Salts and crystalline forms of activin receptor-like kinase inhibitors

Abstract

Various salt forms of Compound (I) represented by the following structural formula and corresponding pharmaceutical compositions thereof are disclosed. Specific single crystalline forms of 1: 1.5 compound (I) succinate, 1: 1 compound (I) hydrochloride, and 1: 1 compound (I) fumarate are characterized by a variety of properties and physical measurements. Methods of making specific crystalline forms are also disclosed. The present disclosure also provides methods of treating or ameliorating progressive ossified fibrous dysplasia in a subject. # imgabs0 #

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 12, 2020, application number 202080057092.0, and invention name “Salts and crystal forms of activin receptor-like kinase inhibitors”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 885,977, filed on August 13, 2019. The entire contents of the foregoing application are incorporated herein by reference. Background Art

[0004] Activin receptor-like kinase-2 (ALK2) is encoded by the activin A receptor form 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). ALK2 inhibitors and mutant forms of ALK2 have the potential to treat a number of diseases, including fibrodysplasia ossificans progressiva (FOP); heterotopic ossification (HO), e.g., induced by major surgery, trauma (such as head or blast injuries), 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.

[0005] U.S. Patent No. 10,233,186 (the entire teaching of which is incorporated herein by reference) discloses potent, highly selective ALK2 inhibitors and mutant forms of ALK2. The structure of one inhibitor disclosed in U.S. Patent No. 10,233,186 is referred to herein as "Compound (I)" and is shown below:

[0006]

[0007] The successful development of pharmaceutically active agents, such as Compound (I), generally requires the identification of a solid form that possesses properties that enable isolation and purification immediately following synthesis, that can be manufactured on a large scale, and that can be stored for long periods of time with minimal water absorption, decomposition, or conversion to other solid forms, that is suitable for formulation, and that can be readily absorbed (e.g., soluble in water and gastric fluid) following administration to a subject. Summary of the invention

[0008] It has now been found that the free base of Compound (I) is physically unstable in a humid environment and tends to become sticky when exposed to water. As a result, Compound (I) has been found to be difficult to isolate when prepared on a production scale.

[0009] It has also been found that 1.5:1 succinate (i.e., sesquisuccinate), 1:1 hydrochloride (1:1 hydrochloride) and 1:1 fumarate (1:1 fumarate) 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 in artificial gastric juice (see Table 2), have a high melting point start and are suitable for large-scale synthesis. Another advantage of 1.5:1 succinate is that it exists in a single polymorph form and does not undergo thermal transition below its melting point, indicating a high degree of crystalline stability (see Example 2.4). The name "1:1" is the molar ratio between the acid (hydrochloric acid or fumaric acid) and compound (I); and the name "1.5:1" is 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 stoichiometries are possible. For example, compound (I) forms both a 1:1 hydrochloride and a 2:1 hydrochloride. The 1:1 hydrochloride salt of Compound (I) is referred to herein as "1:1 Compound (I) HCl"; and the 1.5:1 succinate salt is referred to herein as "1.5:1 Compound (I) sesquisuccinate".

[0010] By using thirteen different acids for salt screening, compound (I) HCl, compound (I) fumarate and compound (I) sesquisuccinate were identified (see Example 1). From this salt screening, only eight crystalline forms were identified. 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. From these eight salts, benzenesulfonate, maleate and 2:1 HCl were found to be unsuitable because of their low crystallinity and instability (deliquesce) under humid environments; benzoate was found to be unsuitable due to poor water solubility and high mass loss during melting; and salicylate was found to be unsuitable due to poor water solubility, high mass loss during melting and possible polymorphs.

[0011] 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) sesquisuccinate".

[0012] 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".

[0013] 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".

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising a 1.5:1 Compound (I) sesquisuccinate salt (or a 1:1 Compound (I) HCl salt or a 1:1 Compound (I) fumarate salt) and a pharmaceutically acceptable carrier or diluent.

[0015] The present disclosure provides a method for treating or ameliorating fibrodysplasia ossificans progressiva in a subject, comprising administering a pharmaceutically effective amount of a salt disclosed herein or a corresponding pharmaceutical composition to a subject in need thereof.

[0016] The present disclosure provides a method for treating or improving diffuse intrinsic pontine glioma in a subject, comprising administering a pharmaceutically effective amount of a salt disclosed herein or a corresponding pharmaceutical composition to a subject in need thereof.

[0017] The present disclosure also provides a method of inhibiting abnormal ALK2 activity 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.

[0018] The present disclosure also provides the use of the salt of the present disclosure or a pharmaceutical composition comprising the salt thereof in any of the methods of the present disclosure described above. In one embodiment, the salt of the present disclosure or a pharmaceutical composition comprising the salt thereof for use in any of the methods of the present disclosure described herein is provided. In another embodiment, the use of the salt of the present disclosure or a pharmaceutical composition comprising the salt thereof for use in the manufacture of a medicament for any of the methods of the disclosure is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the X-ray powder diffraction (XRPD) pattern of 1.5:1 Compound (I) sesquisuccinate salt.

[0020] Figure 2 Shown are the thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) thermograms of 1.5:1 Compound (I) sesquisuccinate salt.

[0021] Figure 3 The 1.5:1 sesquisuccinate of compound (I) is shown 1 H-NMR spectroscopy ( 1 H-NMR).

[0022] Figure 4 The DVS isotherm of the 1.5:1 Compound (I) sesquisuccinate salt is shown.

[0023] Figure 5 Shown are XRPD patterns of 1.5:1 Compound (I) sesquisuccinate salt (Form A) before (lower) and after (upper) DVS measurement.

[0024] Figure 6 Shown are variable humidity XRPD patterns of 1.5:1 Compound (I) sesquisuccinate salt (Form A). From bottom to top, each XRPD diffraction pattern was collected in situ at variable humidity stages of 40% RH, 60% RH, 90% RH, 40% RH, 0% RH and back to 40% RH.

