate salt (i.e., 1:1 fumarate salt) of Compound (I), as well as polymorphic forms of each of the foregoing.

[0018] "Hydrated form" refers to a solid or crystalline form of Compound (I) in the form of a free base or salt in which water is combined with the free base Compound (I) or the corresponding salt in a stoichiometric ratio (e.g., a molar ratio of Compound (I):water of 1:1 or 1:2) as an integral part of the solid or crystal. "Unhydrated form" refers to a form in which there is no stoichiometric ratio between water and the free base of Compound (I) or the corresponding salt of Compound (I), and water is substantially absent from the solid form (e.g., less than 10% by weight by Karl Fischer analysis). The new solid forms disclosed in the present disclosure include hydrated and unhydrated forms.

[0019] As used herein, "crystalline" refers to a solid having a crystalline structure in which the individual molecules have a highly uniform and regular three-dimensional arrangement.

[0020] The disclosed crystalline Compound (I) salts can be crystals of a single crystalline form or a mixture of crystals of different single crystalline forms. By single crystalline form, it is meant that Compound (I) is a single crystal or multiple crystals, each of which has the same crystalline form.

[0021] With respect to the crystalline forms of Compound (I) disclosed herein, at least a certain weight percentage of the 1.5:1 Compound (I) salt is in single crystalline form. The certain weight percentages include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, 70% to 80%, 80% to 90%, or 90% to 100% by weight of the Compound (I) salt is in single crystalline form. It is understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed by the present disclosure.

[0022] When a crystalline Compound (I) salt is defined as a specified percentage of one particular crystalline form of Compound (I) salt, the remainder consists of amorphous forms and / or crystalline forms other than the specified one or more particular forms. Examples of single crystalline forms include 1.5:1 Compound (I) sesquisuccinate (Form A), 1:1 Compound (I) HCl salt (Forms A, D, G, and I), and 1:1 Compound (I) fumarate (Forms A, C, and D), characterized by one or more properties as discussed herein.

[0023] Compound (I) has a chiral center. Compound (I), in the salts and polymorphs disclosed herein, is at least 80%, 90%, 99%, or 99.9% pure by weight with respect to other stereoisomers (i.e., the ratio of the weight of the stereoisomer to the weight of all stereoisomers).

[0024] The crystalline Compound (I) salts disclosed herein exhibit strong, distinctive XRPD patterns with sharp peaks and flat baselines corresponding to the angular peak positions at 2θ, indicative of highly crystalline material (see, e.g., Figure 1). The XRPD patterns disclosed herein are obtained from a copper radiation source (Cu Kα1, λ=1.5406 Å).

[0025] Characterization of the 1.5:1 Compound(I) Sesquisuccinate Crystalline Form In one embodiment, the 1.5:1 Compound (I) sesquisuccinate salt is Form A as a single crystalline form characterized by an X-ray powder diffraction pattern containing peaks at 8.5, 15.4, and 21.3 ± 0.2 degrees 2θ. In another embodiment, Form A is characterized by an X-ray powder diffraction pattern containing at least three peaks (or four peaks) selected from 4.3, 8.5, 14.0, 15.4, and 21.3 ± 0.2 degrees 2θ. In another embodiment, Form A is characterized by an X-ray powder diffraction pattern containing peaks at 4.3, 8.5, 14.0, 15.4, and 21.3 ± 0.2 degrees 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising peaks at 4.3, 6.7, 8.5, 12.8, 14.0, 15.4, 17.0, and 21.3±0.2 degrees 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising peaks at 4.3, 6.7, 8.5, 12.8, 14.0, 15.4, 15.7, 16.6, 17.0, 18.1, 19.4, 19.8, 20.1, 20.7, 21.3, 22.3, 25.0, 29.1, and 34.4±0.2 degrees 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern substantially similar to FIG. 1.

[0026] It is well known in the art of crystallography that for any given crystalline form, the positions of angular peaks may vary slightly due to factors such as temperature fluctuations, sample misalignment, and the presence or absence of an internal standard. In this disclosure, the variability in angular peak positions is ±0.2 in 2θ. In addition, the relative peak intensities for a given crystalline form may vary due to differences in crystallite size and non-random crystallite orientation in sample preparation for XRPD analysis. It is well known in the art that this variability will account for the above factors without preventing unambiguous identification of the crystalline form.

[0027] In another embodiment, Form A as the 1.5:1 Compound (I) sesquisuccinate salt is characterized by a peak phase transition temperature by differential scanning calorimetry (DSC) of 177±2°C.

[0028] Characterization of the crystalline form of 1:1 Compound (I) hydrochloride In one embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 12.9, 17.0, 19.0, 21.1, and 22.8°±0.2 in term of 2θ. In another embodiment, 1:1 Compound (I) hydrochloride salt is Form A as a single crystalline form characterized by an X-ray powder diffraction pattern comprising peaks at 12.9, 17.0, 19.0, 21.1, and 22.8°±0.2 in term of 2θ. In another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising peaks at 12.9, 13.8, 15.1, 17.0, 19.0, 19.6, 21.1, and 22.8°±0.2 in term of 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising peaks at 5.7, 10.1, 12.6, 12.9, 13.8, 15.1, 17.0, 19.0, 19.6, 20.3, 21.1, 22.1, 22.8, 23.4, 24.0, 24.8, 25.5, 26.1, and 28.6°±0.2 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern substantially similar to Figure 8.

[0029] In another embodiment, Form A as the 1:1 Compound (I) hydrochloride salt is characterized by a peak phase transition temperature by differential scanning calorimetry (DSC) of 207±2°C.

[0030] In one embodiment, the 1:1 Compound (I) hydrochloride salt is Form D as a single crystalline form characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 10.8, 16.9, 18.8, 22.1, and 24.7°±0.2 in term of 2θ. In another embodiment, the 1:1 Compound (I) hydrochloride salt is Form D as a single crystalline form characterized by an X-ray powder diffraction pattern comprising peaks at 10.8, 16.9, 18.8, 22.1, and 24.7°±0.2 in term of 2θ.

[0031] In another embodiment, Form D is characterized by an X-ray powder diffraction pattern comprising peaks at 10.8, 13.3, 16.9, 18.8, 22.1, and 24.7°±0.2 in term of 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern comprising peaks at 10.8, 13.1, 13.3, 16.6, 16.9, 17.4, 18.8, 20.8, 22.1, and 24.7°±0.2 in term of 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern substantially similar to Figure 15.

[0032] In another embodiment, Form D as the 1:1 Compound (I) hydrochloride salt is characterized by a peak phase transition temperature by differential scanning calorimetry (DSC) of 207±2°C.

[0033] In one embodiment, the 1:1 Compound (I) hydrochloride salt is Form G as a single crystalline form characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 10.2, 12.8, 16.7, 17.4, 18.4, and 22.5°±0.2 in term of 2θ. In another embodiment, the 1:1 Compound (I) hydrochloride salt is Form G as a single crystalline form characterized by an X-ray powder diffraction pattern comprising peaks at 10.2, 12.8, 16.7, 17.4, 18.4, and 22.5°±0.2 in term of 2θ. In another embodiment, Form G is characterized by an X-ray powder diffraction pattern comprising peaks at 10.2, 12.8, 16.7, 17.4, 18.4, 21.3, 22.0, 22.5, and 24.3°±0.2 in term of 2θ. In yet another embodiment, Form G is characterized by an X-ray powder diffraction pattern comprising peaks at 10.2, 12.8, 14.9, 16.7, 17.4, 18.4, 20.5, 21.3, 22.0, 22.5, and 24.3°±0.2 in term of 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern substantially similar to Figure 18.

[0034] In another embodiment, Form G as the 1:1 Compound (I) hydrochloride salt is characterized by peak phase transition temperatures by differential scanning calorimetry (DSC) of 175±4°C and 197±4°C.