[0025] Figure 7 Variable temperature XRPD patterns of 1.5:1 Compound (I) sesquisuccinate salt (Form A) are shown. From bottom to top, each XRPD diffraction pattern was collected in situ at variable temperature stages of 40°C, 60°C, 80°C, 100°C, 120°C, 140°C, 160°C and back to 25°C.

[0026] Figure 8 Shown is the XRPD pattern of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A).

[0027] Fig. 9 Shown are TGA and DSC thermograms of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A).

[0028] Fig.10 The 1:1 Compound (I) crystalline HCl salt monohydrate (Form A) is shown 1 H-NMR.

[0029] Fig.11 Shown is the DVS isotherm of the 1:1 Compound (I) crystalline HCl salt monohydrate (Form A).

[0030] Fig.12 XRPD patterns of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A) before (lower) and after (upper) DVS measurements are shown. Additional peaks observed after DVS are indicated by arrows.

[0031] Fig.13 Shown are variable humidity XRPD patterns of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A). From bottom to top, each XRPD diffraction pattern was collected in situ at variable humidity stages (40% RH, 90% RH, 0% RH and back to 40% RH) under ambient conditions.

[0032] Fig.14Shown are variable temperature XRPD patterns of 1:1 Compound (I) crystalline HCl salt monohydrate (Form A). From bottom to top, each XRPD diffraction pattern was collected in situ at variable temperature stages of 50°C, 100°C, 160°C and back to 25°C under ambient conditions.

[0033] Fig.15 Shown are XRPD patterns of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form D) observed during initial screening (lower) and scale-up (upper).

[0034] Fig.16 Shown are the TGA and DSC thermograms of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form D).

[0035] Fig.17 The anhydrous 1:1 crystalline HCl salt of Compound (I) (Form D) is shown 1 H-NMR.

[0036] Fig.18 Shown are XRPD patterns of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form G) observed during screening (lower), scale-up (wet) (middle), and drying (upper).

[0037] Fig.19 Shown are TGA and DSC thermograms of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form G).

[0038] Fig. 20 The anhydrous 1:1 Compound (I) crystalline HCl salt (Form G) is shown 1 H-NMR.

[0039] Fig.21 Shown are XRPD patterns of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form I) observed during initial screening (lower) and scale-up (upper).

[0040] Fig. 22 Shown are TGA and DSC thermograms of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form I).

[0041] Fig.23 The anhydrous 1:1 crystalline HCl salt of Compound (I) (Form I) is shown 1 H-NMR.

[0042] Fig.24 The DVS isotherm of the free base of Compound (I) is shown.

[0043] Fig.25 Shown is the XRPD pattern of the 2:1 Compound (I) crystalline HCl salt (Form B).

[0044] Fig.26 Shown are XRPD patterns of the anhydrous 1:1 crystalline fumarate salt of Compound (I) (Form A) observed during initial screening (lower) and scale-up (upper).

[0045] Fig. 27 Shown are TGA and DSC thermograms of anhydrous 1:1 Compound (I) crystalline fumarate salt (Form A).

[0046] Fig.28 The anhydrous 1:1 crystalline fumarate salt of Compound (I) (Form A) is shown. 1 H-NMR.

[0047] Fig.29 Shown is the XRPD pattern of a 1:1 crystalline fumarate salt of Compound (I) (Form C).

[0048] Fig.30 Shown is the XRPD pattern of a 1:1 crystalline fumarate salt of Compound (I) (Form D). DETAILED DESCRIPTION

[0049] The present disclosure relates to a novel succinate salt (ie, 1:1.5 sesquisuccinate salt) of compound (I), a novel hydrochloride salt (ie, 1:1 hydrochloride salt) and a novel fumarate salt (ie, 1:1 fumarate salt) of compound (I), and polymorphs of each of the above.

[0050] "Hydrated form" refers to a solid or crystalline form of compound (I) in the form of a free base or salt, wherein 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. "Non-hydrated 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 (e.g., less than 10% by weight by Karl Fischer analysis) in solid form. The new solid forms disclosed in the present disclosure include hydrated forms and non-hydrated forms.

[0051] As used herein, "crystalline" refers to a solid having a crystal structure in which the individual molecules have a highly uniform and regular three-dimensional configuration.

[0052] The disclosed crystalline Compound (I) salt may be a single crystal form or a mixture of crystals of different single crystal forms. Single crystal form means that Compound (I) is a single crystal or a plurality of crystals, wherein each crystal has the same crystal form.

[0053] For the crystalline forms of Compound (I) disclosed herein, at least a particular weight percentage of the 1.5:1 salt of Compound (I) is in single crystal form. Particular weight percentages include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 70%-80%, 80%-90%, 90%-100% of the salt of Compound (I) is in single crystal form. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed by the present disclosure.

[0054] When a crystalline Compound (I) salt is defined as a specified percentage of one particular crystalline form of a Compound (I) salt, the remainder is made up of amorphous forms and / or crystalline forms other than the specified one or more specific forms. Examples of single crystalline forms include 1.5:1 Compound (I) sesquisuccinate (Form A), 1:1 Compound (I) HCl salt (Form A, Form D, Form G and Form I) and Compound (I) 1:1 fumarate (Form A, Form C and Form D), which are characterized by having one or more of the properties discussed herein.

[0055] Compound (I) has a chiral center. Compound (I) as salts and polymorphs disclosed herein is at least 80%, 90%, 99% or 99.9% pure by weight relative to other stereoisomers, i.e., the weight of a stereoisomer to the weight of all stereoisomers.

[0056] The crystalline Compound (I) salts disclosed herein exhibit a strong, unique XRPD pattern with sharp peaks corresponding to angular peak positions in 2θ and a flat baseline, indicative of a highly crystalline material (see, e.g., Figure 1 ). The XRPD patterns disclosed in this application are from a copper radiation source (Cu Kα1; )get.

[0057] Characterization of the 1.5:1 Compound (I) sesquisuccinate crystalline form

[0058] In one embodiment, the 1.5:1 Compound (I) sesquisuccinate salt is in a single crystalline Form A, characterized by an X-ray powder diffraction pattern comprising peaks at 8.5°, 15.4°, and 21.3°±0.2 in 2θ. In another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising at least three peaks (or four peaks) selected from 4.3°, 8.5°, 14.0°, 15.4°, and 21.3°±0.2 in 2θ. In another embodiment, Form A is characterized by an X-ray powder diffraction pattern comprising peaks at 4.3°, 8.5°, 14.0°, 15.4°, and 21.3°±0.2 in 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, in 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, in 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern substantially similar to Figure 1 .

[0059] It is well known in the art of crystallography that for any given crystalline form, the angular peak position may vary slightly due to factors such as temperature changes, sample displacement, and the presence or absence of an internal standard. In the present disclosure, the change in angular peak position is ±0.2 in 2θ. In addition, the relative peak intensity of 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 hindering the unambiguous identification of the crystalline form.

[0060] In another embodiment, the 1.5:1 Compound (I) sesquisuccinate salt Form A is characterized by a peak phase transition temperature of 177 ± 2 °C by differential scanning calorimetry (DSC).

[0061] Characterization of 1:1 Compound (I) hydrochloride crystalline form

[0062] In one embodiment, Form A is characterized in that the X-ray powder diffraction pattern comprises at least three peaks (or four peaks) at 12.9°, 17.0°, 19.0°, 21.1°, and 22.8°±0.2, in terms of 2θ. In another embodiment, the 1:1 Compound (I) hydrochloride salt is a single crystalline Form A, characterized in that the X-ray powder diffraction pattern comprises peaks at 12.9°, 17.0°, 19.0°, 21.1°, and 22.8°±0.2, in terms of 2θ. In another embodiment, Form A is characterized in that the X-ray powder diffraction pattern comprises peaks at 12.9°, 13.8°, 15.1°, 17.0°, 19.0°, 19.6°, 21.1°, and 22.8°±0.2, in terms 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, in 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern substantially similar to Figure 8 .

[0063] In another embodiment, the 1:1 Compound (I) hydrochloride salt Form A is characterized by a peak phase transition temperature of 207 ± 2 °C by differential scanning calorimetry (DSC).

[0064] In one embodiment, the 1:1 compound (I) hydrochloride salt is a single crystalline form, Form D, characterized in that the X-ray powder diffraction pattern comprises at least three peaks (or four peaks) selected from 10.8°, 16.9°, 18.8°, 22.1°, and 24.7°±0.2 in 2θ. In another embodiment, the 1:1 compound (I) hydrochloride salt is a single crystalline form, Form D, characterized in that the X-ray powder diffraction pattern comprises peaks at 10.8°, 16.9°, 18.8°, 22.1°, and 24.7°±0.2 in 2θ. In another embodiment, Form D is characterized in that the X-ray powder diffraction pattern comprises peaks at 10.8°, 13.3°, 16.9°, 18.8°, 22.1°, and 24.7°±0.2 in 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 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern substantially similar to Fig.15 .

[0065] In another embodiment, the 1:1 Compound (I) hydrochloride salt Form D is characterized by a peak phase transition temperature of 207 ± 2 °C by differential scanning calorimetry (DSC).

[0066] In one embodiment, the 1:1 compound (I) hydrochloride salt is a single crystalline form G, characterized in that the X-ray powder diffraction pattern comprises at least three peaks (or four peaks) at 10.2°, 12.8°, 16.7°, 17.4°, 18.4°, and 22.5°±0.2 in terms of 2θ. In another embodiment, the 1:1 compound (I) hydrochloride salt is a single crystalline form G, characterized in that the X-ray powder diffraction pattern comprises peaks at 10.2°, 12.8°, 16.7°, 17.4°, 18.4°, and 22.5°±0.2 in terms of 2θ. In another embodiment, Form G is characterized in that the X-ray powder diffraction pattern comprises peaks at 10.2°, 12.8°, 16.7°, 17.4°, 18.4°, 21.3°, 22.0°, 22.5°, and 24.3°±0.2 in terms 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 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern substantially similar to Fig.18 .

[0067] In another embodiment, the 1:1 Compound (I) hydrochloride salt Form G is characterized by differential scanning calorimetry (DSC) peak phase transition temperatures of 175±4°C and 197±4°C.

[0068] In one embodiment, the 1:1 compound (I) hydrochloride salt is a single crystalline form, Form I, characterized in that the X-ray powder diffraction pattern comprises 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 terms of 2θ. In another embodiment, the 1:1 compound (I) hydrochloride salt is a single crystalline form, Form I, characterized in that the X-ray powder diffraction pattern comprises peaks at 5.4°, 8.2°, 16.3°, 16.5°, 18.4°, and 21.5°±0.2 in terms of 2θ. In another embodiment, Form I is characterized in that the X-ray powder diffraction pattern comprises peaks at 5.4°, 8.2°, 13.1°, 16.3°, 16.5°, 18.4°, and 21.5°±0.2 in terms 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 in 2θ. In yet another embodiment, Form I is characterized by an X-ray powder diffraction pattern substantially similar to Fig.21 .

[0069] In another embodiment, the 1:1 Compound (I) hydrochloride salt Form I is characterized by peak phase transition temperatures of 187±4°C and 200±4°C by differential scanning calorimetry (DSC).

[0070] Characterization of 2:1 Compound (I) hydrochloride crystalline form

[0071] In one embodiment, the 2:1 compound (I) hydrochloride salt is a single crystalline form B, characterized in that the X-ray powder diffraction pattern comprises at least three peaks (or four peaks) selected from 10.6°, 17.0°, 18.3°, 20.9°, and 21.1°±0.2 in 2θ. In one embodiment, the 2:1 compound (I) hydrochloride salt is a single crystalline form B, characterized in that the X-ray powder diffraction pattern comprises peaks at 10.6°, 17.0°, 18.3°, 20.9°, and 21.1°±0.2 in 2θ. In another embodiment, the 2:1 compound (I) hydrochloride salt form B is characterized in that the X-ray powder diffraction pattern comprises peaks at 10.6°, 12.7°, 15.8°, 17.0°, 18.3°, 18.9°, 20.9°, 21.1°, and 22.0°±0.2 in 2θ. In yet another embodiment, the 2:1 Compound (I) hydrochloride salt Form B 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 in 2θ. In yet another embodiment, the 2:1 Compound (I) hydrochloride salt Form B is characterized by an X-ray powder diffraction pattern substantially similar to Fig.25 .