[0035] In one embodiment, the 1:1 Compound (I) hydrochloride salt is Form I as a single crystalline form characterized by an X-ray powder diffraction pattern containing at least three peaks (or four peaks) selected from 5.4, 8.2, 16.3, 16.5, 18.4, and 21.5°±0.2 in term of 2θ. In another embodiment, the 1:1 Compound (I) hydrochloride salt is Form I as a single crystalline form characterized by an X-ray powder diffraction pattern containing peaks at 5.4, 8.2, 16.3, 16.5, 18.4, and 21.5°±0.2 in term of 2θ. In another embodiment, Form I is characterized by an X-ray powder diffraction pattern containing peaks at 5.4, 8.2, 13.1, 16.3, 16.5, 18.4, and 21.5°±0.2 in term of 2θ. In yet another embodiment, Form I is characterized by an X-ray powder diffraction pattern comprising peaks at 5.4, 8.2, 10.2, 13.1, 16.3, 16.5, 17.1, 18.4, 21.5, and 21.8°±0.2 2θ. In yet another embodiment, Form I is characterized by an X-ray powder diffraction pattern substantially similar to Figure 21.

[0036] In another embodiment, Form I as the 1:1 Compound (I) hydrochloride salt is characterized by peak phase transition temperatures by differential scanning calorimetry (DSC) of 187±4°C and 200±4°C.

[0037] Characterization of the crystalline form of 2:1 Compound (I) hydrochloride salt In one embodiment, the 2:1 Compound (I) hydrochloride salt is Form B as a single crystalline form characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 10.6, 17.0, 18.3, 20.9, and 21.1°±0.2 in term of 2θ. In one embodiment, the 2:1 Compound (I) hydrochloride salt is Form B as a single crystalline form characterized by an X-ray powder diffraction pattern comprising peaks at 10.6, 17.0, 18.3, 20.9, and 21.1°±0.2 in term of 2θ. In another embodiment, Form B as the 2:1 Compound (I) hydrochloride salt is characterized by an X-ray powder diffraction pattern comprising peaks in degrees 2θ at 10.6, 12.7, 15.8, 17.0, 18.3, 18.9, 20.9, 21.1, and 22.0±0.2. In yet another embodiment, Form B as the 2:1 Compound (I) hydrochloride salt is characterized by an X-ray powder diffraction pattern comprising peaks at 7.8, 8.6, 10.6, 11.9, 12.7, 13.3, 15.4, 15.8, 16.5, 17.0, 18.3, 18.9, 19.7, 20.9, 21.1, 22.0, 22.6, 24.5, 26.7, 27.1, 28.9, and 29.7°±0.2 2θ. In yet another embodiment, Form B as the 2:1 Compound (I) hydrochloride salt is characterized by an X-ray powder diffraction pattern substantially similar to Figure 25.

[0038] Characterization of 1:1 Compound(I) fumarate crystalline forms In one embodiment, the 1:1 Compound (I) fumarate salt is Form A as a single crystalline form, characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 5.7, 15.3, 16.9, 22.4, and 23.0°±0.2 in 2θ. In one embodiment, the 1:1 Compound (I) fumarate salt is Form A as a single crystalline form, characterized by an X-ray powder diffraction pattern comprising peaks at 5.7, 15.3, 16.9, 22.4, and 23.0°±0.2 in 2θ. In another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising peaks at 5.7, 7.5, 9.8, 10.3, 12.3, 15.3, 16.9, 17.5, 22.4, and 23.0°±0.2 in 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising peaks at 5.7, 7.5, 9.8, 10.3, 11.2, 12.3, 14.8, 15.3, 16.2, 16.9, 17.2, 17.5, 18.3, 18.8, 19.9, 20.7, 21.5, 22.4, 23.0, 23.5, and 25.8±0.2 degrees 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern substantially similar to Figure 26.

[0039] In another embodiment, Form A as the 1:1 Compound (I) fumarate salt is characterized by a peak phase transition temperature by differential scanning calorimetry (DSC) of 224±2°C.

[0040] In one embodiment, the 1:1 Compound (I) fumarate salt is Form C as a single crystalline form characterized by an X-ray powder diffraction pattern containing at least three peaks (or four peaks) selected from 6.3, 9.0, 13.5, 18.9, and 22.5°±0.2 in 2θ. In one embodiment, the 1:1 Compound (I) fumarate salt is Form C as a single crystalline form characterized by an X-ray powder diffraction pattern containing peaks at 6.3, 9.0, 13.5, 18.9, and 22.5°±0.2 in 2θ. In another embodiment, Form C is characterized by an X-ray powder diffraction pattern containing peaks at 4.5, 6.3, 9.0, 13.5, 14.7, 18.9, 19.7, 21.0, 22.5, and 23.6°±0.2 in 2θ. In yet another embodiment, Form C is characterized by an X-ray powder diffraction pattern comprising peaks at 4.5, 6.3, 7.4, 9.0, 13.5, 14.7, 16.2, 16.8, 17.4, 17.8, 18.4, 18.9, 19.7, 21.0, 22.5, 23.6, 25.5, 26.2, 27.5, and 28.3°±0.2 2θ. In yet another embodiment, Form C is characterized by an X-ray powder diffraction pattern substantially similar to Figure 29.

[0041] In one embodiment, the 1:1 Compound (I) fumarate salt is Form D as a single crystalline form characterized by an X-ray powder diffraction pattern containing at least three peaks (or four peaks) selected from 4.6, 11.0, 18.5, 20.5, and 21.0°±0.2 in term of 2θ. In one embodiment, the 1:1 Compound (I) fumarate salt is Form D as a single crystalline form characterized by an X-ray powder diffraction pattern containing peaks at 4.6, 11.0, 18.5, 20.5, and 21.0°±0.2 in term of 2θ. In another embodiment, Form D is characterized by an X-ray powder diffraction pattern containing peaks at 4.6, 11.0, 15.1, 18.5, 19.4, 20.5, 21.0, and 25.0°±0.2 in term of 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern comprising peaks at 4.6, 11.0, 12.0, 14.3, 15.1, 18.5, 19.4, 20.5, 21.0, 22.8, 23.6, and 25.0°±0.2 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern substantially similar to Figure 30.

[0042] In one embodiment, the 1:1 Compound (I) fumarate salt is Form C as a single crystalline form admixed with Form D, wherein Form C is characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 6.3, 9.0, 13.5, 18.9, and 22.5°±0.2 in 2θ, and Form D is characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 4.6, 11.0, 18.5, 20.5, and 21.0°±0.2 in 2θ.

[0043] In one embodiment, the 1:1 Compound (I) fumarate salt is Form C as a single crystalline form admixed with Form D, wherein Form C is characterized by an X-ray powder diffraction pattern comprising peaks at 6.3, 9.0, 13.5, 18.9, and 22.5°±0.2 in term of 2θ, and Form D is characterized by an X-ray powder diffraction pattern comprising peaks at 4.6, 11.0, 18.5, 20.5, and 21.0°±0.2 in term of 2θ.

[0044] In one embodiment, the 1:1 Compound (I) fumarate salt is Form C as a single crystalline form admixed with Form D, wherein Form C is characterized by an X-ray powder diffraction pattern comprising peaks at 4.5, 6.3, 9.0, 13.5, 14.7, 18.9, 19.7, 21.0, 22.5, and 23.6°±0.2 in term of 2θ, and Form D is characterized by an X-ray powder diffraction pattern comprising peaks at 4.6, 11.0, 15.1, 18.5, 19.4, 20.5, 21.0, and 25.0°±0.2 in term of 2θ.

[0045] In one embodiment, the 1:1 Compound (I) fumarate salt is Form C as a single crystalline form blended with Form D, wherein Form C has angles in degrees 2θ of 4.5°, 6.3°, 7.4°, 9.0°, 13.5°, 14.7°, 16.2°, 16.8°, 17.4°, 17.8°, 18.4°, 18.9°, 19.7°, 21.0°, 22.5°, 23.6°, 25.5°, 26.0°, 27.0°, 28.0°, 29.0°, 30.0°, 31.0°, 32.0°, 33.0°, 34.0°, 35.0°, 36.0°, 37.0°, 38.0°, 39.0°, 40.0°, 41.0°, 42.0°, 43.0°, 44.0°, 45.0°, 46.0°, 47.0°, 48.0°, 49.0°, 50.0°, 51.0°, 52.0°, 53.0°, 54.0°, 55.0°, 56.0°, 57.0°, 58.0°, 59.0°, 60.0°, 61.0°, 62.0°, 63.0°, 64.0°, 65.0°, 66.0°, 67.0°, 68.0°, 69.0°, 70.0°, 71.0°, 72.0°, 73.0°, 74.0°, 75.0°, 76 Form A is characterized by an X-ray powder diffraction pattern containing peaks at 4.6°, 11.0°, 12.0°, 14.3°, 15.1°, 18.5°, 19.4°, 20.5°, 21.0°, 22.8°, 23.6°, and 25.0°±0.2 2θ, and Form B is characterized by an X-ray powder diffraction pattern containing peaks at 4.6°, 11.0°, 12.0°, 14.3°, 15.1°, 18.5°, 19.4°, 20.5°, 21.0°, 22.8°, 23.6°, and 25.0°±0.2 2θ.