[0072] Characterization of 1:1 Compound (I) fumarate salt crystalline form

[0073] In one embodiment, the 1:1 compound (I) fumarate salt is a single crystalline form A, characterized in that the X-ray powder diffraction pattern comprises 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 a single crystalline form A, characterized in that the X-ray powder diffraction pattern comprises peaks at 5.7°, 15.3°, 16.9°, 22.4°, and 23.0°±0.2 in 2θ. In another embodiment, Form A is characterized in that the X-ray powder diffraction pattern comprises 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, in 2θ. In yet another embodiment, Form A is characterized by an X-ray powder diffraction pattern substantially similar to Fig.26 .

[0074] In another embodiment, the 1:1 Compound (I) fumarate salt Form A is characterized by a peak phase transition temperature of 224 ± 2 °C by differential scanning calorimetry (DSC).

[0075] In one embodiment, the 1:1 compound (I) fumarate salt is a single crystalline form C, characterized in that the X-ray powder diffraction pattern comprises 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 a single crystalline form C, characterized in that the X-ray powder diffraction pattern comprises peaks at 6.3°, 9.0°, 13.5°, 18.9°, and 22.5°±0.2 in 2θ. In another embodiment, Form C is characterized in that the X-ray powder diffraction pattern comprises 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 in 2θ. In yet another embodiment, Form C is characterized by an X-ray powder diffraction pattern substantially similar to Fig.29 .

[0076] In one embodiment, the 1:1 compound (I) fumarate salt is a single crystalline form D, characterized in that the X-ray powder diffraction pattern comprises at least three peaks (or four peaks) selected from 4.6°, 11.0°, 18.5°, 20.5°, and 21.0°±0.2 in 2θ. In one embodiment, the 1:1 compound (I) fumarate salt is a single crystalline form D, characterized in that the X-ray powder diffraction pattern comprises peaks at 4.6°, 11.0°, 18.5°, 20.5°, and 21.0°±0.2 in 2θ. In another embodiment, Form D is characterized in that the X-ray powder diffraction pattern comprises peaks at 4.6°, 11.0°, 15.1°, 18.5°, 19.4°, 20.5°, 21.0°, and 25.0°±0.2 in 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, in 2θ. In yet another embodiment, Form D is characterized by an X-ray powder diffraction pattern substantially similar to Fig.30 .

[0077] In one embodiment, the 1:1 Compound (I) fumarate salt is in a single crystalline form, Form C, mixed with Form D, wherein Form C is characterized in that the X-ray powder diffraction pattern comprises at least three peaks (or four peaks) at 6.3°, 9.0°, 13.5°, 18.9°, and 22.5°±0.2, in terms of 2θ; and Form D is characterized in that the X-ray powder diffraction pattern comprises at least three peaks (or four peaks) at 4.6°, 11.0°, 18.5°, 20.5°, and 21.0°±0.2, in terms of 2θ.

[0078] In one embodiment, the 1:1 Compound (I) fumarate salt is in a single crystalline form, Form C, mixed with Form D, wherein Form C is characterized in that an X-ray powder diffraction pattern comprises peaks at 6.3°, 9.0°, 13.5°, 18.9°, and 22.5°±0.2, in 2θ; and Form D is characterized in that an X-ray powder diffraction pattern comprises peaks at 4.6°, 11.0°, 18.5°, 20.5°, and 21.0°±0.2, in 2θ.

[0079] In one embodiment, the 1:1 Compound (I) fumarate salt is in a single crystalline form, Form C, mixed 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 terms 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 terms of 2θ.

[0080] In one embodiment, the 1:1 fumarate salt of Compound (I) is in a single crystalline form, Form C, mixed with Form D, wherein Form C is characterized by an X-ray powder diffraction pattern 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; and Form D is characterized by an X-ray powder diffraction pattern comprising peaks, in terms of 2θ, 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.

[0081] Pharmaceutical composition

[0082] 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 carriers or diluents. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting any subject composition or its components, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense that it is compatible with the subject composition and its components and does not harm the subject. Some examples of materials that can be used 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, Safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, 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) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0083] The compositions of the present disclosure can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implantable kits. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In one embodiment, the compositions of the present disclosure are administered orally, intraperitoneally, or intravenously. The sterile injectable form of the compositions of the present disclosure may be an aqueous or oily suspension. These suspensions may be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenteral acceptable diluents or solvents, for example, in the form of solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be employed are particularly water, Ringer's solution, and isotonic sodium chloride solution. In addition, conventionally sterile, non-volatile oils are used as solvents or suspension media.

[0084] For this purpose, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives can be used to prepare injectables, and natural pharmaceutically acceptable oils, such as olive oil or castor oil, particularly its polyoxyethylated form, can also be used to prepare injectables. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants, which are generally used to prepare pharmaceutically acceptable dosage forms, including emulsions and suspensions. For formulation purposes, other conventional surfactants can also be used, such as Tween, Spans and other emulsifiers or bioavailability enhancers, which are generally used to manufacture pharmaceutically acceptable solids, liquids or other dosage forms.

[0085] The pharmaceutically acceptable compositions of the present disclosure can be orally administered in any oral acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, conventional carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in capsule form, available diluents include lactose and dry corn starch. When aqueous suspensions are required for oral use, active ingredients are combined with emulsifiers and suspending agents. If desired, certain sweeteners, flavorings or coloring agents may also be added.

[0086] Alternatively, the pharmaceutically acceptable compositions of the present disclosure can be administered in the form of suppositories for rectal administration. They can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0087] Pharmaceutically acceptable compositions of the present disclosure may also be applied topically, particularly when the therapeutic target includes an area or organ that is easily accessible to topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application of the lower intestinal tract may be carried out in the form of a rectal suppository formulation (see above) or a suitable enema formulation. Topical transdermal patches may also be used.