[0046] Pharmaceutical Compositions The pharmaceutical compositions of the present disclosure comprise a salt of Compound (I) described herein, or a crystalline form thereof, and one or more pharmaceutically acceptable carrier(s) or diluent(s). The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting any subject composition or its components. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components, and not harmful to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and (10) glycols such as propylene glycol, (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

[0047] The compositions of the present disclosure may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In certain embodiments, the compositions of the present disclosure are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present disclosure may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media.

[0048] For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful for preparing injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used to formulate pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Spans, and other emulsifiers or bioavailability enhancers, which are commonly used to prepare pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0049] The pharmaceutically acceptable compositions of the present disclosure can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or aqueous solutions.For tablets for oral use, commonly used carriers include lactose and corn starch.Lubricants such as magnesium stearate are also typically added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents.If desired, certain sweeteners, flavorings or coloring agents can also be added.

[0050] Alternatively, the pharmaceutically acceptable compositions of the present disclosure may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0051] The pharmaceutically acceptable compositions of the present disclosure can also be administered locally, especially when the therapeutic target comprises areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract.Suitable topical formulations for each of these areas or organs can be easily prepared.Topical application for the lower intestinal tract can be achieved with a rectal suppository formulation (see above) or a suitable enema formulation.Topical transdermal patches can also be used.

[0052] For topical application, pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of the present disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water.Alternatively, pharmaceutically acceptable compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0053] Pharmaceutically acceptable compositions of the present disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0054] The amount of compounds of the present disclosure that may be combined with the carriers to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration, and other factors determined by the person administering the single dosage form.

[0055] dosage The toxicity and therapeutic efficacy of the salts of Compound (I) described herein, or crystalline forms thereof, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. 50 is the dose that is lethal to 50% of the population. 50 is the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects (LD 50 / ED 50) is the therapeutic index. Salts of Compound (I), or crystalline forms thereof, that exhibit large therapeutic indices are preferred. Salts of Compound (I), or crystalline forms thereof, described herein that exhibit toxic side effects may also be used, although care should be taken to design a delivery system that targets such salts or crystalline forms to the site of affected tissue to minimize the potential for damage to non-infected cells, thereby reducing side effects.

[0056] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such salts or crystalline forms can be determined to be ED 100 with little or no toxicity. 50 The therapeutically effective dose may fall within a circulating concentration range including the IC (IC ) as determined in cell culture. The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For any salt of Compound (I) described herein, or a crystalline form thereof, the therapeutically effective dose may be initially estimated from cell culture assays. The dose may be determined based on the IC (IC ) as determined in cell culture. 50 The compounds may be formulated in animal models to achieve a circulating plasma concentration range that includes (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels may be measured, for example, by high performance liquid chromatography.

[0057] It should also be understood that the specific dosage and treatment regimen for any particular subject will depend on a variety of factors, including, but not limited to, the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, as well as the judgment of the treating physician and the severity of the particular disease being treated. The amount of a salt or crystalline form of Compound (I) of the present disclosure in a composition will also depend on the particular compound in the composition.

[0058] Treatment method A "subject" is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, such as companion animals (e.g., dogs, cats, etc.), livestock animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0059] A "treatment" regimen for a subject with an effective amount of a compound of the present disclosure may consist of a single administration, or alternatively, may include a series of applications. For example, 1:1 Compound (I) fumarate and 1:1 Compound (I) maleate may be administered at least once a week. However, in another embodiment, the compound may be administered to a subject from about once a week to once a day for a given treatment. The length of the treatment period depends on various factors, such as the severity of the disease, the age of the subject, the concentration and activity of the compound of the present disclosure, or a combination thereof. It will also be understood that the effective dosage of the compound used for treatment or prevention may increase or decrease over the course of a particular treatment or prevention regimen. Changes in dosage may occur as a result of or be evident from standard diagnostic assays known in the art. In some cases, chronic administration may be required.

[0060] Mutations in ALK2 cause the kinase to become inappropriately activated and are associated with various diseases. Compound (I), its salts, and crystalline forms disclosed herein inhibit mutant ALK2 genes, for example, mutant ALK2 genes that result in the expression of ALK2 enzymes with amino acid modifications. In another aspect, Compound (I), its salts, and crystalline forms disclosed herein inhibit both wild-type (WT) ALK2 protein and mutant ALK2 protein. For purposes of this disclosure, the sequence information for ALK2 can be found on the National Center for Biotechnology Information (NCBI) webpage (https: / / www.ncbi.nlm.nih.gov / ) under ACVR1 activin A receptor type 1 [Homo sapiens (human)]; Entrez Gene ID (NCBI):90. It is also known as FOP, ALK2, SKR1, TSRI, ACTRI, ACVR1A, and ACVRLK2, and the sequence information is incorporated herein by reference.

[0061] In one embodiment, the present disclosure provides a method for inhibiting abnormal ALK2 activity in a subject, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of Compound (I), or a salt, crystalline form, or pharmaceutical composition described herein. In one embodiment, the abnormal ALK2 activity is caused by a mutation in the ALK2 gene that results in expression of an ALK2 enzyme having an amino acid modification selected from one or more of L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328V, G328W, G328E, G328R, G356D, and R375P. In one embodiment, the ALK2 enzyme has the amino acid modification R206H.

[0062] Due to their activity against ALK2, Compound (I), or a salt, crystalline form, or pharmaceutical composition described herein, can be used to treat subjects with conditions associated with abnormal ALK2 activity. In one embodiment, the condition associated with abnormal ALK2 activity is fibrodysplasia ossificans progressiva. Diagnosis of FOP is based on the presence of a congenital malformation of the great toe (hallux valgus) and the formation of fibrous nodules in soft tissue. These nodules may or may not transform into heterotopic bone. These soft tissue lesions often first appear on the head, neck, or back. Approximately 97% of subjects with FOP have the same c.617G>A;R206H mutation in the ACVR1 (ALK2) gene. Genetic testing is available through the University of Pennsylvania (Kaplan et al., Pediatrics 2008, 121(5):e1295-e1300).

[0063] Other common congenital anomalies include deformed thumbs, short and wide femoral necks, osteochondromas of the tibia, and fused facet joints in the cervical spine. Fused facet joints in the neck often cause infants to drag their hips rather than crawl. FOP is often misdiagnosed (approximately 80% of the time, due to cancer or fibromatosis), and subjects frequently undergo inappropriate diagnostic procedures, such as biopsies, which worsen the disease and cause permanent disability.

[0064] In certain embodiments, the present disclosure provides a method of treating or ameliorating fibrodysplasia ossificans progressiva in a subject, comprising administering to a subject in need thereof a pharmaceutically effective amount of Compound (I), or a salt, crystalline form, or pharmaceutical composition described herein.

[0065] In one embodiment, the condition associated with aberrant ALK2 activity is fibrodysplasia ossificans progressiva (FOP), and the subject has a mutation in the ALK2 gene that results in expression of an ALK2 enzyme with an amino acid modification selected from one or more of: L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328W, G328E, G328R, G356D, and R375P. In one aspect of this embodiment, the ALK2 enzyme has the amino acid modification R206H.