[0088] For topical application, the pharmaceutically acceptable composition can be formulated into a suitable ointment containing an active component suspended or dissolved in one or more carriers. The carrier for topical application of the disclosed compound includes but is not limited to mineral oil, liquid paraffin, white paraffin, propylene glycol, polyethylene oxide, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutically acceptable composition can be formulated into a suitable lotion or cream containing an active component 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 ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0089] The 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 improve bioavailability), fluorocarbons and / or other conventional solubilizing or dispersing agents.

[0090] The amount of a compound of the disclosure that may be combined with a carrier to produce a composition in a single dosage form will vary depending on the host treated, the particular mode of administration, and other factors as determined by the person administering the single dosage form.

[0091] dose

[0092] The toxicity and therapeutic efficacy of the salts of Compound (I) or its crystalline forms described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. 50 The dose that is lethal to 50% of the population. 50 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 a large therapeutic index are preferred. Although salts of compound (I) or crystalline forms thereof described herein that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such salts or crystalline forms to the affected tissue site in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0093] The data obtained from the 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 within a range of circulating concentrations that include the ED with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any salt of Compound (I) or crystalline form thereof described herein, the therapeutically effective dose can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC as determined in cell culture.50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0094] 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 employed, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the attending physician, as well as the severity of the specific disease being treated. The amount of the salt or crystalline form of Compound (I) of the present disclosure in the composition will also depend on the specific compound in the composition.

[0095] Treatment

[0096] A "subject" is a mammal, preferably a human, but may also be an animal in need of veterinary treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0097] The "treatment" regimen of 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, for a given treatment, the compound may be administered to the subject from about once a week to once a day. 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 disclosed compound, or a combination thereof. It should also be understood that the effective dose of the compound for treatment or prevention may increase or decrease during a specific treatment or prevention regimen. The change in dosage may be obtained and become apparent by standard diagnostic assays known in the art. In some cases, long-term administration may be required.

[0098] Mutations in ALK2 can cause abnormal activity of the kinase and are associated with various diseases. Compound (I), its salt and crystalline form disclosed herein inhibit mutant ALK2 genes, such as mutant ALK2 genes that cause expression of ALK2 enzymes with amino acid modifications. In another aspect, compound (I), its salt and crystalline form disclosed herein inhibit both wild-type (WT) ALK2 protein and mutant forms of ALK2 protein. For the purpose of this disclosure, the sequence information of ALK2 is found under ACVR1 activin A receptor type 1 [Homo sapiens (human)] on the National Center for Biological Information (NCBI) webpage (https: / / www.ncbi.nlm.nih.gov / ); Entrez gene ID (NCBI): 90. Also known as: FOP; ALK2; SKR1; TSRI; ACTRI; ACVR1A; ACVRLK2; the sequence information is incorporated herein.

[0099] In one embodiment, the present disclosure provides a method for inhibiting abnormal ALK2 activity in a subject, the method comprising the following steps: administering a pharmaceutically effective amount of a compound (I) or salt, a crystalline form or a pharmaceutical composition as described herein to a subject in need thereof. In one embodiment, abnormal ALK2 activity is caused by a mutation in the ALK2 gene, which results in the expression of an ALK2 enzyme having an amino acid modification selected from one or more of the following: L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328V, G328W, G328E, G328R, G356D and R375P. In one embodiment, the ALK2 enzyme has an amino acid modification R206H.

[0100] Due to the activity against ALK2, the compound (I) or salt, crystalline form or pharmaceutical composition described herein can be used to treat subjects suffering from a condition associated with abnormal ALK2 activity. In one embodiment, the condition associated with abnormal ALK2 activity is progressive fibrous dysplasia ossificans. The diagnosis of FOP is based on the presence of congenital malformations of the big toe (hallus valgus) and the formation of fibrous nodules in the soft tissue. The nodules may or may not transform into ectopic bone. These soft tissue lesions often first appear on the head, neck or back. About 97% of FOP subjects have the same c.617G>A; R206H mutation in the ACVR1 (ALK2) gene. The University of Pennsylvania has a genetic test (Kaplan et al., Pediatrics 2008, 121(5): e1295-e1300).

[0101] Other common congenital anomalies include deformed thumbs, short and narrow femurs, osteochondromas of the tibia, and fused facet joints of the cervical spine. Fused facet joints of the neck often cause young children to step on their hips instead of crawling. FOP is often misdiagnosed (about 80%; cancer or fibromatosis) and subjects often undergo inappropriate diagnostic procedures such as biopsies that worsen the disease and cause permanent disability.

[0102] In one embodiment, the present disclosure provides a method for treating or improving fibrodysplasia ossificans progressiva in a subject, comprising administering a pharmaceutically effective amount of compound (I) or salt, crystalline form or pharmaceutical composition described herein to the subject in need thereof.

[0103] In one embodiment, the disorder associated with abnormal ALK2 activity is fibrodysplasia ossificans progressiva (FOP), and the subject has a mutation in the ALK2 gene that results in the 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. In one aspect of this embodiment, the ALK2 enzyme has the amino acid modification R206H.

[0104] The present disclosure includes methods for identifying and / or diagnosing subjects, which are treated with compounds (I) or salts, crystalline forms or pharmaceutical compositions described herein. In one embodiment, the present disclosure provides a method for detecting a condition associated with abnormal ALK2 activity in a subject, such as FOB, wherein the method comprises: a. obtaining a sample, such as plasma, from a subject, such as a human subject; and b. detecting the presence or absence of one or more mutations in the ALK2 gene as described herein 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 a subject; b. detecting the presence or absence of one or more mutations in the ALK2 gene as described herein in the sample using the detection method described herein; and c. when the presence of one or more mutations is detected, diagnosing the subject with the condition. 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 blot and Southern blot. 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 the presence of one or more mutations in the ALK2 gene as described herein in the sample; diagnosing the subject as having the condition when one or more mutations in the sample are detected; and administering an effective amount of compound (I), or salt, crystalline form, or pharmaceutical composition described herein to the diagnosed subject. In one embodiment, the present disclosure provides a method for treating a condition associated with abnormal ALK2 activity in a subject, the method comprising a. determining whether, having determined whether, or receiving the following information: the subject has one or more mutations in the ALK2 gene as described herein; b. the subject is identified as responsive to one or more compounds or pharmaceutical compositions described herein; and c. administering an effective amount of compound (I), or salt, crystalline form, or pharmaceutical composition to the subject.

[0105] In one embodiment, the disorder associated with abnormal ALK2 activity is a brain tumor, for example, a glioma. In one embodiment, the glioma is a diffuse intrinsic pontine glioma (DIPG). In one embodiment, the present disclosure provides a method for treating or improving diffuse intrinsic pontine glioma in a subject, comprising administering a pharmaceutically effective amount of a compound (I), or a salt, a crystalline form, or a pharmaceutical composition described herein to a subject in need thereof.

[0106] In one embodiment, the disorder associated with aberrant ALK2 activity is diffuse intrinsic pontine glioma, and the subject has a mutation in the ALK2 gene that results in the 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.

[0107] In one embodiment, the disorder associated with aberrant ALK2 activity is anemia associated with inflammation, cancer, or a chronic disease.

[0108] In one embodiment, the disorder associated with aberrant ALK2 activity is trauma- or surgery-induced heterotopic ossification.

[0109] In one embodiment, the compounds of the present disclosure are co-administered with a second therapeutic agent useful for treating the disease to be treated, e.g., FOP (either as part of a combined dosage form or as a separate dosage form administered prior to, sequentially, or after administration). In one aspect of this embodiment, the compounds of the present disclosure are co-administered with a steroid (e.g., prednisone) or other anti-allergic agent such as omalizumab.

[0110] In one embodiment, the compounds of the present disclosure are co-administered with a RAR-γ agonist or an antibody to activin for the treatment of the disease to be treated, e.g., FOP. In one embodiment, the RAR-γ agonist to be co-administered is palovarotene. In one embodiment, the antibody to activin to be co-administered is REGN2477.

[0111] In one embodiment, the compounds of the present disclosure are co-administered with a therapy targeting mast cells that can be used to treat FOP. In one embodiment, the compounds of the present disclosure are co-administered with a mast cell inhibitor, including but not limited to a KIT inhibitor. In one embodiment, the mast cell inhibitor to be co-administered is selected from cromolyn sodium (or sodium cromoglicate); Ibrutinib Omalizumab Antileukotriene agents (eg, montelukast or Qiliutong ( or ZYFLO )); and KIT inhibitors (eg, imatinib , midostaurin (PKC412A), masitinib ( or ), avapritinib, DCC-2618, PLX9486).

[0112] The present invention specifically relates to the following embodiments:

[0113] Embodiment 1. A succinate salt of compound (I), which is represented by the following structural formula:

[0114]

[0115] The molar ratio of compound (I) to succinic acid is 1:1.5.

[0116] Embodiment 2. The succinate salt of embodiment 1, wherein the succinate salt is crystalline.

[0117] Embodiment 3. The succinate salt of embodiment 1, wherein the succinate salt is in a single crystalline form.

[0118] Embodiment 4. The succinate salt of any one of embodiments 1-3, wherein the succinate salt is unsolvated.

[0119] Embodiment 5. The succinate salt of any one of Embodiments 1-4, wherein the succinate salt is in a single crystalline form, Form A, characterized by an X-ray powder diffraction pattern comprising peaks at 8.5°, 15.4°, and 21.3°±0.2 in 2θ.

[0120] Embodiment 6. The succinate salt of any one of embodiments 1-4, wherein the succinate salt is in a single crystalline form, Form A, characterized in that the X-ray powder diffraction pattern comprises at least three peaks at positions selected from 4.3°, 8.5°, 14.0°, 15.4°, and 21.3°±0.2 in 2θ.

[0121] Embodiment 7. The succinate salt of any one of Embodiments 1-4, wherein the succinate salt is in a single crystalline form, Form A, characterized in that the X-ray powder diffraction pattern comprises peaks at 4.3°, 8.5°, 14.0°, 15.4°, and 21.3°±0.2 in 2θ.

[0122] Embodiment 8. The succinate salt of any one of Embodiments 1-4, wherein the succinate salt is in a single crystalline form, Form A, characterized in that the X-ray powder diffraction pattern comprises peaks at 4.3°, 6.7°, 8.5°, 12.8°, 14.0°, 15.4°, 17.0°, and 21.3°±0.2 in 2θ.

[0123] Embodiment 9. The succinate salt of any one of Embodiments 1-4, wherein the succinate salt is in a single crystalline form, 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, in 2θ.

[0124] Embodiment 10. The succinate salt of any one of embodiments 2-9, wherein the succinate salt is in a single crystalline form, Form A, characterized by a peak phase transition temperature of 177±2°C by differential scanning calorimetry (DSC).

[0125] Embodiment 11. The succinate salt of any one of embodiments 5-10, wherein at least 90% by weight of the succinate salt is in single crystalline Form A.

[0126] Embodiment 12. A hydrochloride salt of compound (I), which is represented by the following structural formula:

[0127]

[0128] The molar ratio between compound (I) and hydrochloric acid is 1:1.

[0129] Embodiment 13. The hydrochloride salt of Embodiment 12, wherein the salt is crystalline.

[0130] Embodiment 14. The hydrochloride salt of Embodiment 12, wherein the salt is in single crystal form.

[0131] Embodiment 15. The hydrochloride salt of Embodiment 13 or 14, wherein the salt is a monohydrate.

[0132] Embodiment 16. The hydrochloride salt of embodiment 13 or 14, wherein the salt is unsolvated.

[0133] Embodiment 17. The hydrochloride salt of Embodiment 15, wherein the hydrochloride salt is in single crystalline Form A, characterized in that the X-ray powder diffraction pattern comprises at least three peaks at 12.9°, 17.0°, 19.0°, 21.1°, and 22.8°±0.2 in 2θ.