[0066] The present disclosure includes methods for identifying and / or diagnosing a subject for treatment with Compound (I), or a salt, crystalline form, or pharmaceutical composition described herein. In one embodiment, the present disclosure provides a method for detecting a condition associated with abnormal ALK2 activity, e.g., FOB, in a subject, the method comprising: a. obtaining a sample, e.g., plasma, from the subject, e.g., a human subject; and b. detecting whether one or more mutations in the ALK2 gene, as described herein, are present in the sample. In another embodiment, the present disclosure provides a method for diagnosing a condition associated with abnormal ALK2 activity in a subject, the method comprising: a. obtaining a sample from the subject; b. detecting whether one or more mutations in the ALK2 gene, as described herein, are present in the sample using a detection method described herein; and c. diagnosing the subject with the condition if the presence of the one or more mutations is detected. Methods for detecting mutations include, but are not limited to, hybridization-based methods, amplification-based methods, microarray analysis, flow cytometry analysis, DNA sequencing, next-generation sequencing (NGS), primer extension, PCR, in situ hybridization, dot blotting, and Southern blotting. In one embodiment, the present disclosure provides a method for diagnosing and treating a condition associated with abnormal ALK2 activity in a subject, the method comprising: a. obtaining a sample from the subject; b. detecting whether one or more mutations in the ALK2 gene as described herein are present in the sample; and diagnosing the subject with the condition if one or more mutations are detected in the sample; and administering an effective amount of Compound (I), or a salt, crystalline form, or pharmaceutical composition as described herein, to the diagnosed subject.In certain embodiments, the present disclosure provides a method of treating a condition associated with aberrant ALK2 activity in a subject, the method comprising: a. determining whether the subject has, has been determined to have, or receives information that the subject has one or more mutations in the ALK2 gene as described herein; b. identifying the subject as being responsive to one or more compounds or pharmaceutical compositions described herein; and c. administering to the subject an effective amount of Compound (I), or a salt, crystalline form, or pharmaceutical composition.

[0067] In some embodiments, the pathology associated with abnormal ALK2 activity is a brain tumor, such as a glial tumor. In some embodiments, the glial tumor is a diffuse intrinsic pontine glioma (DIPG). In some embodiments, the present disclosure provides a method for treating or ameliorating diffuse intrinsic pontine glioma in a subject, comprising administering to a subject in need thereof a pharmaceutically effective amount of Compound (I), or a salt, crystalline form, or pharmaceutical composition as described herein.

[0068] In one embodiment, the condition associated with aberrant ALK2 activity is diffuse intrinsic pontine glioma, and the subject has a mutation in the ALK2 gene that results in expression of an ALK2 enzyme having an amino acid modification selected from one or more of R206H, G328V, G328W, G328E, and G356D. In one aspect of this embodiment, the ALK2 enzyme has the amino acid modification R206H.

[0069] In certain embodiments, the condition associated with aberrant ALK2 activity is anemia associated with inflammation, cancer, or chronic disease.

[0070] In one embodiment, the condition associated with aberrant ALK2 activity is trauma- or surgery-induced heterotopic ossification.

[0071] In certain embodiments, a compound of the present disclosure is co-administered (either as part of a combined dosage form or as a separate dosage form administered before, consecutively with, or after) a second therapeutic agent useful in treating the disease being treated, e.g., FOP. In one aspect of this embodiment, a compound of the present disclosure is co-administered with another anti-allergy agent, such as a steroid (e.g., prednisone) or omalizumab.

[0072] In some embodiments, the compound of the present disclosure is co-administered with RAR-γ agonist or antibody to activin for treating the disease to be treated, such as FOP.In some embodiments, the RAR-γ agonist to be co-administered is palovarotene.In some embodiments, the antibody to activin to be co-administered is REGN2477.

[0073] In some embodiments, compounds of the present disclosure are co-administered with mast cell-targeting therapeutic agents useful in treating FOP, hi some embodiments, compounds of the present disclosure are co-administered with mast cell inhibitors, including but not limited to, KIT inhibitors. In certain embodiments, the mast cell inhibitor to be co-administered is selected from cromolyn sodium (or sodium cromoglycate), brentuximab (ADCETRIS®), ibrutinib (IMBRUVICA®), omalizumab (XOLAIR®), antileukotrienes (e.g., montelukast (SINGULAIR®) or zileuton (ZYFLO® or ZYFLO CR®)), and KIT inhibitors (e.g., imatinib (GLEEVEC®), midostaurin (PKC412A), masitinib (MASIVET® or KINAVET®), avapritinib, DCC-2618, PLX9486).

[0074] The following examples are intended to illustrate, but not to limit the scope of the present disclosure in any way. [Example]

[0075] [Table 1] [Table 2] [Table 3]

[0076] Analysis conditions X-ray powder diffraction (XRPD) Powder X-ray diffraction was performed using a Rigaku MiniFlex 600 or Bruker D8 Advance equipped with a Lynxeye detector in reflectance mode (i.e., Bragg-Brentano geometry). Samples were prepared on Si zero-return wafers. Typical scans were from 4 to 30 degrees 2θ using a 0.05 degree step size over 5 minutes at 40 kV and 15 mA. High-resolution scans were from 4 to 40 degrees 2θ using a 0.05 degree step size over 30 minutes at 40 kV and 15 mA. Typical parameters for XRPD are listed below. [Table 4]

[0077] Thermogravimetric analysis and differential scanning calorimetry (TGA and DSC) Thermogravimetric analysis and differential scanning calorimetry were performed using a Mettler Toledo TGA / DSC 3+ The measurements were carried out simultaneously on the same sample using a DSC / TGA. The desired amount of sample is weighed directly into an airtight aluminum pan with small holes. Typical sample mass for the measurements is 5-10 mg. Typical temperature range is 30-300°C at a heating rate of 10°C per minute (total time of 27 minutes). The guard and purge gas is nitrogen (20-30 mL / min and 50-100 mL / min). Typical parameters for DSC / TGA are listed below: [Table 5]

[0078] Differential scanning calorimetry (DSC) 1-5 mg of material was weighed into an aluminum DSC pan and non-hermetically sealed with an aluminum lid. The sample pan was then loaded into a TA Instruments Q2000 (equipped with a condenser). Once a stable heat flow response was obtained at 30°C, the sample and reference material were heated to 300°C at a rate of 10°C / min, and the resulting heat flow response was monitored. Prior to analysis, the instrument was calibrated for temperature and heat flow using an indium reference standard. Sample analysis was performed with the aid of TA Universal Analysis 2000 software, referencing the temperature of the thermal event as the onset and peak temperatures measured according to the manufacturer's specifications. Method gas: N2 at 60.00 mL / min.

[0079] 1 H-nuclear magnetic resonance spectroscopy ( 1 H-NMR) Proton NMR was performed on a Bruker Avance 300 MHz spectrometer. Solids were dissolved in 0.75 mL of deuterated solvent in 4 mL vials and transferred to NMR tubes (Wilmad 5 mm thin-walled 8", 200 MHz, 506-PP-8). A typical run is usually 16 scans. Typical parameters for NMR are listed below: [Table 6]

[0080] Dynamic Vapor Sorption (DVS) Dynamic vapor sorption (DVS) was performed using a DVS Intrinsic 1. Samples were loaded into a sample pan and suspended from a microbalance. A typical sample mass for DVS measurements is 25 mg. Nitrogen gas bubbled through distilled water provides the desired relative humidity. Samples were held at each level for a minimum of 5 minutes and advanced to the next humidity level only if there was a change in weight of <0.002% between measurements (60-second interval) or 240 minutes had elapsed. A typical measurement involves the following steps: 1- Equilibrate at 50% RH 2-50%~2%. (50%, 40%, 30%, 20%, 10%, and 2%) a. Hold at each humidity level for a minimum of 5 minutes and a maximum of 60 minutes. The pass criterion is a change of less than 0.002%. 3-2%~95% (2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%) a. Hold at each humidity level for a minimum of 5 minutes and a maximum of 60 minutes. The pass criterion is a change of less than 0.002%. 4-95%~2% (95%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 2%) a. Hold at each humidity level for a minimum of 5 minutes and a maximum of 60 minutes. The pass criterion is a change of less than 0.002%. 5-2%~50% (2%, 10%, 20%, 30%, 40%, 50%) a. Hold at each humidity level for a minimum of 5 minutes and a maximum of 60 minutes. The pass criterion is a change of less than 0.002%.

[0081] High-performance liquid chromatography (HPLC) Agilent 1220 Infinity LC: High-performance liquid chromatography (HPLC) was performed using an Agilent 1220 Infinity LC. The flow rate range was 0.2–5.0 mL / min, the operating pressure range was 0–600 bar, the temperature range was 5°C above ambient to 60°C, and the wavelength range was 190–600 nm. [Table 7]

[0082] Karl Fischer titration Karl Fischer titration for water content determination was performed using a Mettler Toledo C20S coulometric KF titrator equipped with a diaphragm-equipped current generator cell and double platinum pin electrodes. Aquastar™ CombiCoulomat fritless reagents were used in both the anode and cathode compartments. Approximately 0.03-0.10 g of sample was dissolved in the anode compartment and titrated until the solution potential dropped below 100 mV. A 1 wt% Hydranal water standard was used for sample verification prior to analysis.

[0083] Microscopic observation Optical microscopy was performed using a Zeiss AxioScope A1 equipped with 2.5x, 10x, 20x, and 40x objectives and polarizers. Images were acquired with a built-in Axiocam 105 digital camera and processed using ZEN2 (blue edition) software provided by Zeiss.

[0084] Example 1: Combinatorial salt screening 1.1.Salt screening According to Marvin Sketch software predictions, the free base of Compound (I) has multiple pKa values. This compound has three basic nitrogens with theoretical pKa values ​​of 8.95, 3.57, and 2.86. The theoretical logP is 2.98.

[0085] Salt screening was performed using 13 different counterions. All counterions were tested at 1.1 equivalents. HCl was also tested using 2.2 equivalents of counterion, and sulfuric acid was tested using 0.5 equivalents of counterion. A list of counterions is provided in Table 1.

[0086] A stock solution of Compound (I) was prepared in absolute EtOH (20 wt %, density 0.8547 g / mL). Stock solutions of all counterions were also prepared in EtOH. Solid counterions were prepared at 0.02 g / mL, and liquid counterions were prepared at 10% by volume.

[0087] Salt formation was carried out in 2 mL vials at room temperature. 25 mg of Compound (I) (145.6 μL stock solution) and 1.1 equivalents of counterion were added to each vial. In the case of sulfuric acid, 0.55 and 1.1 equivalents of counterion were added. In the case of HCl, 1.1 and 2.2 equivalents of counterion were added. The solvent was evaporated overnight with stirring at 30° C., and then placed under vacuum at 50° C. for 4 hours to completely dry.

[0088] For screening, approximately 25 volumes of solvent (0.625 mL) were added to each vial. The three solvents chosen were EtOH, EtOAc, and IPA:water (9:1 by volume). Once the solvent was added, the mixture (or solution) was heated to 45° C., held for 1.5 hours, cooled to room temperature, and stirred overnight. Once a slurry was formed, the solid was filtered for XRPD analysis.

[0089] XRPD analysis was performed in three stages. Wet cake XRPD was performed for all samples (if a solid was observed). The specific solid was then left on the XRPD plate and dried under vacuum at 50°C for at least 3 hours. XRPD was then performed on the specific dried solid. The solid was then exposed to >90% relative humidity for 1 day, and XRPD was performed on the resulting solid. The humid environment was created by placing a beaker of saturated aqueous potassium sulfate solution in a sealed container. All XRPD patterns were compared to the XRPD patterns of the counterions and the known free molecule.

[0090] If no solids formed with the first three screening solvents (EtOH, EtOAc, IPA:water), the cap was opened and the solvent was evaporated at 30°C with stirring. The solids were evaporated to dryness by placing under vacuum at 50°C for 3-4 hours, and a second round of solvents was added (IPOAc, MBK, MtBE). If no solids formed with the second round of solvents, the solvent was evaporated to dryness again and DEE was added. [Table 8]

[0091] Crystalline solids were observed when screened with benzenesulfonic acid (BSA), benzoic acid, fumaric acid, HCl (1 and 2 equivalents), maleic acid, salicylic acid, and succinic acid. One unique XRPD pattern was observed with BSA, benzoic acid, HCl (2 equivalents), salicylic acid, and succinic acid. Multiple patterns were observed with HCl (1 equivalent) and fumaric acid. Two patterns were observed with maleic acid, both of which deliquesced upon exposure to humidity. Of the crystalline solids, solids resulting from screens with benzoic acid, fumaric acid, HCl (1 equivalent), salicylic acid, and succinic acid did not deliquesce upon exposure to humidity.

[0092] The crystalline salts were characterized and evaluated for potential utility based on melting point, crystallinity, stability upon dry and wet exposure, solubility in water, polymorphism, and counterion acceptability.

[0093] Given their acceptable physicochemical properties, the mono-HCl, succinate, and fumarate salts were selected for further development. For comparison, the free base was also included for further characterization.

[0094] The benzoate salt was not selected due to its low solubility in water and high mass loss upon melting. The salicylate salt was not selected due to its low solubility in water, high mass loss upon melting, and potential for polymorphism. The besylate salt, maleate salt, and bis-HCl salts were not selected due to their low crystallinity and instability in humid environments (deliquescence).

[0095] A sample of the free base showed an onset of melting at 116.19°C in DSC. The TGA thermogram showed a gradual mass loss of 0.16 wt% before melting and a step mass loss of 0.05 wt% upon melting. The solid was finely divided by microscopic observation. Karl Fischer titration of the free base showed a water content of 0.37 wt%.

[0096] The free base exhibited high solubility in many organic solvent systems (>200 mg / mL at room temperature in most organic solvents tested), high solubility in simulated fluids (0.08 mg / mL in water, approximately 17 mg / mL in fasted simulated gastric fluid, and approximately 7 mg / mL in fasted simulated intestinal fluid), acceptable melting (onset 116°C), and low residual solvent (<0.20 wt% by thermogravimetric analysis). Disadvantages of the free base are that it is polymorphic (four patterns were observed during limited screening), is physically unstable in humid environments (>90% relative humidity), becoming a sticky rubber within four days, and it becomes rubbery in water. Laboratory-scale results also indicated that the free base would be difficult to isolate as a crystalline solid on a manufacturing scale.

[0097] The mono-HCl salt exhibits high melting (onset 203°C), is a hydrate (channel hydrate), and has high crystallinity by X-ray powder diffraction. It has high solubility in water and simulated fluids (>30 mg / mL in water and fasted simulated gastric fluid, approximately 7 mg / mL in fasted simulated intestinal fluid). Disadvantages of the mono-HCl salt include sensitivity to added equivalents (as little as 1.3 molar equivalents of HCl will form the bis-HCl salt) and susceptibility to drying.

[0098] The succinate salt exhibited only one pattern during screening, being stable upon dry and wet exposure, less hygroscopic than the mono-HCl salt and the free base, high solubility in water and simulated fluids (>22 m / mL in all fluids), high melting (onset 173 °C), and acceptable mass loss upon melting (0.27 wt%) by thermogravimetric analysis.

[0099] The fumarate salt exhibited high solubility in water and simulated fluids (>15 m / mL in all fluids), and a hypothetical hydrate, designated Form B, was stable upon dry and wet exposure. Form A (anhydrous) exhibited high melting (onset 221°C).

[0100] A summary of the physicochemical properties of the free base and selected salts is provided in Table 2 below. [Table 9]

[0101] 1.2. Wet exposure of the free base The crystalline form of the free base was exposed to high humidity (RH>90%) overnight. The humid environment was created by placing a beaker of saturated aqueous potassium sulfate solution in a sealed container.

[0102] The solid remained in the same crystalline form after overnight humidity exposure, but lost some crystallinity. After XRPD analysis, the same sample was placed in a humid environment again. After one week, the sample was observed to have deliquesced on the XRPD plate. A second experiment was initiated under the same conditions. The solid became darker and sticky. An XRPD was taken of the sample on the sixth day. The peaks were less intense and a baseline shift was observed, indicating increased amorphous content.