[0134] Embodiment 18. The hydrochloride salt of Embodiment 15, wherein the hydrochloride salt is in a single crystalline form, Form A, characterized in that the X-ray powder diffraction pattern comprises peaks at 12.9°, 17.0°, 19.0°, 21.1°, and 22.8°±0.2 in 2θ.

[0135] Embodiment 19. The hydrochloride salt of Embodiment 15, wherein the hydrochloride salt is in single crystalline Form A, characterized in that the X-ray powder diffraction pattern comprises peaks at 12.9°, 13.8°, 15.1°, 17.0°, 19.0°, 19.6°, 21.1°, and 22.8°±0.2 in 2θ.

[0136] Embodiment 20. The hydrochloride salt of Embodiment 15, wherein the hydrochloride salt is in single crystalline Form A 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 in 2θ.

[0137] Embodiment 21. The hydrochloride salt of any one of Embodiments 17-20, wherein the hydrochloride salt is in a single crystalline form, Form A, characterized by a peak phase transition temperature of 207±2°C by differential scanning calorimetry (DSC).

[0138] Embodiment 22. The hydrochloride salt of any one of Embodiments 12, 13, and 15, wherein the hydrochloride salt is in a single crystalline form, Form I, characterized in that the X-ray powder diffraction pattern comprises at least three peaks at positions selected from 5.4°, 8.2°, 16.3°, 16.5°, 18.4°, and 21.5°±0.2 in 2θ.

[0139] Embodiment 23. The hydrochloride salt of any one of Embodiments 12, 13, and 15, wherein the hydrochloride salt is in a single crystalline form, Form I, characterized in that the X-ray powder diffraction pattern comprises peaks at 5.4°, 8.2°, 16.3°, 16.5°, 18.4°, and 21.5°±0.2 in 2θ.

[0140] Embodiment 24. The hydrochloride salt of any one of Embodiments 12, 13, and 15, wherein the hydrochloride salt is in a single crystalline form, Form I, characterized in that the X-ray powder diffraction pattern comprises peaks at 5.4°, 8.2°, 13.1°, 16.3°, 16.5°, 18.4°, and 21.5°±0.2 in 2θ.

[0141] Embodiment 25. The hydrochloride salt of any one of Embodiments 12, 13, and 15, wherein the hydrochloride salt is in a single crystalline form, Form I, characterized in that the X-ray powder diffraction pattern comprises 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 in 2θ.

[0142] Embodiment 26. The hydrochloride salt of any one of Embodiments 22-25, wherein the hydrochloride salt is in a single crystalline form, Form I, characterized by a peak phase transition temperature of 187±4°C and 200±4°C by differential scanning calorimetry (DSC).

[0143] Embodiment 27. The hydrochloride salt of any one of Embodiments 17-26, wherein at least 90% by weight of the hydrochloride salt is in a single crystalline form selected from Form A and Form I.

[0144] Embodiment 28. A fumarate salt of compound (I) represented by the following structural formula:

[0145]

[0146] The molar ratio between compound (I) and fumaric acid is 1:1.

[0147] Embodiment 29. The fumarate salt of Embodiment 28, wherein the salt is crystalline.

[0148] Embodiment 30. The fumarate salt of Embodiment 28, wherein the salt is in a single crystalline form.

[0149] Embodiment 31. A fumarate salt as described in Embodiment 29, wherein the fumarate salt is in a single crystalline form A, characterized in that the X-ray powder diffraction pattern comprises at least three peaks at 5.7°, 15.3°, 16.9°, 22.4° and 23.0°±0.2 in 2θ.

[0150] Embodiment 32. The fumarate salt of Embodiment 29, wherein the fumarate salt is in a single crystalline form, Form A, characterized in that the X-ray powder diffraction pattern comprises peaks at 5.7°, 15.3°, 16.9°, 22.4°, and 23.0°±0.2 in 2θ.

[0151] Embodiment 33. A fumarate salt as described in Embodiment 29, wherein the fumarate salt is in a single crystalline form A, characterized in that the X-ray powder diffraction pattern comprises 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.

[0152] Embodiment 34. The fumarate salt of Embodiment 29, wherein the fumarate salt is in single crystalline Form A, characterized in that the X-ray powder diffraction pattern comprises 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, in 2θ.

[0153] Embodiment 35. A fumarate salt as described in any of Embodiments 31-34, wherein the fumarate salt is in a single crystalline form A, characterized by a peak phase transition temperature of 224±2°C by differential scanning calorimetry (DSC).

[0154] Embodiment 36. The fumarate salt of Embodiment 29, wherein the fumarate salt is in a single crystalline form, Form C, characterized in that the X-ray powder diffraction pattern comprises at least three peaks at 6.3°, 9.0°, 13.5°, 18.9°, and 22.5°±0.2 in 2θ.

[0155] Embodiment 37. The fumarate salt of Embodiment 29, wherein the fumarate salt is in a single crystalline form, Form C, characterized in that the X-ray powder diffraction pattern comprises peaks at 6.3°, 9.0°, 13.5°, 18.9°, and 22.5°±0.2 in 2θ.

[0156] Embodiment 38. The fumarate salt of Embodiment 29, wherein the fumarate salt is in single crystalline Form C, characterized in that the X-ray powder diffraction pattern comprises 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.

[0157] Embodiment 39. The fumarate salt of Embodiment 29, wherein the fumarate salt is in single crystalline Form C, characterized in that the X-ray powder diffraction pattern comprises 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 in 2θ.

[0158] Embodiment 40. The fumarate salt of Embodiment 34, wherein the fumarate salt is in single crystalline Form D, characterized in that the X-ray powder diffraction pattern comprises at least three peaks at 4.6°, 11.0°, 18.5°, 20.5°, and 21.0°±0.2 in 2θ.

[0159] Embodiment 41. The fumarate salt of Embodiment 34, wherein the fumarate salt is in a single crystalline form, Form D, characterized in that the X-ray powder diffraction pattern comprises peaks at 4.6°, 11.0°, 18.5°, 20.5°, and 21.0°±0.2 in 2θ.