[0103] Example 2: Preparation and Characterization of a Crystalline Form of 1.5:1 Compound (I) Sesquisuccinate Salt (Form A) 2.1. Preparation Method A: Compound (I) in the free base form was weighed into a 4 mL vial and 1.1 equivalents of succinic acid was added. EtOH (15 volumes) was then added at room temperature. The solid dissolved and remained in solution. The slurry was heated to 45°C and held with stirring for 2 hours, followed by natural cooling to room temperature. Since the solid still remained in solution, the solution was seeded with a sample succinate salt obtained from screening. The seed crystals were retained, and a white slurry quickly formed. The slurry was stirred overnight at room temperature. Before filtration, the slurry was a medium-thickness beige / off-white slurry. The slurry was filtered, washed twice with 2 volumes of EtOH, and then dried overnight under vacuum at 50°C. The purity by HPLC was 99.79 area %. The resulting solid was analyzed by XRPD (see Figure 1 and Table 3), TGA-DSC (Figure 2), and HPLC. 1 It was further characterized by 1 H NMR (Figure 3) and single crystal X-ray crystallography.

[0104] The initial mass loss by TGA was 0.19 wt %, followed by 0.30 wt % upon melting, see Figure 2. The DSC thermogram showed melting starting at 172.9°C, followed by decomposition of the sample above 200°C. [Table 10]

[0105] Method B: Salt formation was carried out using several different solvent conditions using Compound (I) free base and 1.6 equivalents of succinic acid. Approximately 30 mg of free base was weighed into a 2 mL vial and 10 volumes of solvent were added. In all solvents except MtBE, the free base dissolved at room temperature. Succinic acid was then added as a stock solution in EtOH, such that each solvent composition was approximately 40% EtOH by volume. The solution / slurry was stirred at room temperature until precipitation was observed, and the solid was then collected for XRPD analysis. A summary of the solids obtained from the salt formation experiments is provided in Table 4. [Table 11]

[0106] Method C: Amorphous Slurry Approximately 30 mg of Compound (I) sesquisuccinate was melted in a 2 mL vial to produce an amorphous glass-like solid. Solvent (450 μL) was added to each vial at room temperature along with a stir bar. In all cases, the glass-like solid adhered to the bottom of the vial, so a spatula was used to loosen the solid and ensure proper mixing. In many cases, a light brown slurry formed immediately after loosening the solid. When precipitation was observed, the slurry was sampled for XRPD analysis. The earliest time point for sampling was approximately 30 minutes after solvent addition. The results and observations from the amorphous slurry experiments are summarized in Table 5. [Table 12]

[0107] Method D: Amorphous Vapor Diffusion Approximately 10 mg of amorphous Compound (I) sesquisuccinate was placed in a 4 mL vial. Each 4 mL vial was then placed in a 20 mL vial containing 3 mL of solvent and sealed. The vials were kept at room temperature over the weekend before the solids were sampled for XRPD. Most solids changed appearance from a light beige glass (crushed from an amorphous foam) to a white / off-white powder. Amorphous solids exposed to a humid atmosphere (water was the solvent) became a yellow paste. A summary of the solids obtained from the amorphous vapor diffusion experiments is outlined in Table 6. [Table 13]

[0108] In polymorph screening for Compound (I) sesquisuccinate, over 10 crystallization or salt formation methods were used to generate solids, including experiments utilizing amorphous solids. Throughout the polymorph screening of Compound (I) sesquisuccinate, only crystalline Form A and amorphous solids were observed.

[0109] A sample of the amorphous solid (Compound (I) sesquisuccinate salt) was heated to 140° C. and then allowed to cool to room temperature. The resulting solid was crystalline Form A by XRPD.

[0110] The amorphous solid (Compound (I) sesquisuccinate salt) was exposed to 75% RH / 40°C for 1 week. The solid changed in appearance from a light beige-yellow solid to a hard yellow glass. XRPD of the solid showed crystalline Form A.

[0111] Form A was found to be crystalline with a melting onset of 173°C, was stable upon dry and wet exposure, and exhibited high solubility in water and simulated fluids (>22 mg / mL in all fluids.

[0112] Method E: The intermediate 6-(5-(4-ethoxy-1-isopropylpiperidin-4-yl)pyridin-2-yl)-4-(piperazin-1-yl)pyrrolo[1,2-b]pyridazine (3.5 kg, 7.8 mol) (disclosed in U.S. Pat. No. 10,233,186) was dissolved in isopropyl acetate (IPAc, 2.75 volumes) containing (R)-tetrahydrofuran-3-yl 1H-imidazole-1-carboxylate (1.2 equivalents). The mixture was heated and stirred until complete conversion. The reaction was quenched with aqueous ammonia (2 volumes), at which time additional IPAc (4 volumes) was added. Phase separation, water washing, and distillation afforded a solution of Compound (I) in anhydrous IPAc (approximately 3.5 kg in 3 volumes). Succinic acid in ethanol (1.45 equivalents in 10 volumes) was added while heating at 40-60°C. The mixture was heated to 75-85°C for 30 minutes. After cooling to 70-75°C, the solution was seeded with Compound (I) sesquisuccinate and cooled to 10°C over 8 hours. The suspension was isolated by filtration and washed with ethanol (2 x 3 volumes) to obtain Compound (I) sesquisuccinate Form A.

[0113] 2.2. Wet exposure The sesquisuccinate salt obtained in Example 2.1 was exposed to 75% relative humidity at 40°C for one week. The sample was placed in a 4 mL vial coated with Kimwipe® and then placed in a 20 mL vial containing 3-4 mL of saturated aqueous NaCl. The 20 mL vial was sealed and kept at 40°C. After one week, the solid was collected for XRPD analysis. Form A was physically stable by XRPD after one week under humid conditions.

[0114] DVS DVS showed a mass change of 0.59-0.60 wt% from 2 to 95% relative humidity at 25 °C (Figure 4). Of this mass change, 0.34-0.35 wt% occurred at relative humidity above 80%. XRPD after DVS measurement remained Form A (Figure 5).

[0115] DVS was also performed on the free base of Compound (I) (Figure 24), which showed a reversible mass change of 0.88-0.92 wt% at 2-95% relative humidity at 25°C, of ​​which 0.46-0.53 wt% of the mass change occurred at relative humidity above 70%.

[0116] 2.4.VT-XRPD and VH-XRPD Variable humidity experiments on Compound (I) Form A as the sesquisuccinate salt performed using XRPD indicate that no changes in crystalline structure are observed with humidity (see Figure 6).

[0117] Variable temperature experiments of Compound (I) Form A as the sesquisuccinate salt performed using XRPD indicate that no change in crystalline structure is observed below 160°C (i.e., the melting point) (see Figure 7).

[0118] Example 3: Preparation and Characterization of 1:1 Compound (I) Crystalline HCl Salt Monohydrate 3.1. Preparation Method A: First, 25-35 mg of Compound (I) free base was weighed into a 2 mL vial. Then, solvent was added to the vial (25 volumes or 5 volumes), followed by 0.9, 1.1, 1.5, 2.2, and 3.5 molar equivalents of HCl stock solution in IPA.

[0119] First, IPA:water (9:1 by volume) was added to a total of 25 volumes (including the volume of the HCl stock solution). Initially, all formed solutions. The 1.1 equivalent run showed precipitation overnight, while all others remained in solution. This was likely due to differences in solvent composition, so the remaining solutions (0.9, 1.5, 2.2, and 3.5 equivalents) were evaporated to dryness in air at 50°C, then under active vacuum at 50°C for approximately 3 hours. Additional runs at 1.1 equivalents were prepared in a similar manner by adding 5 volumes of IPA and the appropriate amount of HCl stock solution, followed by evaporation to dryness under low vacuum at 50°C, then under active vacuum at 50°C for approximately 3 hours.

[0120] To the evaporated solid, 25 volumes of EtOAc was added and stirred at room temperature overnight. In all cases, slurries formed. The color of the slurries varied from a white slurry (0.9 equiv.) to a bright / dark yellow slurry (1.5 equiv. and above).

[0121] The slurry was then filtered and the solid collected for XRPD analysis. The salt formed with 0.9 and 1.1 equivalents showed Form A (mono-HCl) by XRPD (FIG. 8). The use of 1.3 and 1.5 equivalents resulted in a mixture of Form A (mono-HCl) and Form B (bis-HCl). The use of 2.2 and 3.5 equivalents resulted in Form B (bis-HCl).

[0122] Compound (I) Form A as a crystalline HCl salt was confirmed by TGA-DSC (FIG. 9). 1 The sample was further characterized by H NMR (FIG. 10) and single-crystal X-ray crystallography (Table 7). The sample showed an onset of melting at 202.86°C in DSC (FIG. 9). The TGA thermogram showed a mass loss of 2.81 wt% before melting (associated with a very broad endotherm in DSC) and a step mass loss of 0.44 wt% upon melting. Karl Fischer titration of the HCl salt showed a water content of 3.17 wt%, supporting the fact that the resulting crystalline HCl salt is a monohydrate. The theoretical amount of water in a monohydrate of the HCl salt is 3.0 wt%. [Table 14] Method B: Compound (I) free base (4.3 kg) was dissolved in isopropyl acetate (5.5 volumes) and isopropyl alcohol (2.5 volumes). The mixture was heated to reflux and HCl (16.5% (w / w) in water, 0.95 equivalents) was charged over 0.75 hours. After refluxing for 1 hour, the solution was cooled to 20-25°C over 2 hours and held for 0.5 hours. The crystalline product was isolated by filtration and washed with a mixture of IPAc, IPA, and water to obtain Compound (I) Form A as a 1:1 crystalline HCl salt monohydrate.

[0123] 3.2. Wet exposure Form A as the HCl salt monohydrate was placed at 40°C / 75% relative humidity for one week. Approximately 10 mg of sample was placed in a 4 mL vial coated with Kimwipe®. The vial was then placed in a 20 mL sealed vial containing saturated aqueous sodium chloride. After one week of wet exposure, several small peak shifts were observed in the XRPD. Peak shifts were also observed in the XRPD patterns of some long-term slurries, indicating that Form A as the HCl salt monohydrate is a channel hydrate and that the peak shifts may be due to variations in water content.

[0124] The sampling day after 1 week of humid exposure (RH 75% and 40°C) was low humidity (RH < 25%) in the laboratory.

[0125] After standing in the sealed vial (ambient conditions) for 14 days, the solid was sampled again.

[0126] The solid was 1:1 Compound (I) crystalline HCl salt monohydrate (Form A) by XRPD.

[0127] 3.3. DVS Form A as HCl Salt Monohydrate DVS was performed on Form A as the HCl salt monohydrate (Figure 11), which showed a mass change of 1.30-1.43 wt% from 2-95% relative humidity at 25 °C, of ​​which 0.99-1.11 wt% of the mass change occurred below 20% relative humidity.

[0128] The sample was analyzed by XRPD after DVS measurement (Figure 12). All peaks of Form A as the HCl salt monohydrate were present in the XRPD, but extra peaks were observed.

[0129] 3.4.VT-XRPD and VH-XRPD A variable humidity experiment performed on Form A as the HCl salt monohydrate using XRPD is shown in Figure 13. The small shift in the peaks at approximately 10 and 13° 2θ towards higher angles (i.e., smaller d-spacing) observed at 0% RH is consistent with contraction of the crystal structure after water loss and is therefore consistent with a channel hydrate.

[0130] A variable temperature experiment performed using XRPD is shown in Figure 14. It confirms that the changes observed in the diffractogram above 100°C are essentially due to thermal expansion of the unit cell and simultaneous contraction due to the removal of water molecules. The crystalline structure does not appear to collapse and / or reorganize as in the presence of bound / crystallized water, again consistent with channel hydrates.

[0131] 3.5. Exposure to dry conditions and rehumidification The 1:1 Compound (I) crystalline HCl salt monohydrate obtained from Example 3.1 was exposed to various drying conditions and then analyzed by XRPD.

[0132] The conditions were as follows: (1) room temperature in a vial containing P2O5 at 50°C, (2) 60°C under vacuum, and (3) heated to 140°C in the DSC.

[0133] In all three cases, new XRPD patterns were observed, which were identified as the anhydrous form of the 1:1 Compound (I) HCl salt. The relative humidity in the laboratory was sufficient to rehydrate the samples on the bench. The DVS did not show significant mass loss until the relative humidity dropped below 20%.

[0134] At the 7 day mark, the HCl salt exposed to P2O5 at 50°C was sampled for XRPD. XRPD immediately after sampling showed Form D. The sample was left on the bench (22-23°C, 28% RH) for 2.25 hours and analyzed by XRPD. The solid had converted to Form A. After leaving it on the bench overnight, the same sample was analyzed by XRPD and remained Form A by XRPD. The XRPD pattern is shown in Figure 12.

[0135] Example 4: Preparation and characterization of anhydrous 1:1 Compound (I) crystalline HCl salt (Form D) 4.1. Preparation Anhydrous 1:1 Compound (I) crystalline HCl salt (Form D) was prepared by extended drying of Form A (monohydrate) in a sealed vial containing phosphorus pentoxide at 50° C. Specifically, 100 mg of Form A (monohydrate) obtained from Example 4.1 was placed in a dry environment for 4 days. A 4 mL open vial containing the sample was placed in a 20 mL sealed vial containing PO at 50° C. for 2 days before sampling. It was identified as a new crystalline form (Form D) by XRPD (FIG. 15).

[0136] It was observed that Form D converted to Form A upon exposure to ambient conditions (22° C., RH 35%) for 2.25 hours. Therefore, Form D was characterized with minimal exposure to ambient conditions.

[0137] The DSC thermogram of a Form D sample showed an endotherm onset at 202.4°C (Figure 16). The TGA of a Form D sample showed a total mass loss of 1.10 wt% (Figure 16). Form A (monohydrate) also shows a DSC endotherm with an onset at 202-203°C after dehydration.

[0138] Karl Fischer titration indicated a water content of 0.72% by weight for the Form D sample. The Form D sample exhibited a cubic morphology by microscopic observation. This morphology was not significantly different from the starting material (Form A). The purity of the Form D sample was 98.82 area percent by HPLC.

[0139] Form D converted to Form A (monohydrate) upon humidity exposure (RH>90% overnight and RH74% / 40° C. for 1 week).

[0140] Compound (I) Form D as a crystalline HCl salt 1 It was further characterized by 1 H NMR (Figure 17). [Table 15]

[0141] Example 5: Preparation and characterization of anhydrous 1:1 Compound (I) crystalline HCl salt (Form G) 5.1. Preparation Form G was observed during rapid cooling from IPA solution and from an amorphous slurry (low crystallinity) in EtOAc and MtBE. Form G was scaled up by rapid cooling in IPA. Approximately 200 mg of the raw HCl salt was weighed into a 20 mL vial and 60 volumes of IPA was added with stirring at 50°C. The solid dissolved and the solution was transferred to a beaker of ice water (0°C). The solution was seeded with a sample of Form G at 0°C. The seed crystals were retained but a thick slurry did not form. The sample was transferred to a -20°C freezer where the solid was allowed to settle over the weekend.

[0142] The resulting slurry appeared to be spattery. Filtration was very slow and the solids were somewhat sticky. The collected solids were quite wet due to insufficient filtration. The collected solids were dried under vacuum overnight at 50° C. The solids obtained from the scale-up were less crystalline than those observed during screening (FIG. 18).

[0143] The DSC thermogram of Form G shows two endotherms with onsets at 163.1°C and 189.6°C, followed by decomposition (Figure 19). The TGA thermogram shows a gradual initial mass loss of 2.62 wt% before the first endothermic event, followed by smaller mass losses (0.35% wet and 0.07% wet) during the endothermic events. The stand-alone DSC agrees well with the combined DSC-TGA data and also shows a broad endotherm between 80 and 130°C. A Form G sample was heated above the broad endotherm in the DSC and then cooled to room temperature. No changes were observed in the XRPD.

[0144] Karl Fischer titration indicated a water content of 2.79 wt% for the sample.

[0145] Microscopic examination of the Form G sample showed solid clumps and some irregular / fine particles. The purity of the Form G sample was 98.89 area percent by HPLC.

[0146] Form G samples partially converted to Form A overnight in a high humidity environment (RH>90%). Form G was stable (by XRPD) after 1 week of humidity exposure (RH75% / 40° C.). [Table 16]

[0147] Example 6: Preparation and Characterization of Anhydrous 1:1 Compound (I) Crystalline HCl Salt (Form I) 6.1. Preparation Form I was observed when salt formation experiments were performed in anhydrous solvent systems (MtBE:IPA and cyclohexane:IPA). Form I was scaled up by performing salt formation in cyclohexane:IPA. First, approximately 200 mg of Compound (I) free base was weighed into a 4 mL vial, and 15 volumes of cyclohexane were added to form a slurry. 1.1 molar equivalents of HCl were added as a 0.55 M solution in IPA over a 30 minute period. The HCl solution was dispensed dropwise into three aliquots. After the first aliquot, a yellow slurry formed, followed by rubberization. The rubberization remained upon addition of the final two aliquots. The vial was then heated to 45°C for 1 hour and seeded with Form I sample. After seeding, the sample was allowed to cool to room temperature. After seeding, a white solid was observed, and after cooling to room temperature, the sample was primarily a white slurry with some yellow rubber on the vial walls. The slurry was filtered and washed twice with 2 volumes of cyclohexane.

[0148] The DSC thermogram of the Form I sample shows an endotherm with an onset at 180.5° C. followed by a small endotherm with an onset at 198° C. (FIG. 22). The TGA thermogram shows a gradual mass loss of 2.34 wt % before melting and a mass loss of 0.26 wt % upon melting.

[0149] The stand-alone DSC is in good agreement with the combined DSC-TGA data and also shows an endotherm between 90 and 120°C. A Form I sample was heated to 150°C in the DSC and subsequently cooled to room temperature for XRPD analysis. No changes were observed in the XRPD pattern. All peaks were shifted slightly higher 2-theta, which could be due to sample misalignment.

[0150] Karl Fischer titration indicated a water content of 2.64 wt % for sample Form I.

[0151] Microscopic observation showed fines (needles) and aggregates. The purity of Form I was 99.51 area percent by HPLC.

[0152] The X-ray powder diffraction (XRPD) pattern of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form I) is shown in FIG.

[0153] Form I samples partially converted to Form A overnight in a high humidity environment (RH>90%). [Table 17]

[0154] Example 7: Preparation and Characterization of Anhydrous 1:1 Compound (I) Crystalline Fumarate Salt (Form A) 7.1. Preparation Form A as the fumarate salt was scaled up by weighing the free base of Compound (I) into a 4 mL vial and adding 1.1 equivalents of fumaric acid. EtOAc (15 volumes) was then added at room temperature. The solid mostly dissolved (the slurry became very thin), and then the solid precipitated to form a thick white slurry. An additional 5 volumes of EtOAc were added to improve mixing. The slurry was heated to 45°C and held with stirring for 2 hours, then allowed to cool to room temperature. The slurry was stirred overnight at room temperature. Before filtration, the slurry was a thick white slurry. The slurry was filtered, washed twice with 2 volumes of EtOAc, and then dried under vacuum at 50°C overnight. The resulting solid was analyzed by XRPD (see Figure 26 and Table 11), TGA-DSC (Figure 27), and HPLC. 1 It was further characterized by 1 H NMR (Figure 28). [Table 18]

[0155] Example 8: Preparation and characterization of anhydrous 1:1 Compound (I) crystalline fumarate salt (Form C) 8.1. Preparation To the free base of Compound (I) in a 4 mL vial, 1.1 equivalents of fumaric acid was added. IPAc (15 volumes) was then added at room temperature. The solid mostly dissolved (the slurry became very thin), and then the solid precipitated as an off-white slurry. The slurry was heated to 45°C and held with stirring for 2 hours, then allowed to cool to room temperature. The slurry was stirred overnight at room temperature. Before filtration, the slurry was a thick white slurry. The slurry was filtered, washed twice with 2 volumes of IPAc, and then dried under vacuum at 50°C overnight. The resulting solid was further slurried in EtOH and EtOAc and characterized by XRPD (see Figure 29 and Table 12). [Table 19]

[0156] Example 9: Preparation and Characterization of Anhydrous 1:1 Compound (I) Crystalline Fumarate Salt (Form D) 9.1. Preparation To the free base of Compound (I) in a 4 mL vial, 1.1 equivalents of fumaric acid were added. Then, IPAc (15 volumes) was added at room temperature. The solid mostly dissolved (the slurry became very thin), and then the solid precipitated as an off-white slurry. The slurry was heated to 45°C and kept under stirring for 2 hours, and then allowed to cool to room temperature. The slurry was stirred overnight at room temperature. Before filtration, the slurry was a thick white slurry. The slurry was filtered, washed twice with 2 volumes of IPAc, and then dried under vacuum at 50°C overnight. The resulting solid was further slurried in a mixture of IPA:water (95:5 volumes) and characterized by XRPD (see Figure 30 and Table 13). [Table 20]

Claims

1. The following structural formula: 【Chemistry 1】 The succinate salt of compound (I) represented by: wherein the molar ratio between compound (I) and succinic acid is 1:1.

5.

2. 2. The crystalline succinate salt of claim 1, wherein the crystalline succinate salt is a single crystal of Form A characterized by an X-ray powder diffraction pattern comprising peaks at 8.5°, 15.4°, and 21.3°±0.2 2θ.

3. 2. The crystalline succinate salt of claim 1, wherein the crystalline succinate salt is a single crystal of Form A characterized by an X-ray powder diffraction pattern containing at least three peaks selected from 4.3°, 8.5°, 14.0°, 15.4°, and 21.3°±0.2 2θ.

4. 3. The crystalline succinate salt of claim 2, wherein the crystalline succinate salt is a single crystal of Form A characterized by an X-ray powder diffraction pattern comprising peaks at 4.3°, 8.5°, 14.0°, 15.4°, and 21.3°±0.2 2θ.

5. 3. The crystalline succinate salt of claim 2, wherein the crystalline succinate salt is a single crystal of Form A characterized by an X-ray powder diffraction pattern comprising peaks at 4.3°, 6.7°, 8.5°, 12.8°, 14.0°, 15.4°, 17.0°, and 21.3°±0.2 2θ.

6. 3. The crystalline succinate salt of claim 2, wherein the crystalline succinate salt is a single crystal of Form A characterized by an X-ray powder diffraction pattern comprising peaks at 4.3°, 6.7°, 8.5°, 12.8°, 14.0°, 15.4°, 15.7°, 16.6°, 17.0°, 18.1°, 19.4°, 19.8°, 20.1°, 20.7°, 21.3°, 22.3°, 25.0°, 29.1°, and 34.4°±0.2 2θ.

7. 7. The crystalline succinate salt according to any one of claims 2 to 6, wherein the crystalline succinate salt is a single crystal of Form A characterized by a peak phase transition temperature of 177±2°C as measured by differential scanning calorimetry (DSC).

8. A pharmaceutical composition comprising the succinate salt according to claim 1 or a crystal of the succinate salt according to any one of claims 2 to 7, and a pharmaceutically acceptable carrier or diluent.

9. 10. A pharmaceutical composition for use in treating or ameliorating fibrodysplasia ossificans progressiva in a subject, the pharmaceutical composition comprising the succinate salt of claim 1 or a crystal of the succinate salt of any one of claims 2 to 7, wherein the subject has a mutation in the ALK2 gene that results in expression of an ALK2 enzyme having an amino acid modification selected from one or more of L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328W, G328E, G328R, G356D, and R375P.

10. 10. A pharmaceutical composition for treating or ameliorating diffuse intrinsic pontine glioma in a subject, the pharmaceutical composition comprising the succinate salt of claim 1 or a crystal of the succinate salt of any one of claims 2 to 7, wherein the subject has a mutation in the ALK2 gene that results in expression of an ALK2 enzyme having an amino acid modification selected from one or more of R206H, G328V, G328W, G328E, and G356D.

11. 10. A pharmaceutical composition for inhibiting abnormal ALK2 activity in a subject, the pharmaceutical composition comprising the succinate salt of claim 1 or a crystal of the succinate salt of any one of claims 2 to 7, wherein the abnormal ALK2 activity is caused by a mutation in the ALK2 gene that results in expression of an ALK2 enzyme having an amino acid modification selected from one or more of L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328V, G328W, G328E, G328R, G356D, and R375P.

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