[0160] Embodiment 42. The fumarate salt of Embodiment 34, wherein the fumarate salt is in single crystalline Form D, characterized in that the X-ray powder diffraction pattern comprises peaks at 4.6°, 11.0°, 15.1°, 18.5°, 19.4°, 20.5°, 21.0°, and 25.0°±0.2 in 2θ.

[0161] Embodiment 43. A fumarate salt as described in Embodiment 34, wherein the fumarate salt is in a single crystalline form D, characterized in that the X-ray powder diffraction pattern comprises 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 in 2θ.

[0162] Embodiment 44. A fumarate salt as described in Embodiment 36, wherein the fumarate salt Form C is mixed with Form D, characterized in that the X-ray powder diffraction pattern comprises at least three peaks at 4.6°, 11.0°, 18.5°, 20.5° and 21.0°±0.2 in 2θ.

[0163] Embodiment 45. A fumarate salt as described in Embodiment 37, wherein the fumarate salt Form C is mixed with Form D, characterized in that the X-ray powder diffraction pattern comprises peaks at 4.6°, 11.0°, 18.5°, 20.5° and 21.0°±0.2 in 2θ.

[0164] Embodiment 46. A fumarate salt as described in Embodiment 38, wherein the fumarate salt Form C is mixed with Form D, characterized in that the X-ray powder diffraction pattern comprises peaks at 4.6°, 11.0°, 15.1°, 18.5°, 19.4°, 20.5°, 21.0° and 25.0°±0.2 in 2θ.

[0165] Embodiment 47. A fumarate salt as described in Embodiment 39, wherein the fumarate salt Form C is mixed with Form D, characterized in that the X-ray powder diffraction pattern comprises 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 in 2θ.

[0166] Embodiment 48. The fumarate salt of any one of Embodiments 31-47, wherein at least 90% by weight of the fumarate salt is in a single crystalline form selected from Form A, Form C, and Form D.

[0167] Embodiment 49. A pharmaceutical composition comprising the salt of any one of Embodiments 1-48 and a pharmaceutically acceptable carrier or diluent.

[0168] Embodiment 50. A method for treating or ameliorating fibrodysplasia ossificans progressiva in a subject, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of a salt according to any one of embodiments 1-48 or a pharmaceutical composition according to embodiment 49.

[0169] Embodiment 51. A method as described in Embodiment 50, wherein the subject has a mutation in the ALK2 gene, which results in the expression of an ALK2 enzyme with an amino acid modification selected from one or more of the following: L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328W, G328E, G328R, G356D and R375P.

[0170] Embodiment 52. A method as described in Embodiment 51, wherein the ALK2 enzyme has an amino acid modification R206H.

[0171] Embodiment 53. A method for 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 at least one compound as described in any one of Embodiments 1-48 or a pharmaceutical composition as described in Embodiment 49.

[0172] Embodiment 54. A method as described in Embodiment 53, wherein the subject has a mutation in the ALK2 gene, which mutation results in the expression of an ALK2 enzyme with an amino acid modification selected from one or more of the following: R206H, G328V, G328W, G328E and G356D.

[0173] Embodiment 55. A method as described in Embodiment 54, wherein the ALK2 enzyme has an amino acid modification R206H.

[0174] Embodiment 56. A method of inhibiting abnormal ALK2 activity in a subject, the method comprising the following steps: administering a pharmaceutically effective amount of at least one compound as described in any one of Embodiments 1-48 or a pharmaceutical composition as described in Embodiment 49 to a subject in need thereof.

[0175] Embodiment 57. A method as described in Embodiment 56, wherein the abnormal ALK2 activity is caused by a mutation in the ALK2 gene, which mutation results in the expression of an ALK2 enzyme with an amino acid modification selected from one or more of the following: L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328V, G328W, G328E, G328R, G356D and R375P.

[0176] Embodiment 58. A method as described in Embodiment 57, wherein the ALK2 enzyme has an amino acid modification R206H.

[0177] Embodiment 59. The method of any one of Embodiments 56-58, wherein the subject has fibrodysplasia ossificans progressiva or diffuse intrinsic pontine glioma.

[0178] The following examples are intended to be illustrative and are not intended to limit the scope of the present disclosure in any way.

[0179] experiment

[0180] abbreviation:

[0181]

[0182]

[0183]

[0184]

[0185] Analysis conditions

[0186] X-ray powder diffraction (XRPD)

[0187] Powder X-ray diffraction was performed using a Rigaku MiniFlex 600 or a Bruker D8 Advance equipped with a Lynxeye detector in reflection mode (i.e., Bragg-Brentano geometry). Samples were prepared on Si zeroed wafers. Typical scans were from 4 to 30 degrees 2θ at 40 kV and 15 mA with a step size of 0.05 degrees in five minutes. High resolution scans were from 4 to 40 degrees 2θ at 40 kV and 15 mA with a step size of 0.05 degrees in 30 minutes. Typical parameters for XRPD are listed below.

[0188]

[0189] Thermogravimetric analysis and differential scanning calorimetry (TGA & DSC)

[0190] Using Mettler Toledo TGA / DSC 3+ Thermogravimetric analysis and differential scanning calorimetry were performed on the same sample at the same time. The required amount of sample was weighed directly in a sealed aluminum pan with a pinhole. The typical sample mass for the measurement was 5-10 mg. The typical temperature range was 30°C to 300°C with a heating rate of 10°C per minute (total time of 27 minutes). The protective gas and purge gas were nitrogen (20-30 mL / min and 50-100 mL / min). The typical parameters of DSC / TGA are listed below.

[0191]

[0192] Differential Scanning Calorimetry (DSC)

[0193] 1-5mg material is weighed into an aluminum DSC pan and sealed non-airtightly with an aluminum lid. The sample pan is then loaded into a TA Instruments Q2000 (equipped with a cooler). After a stable heat flow response is obtained at 30°C, the sample and reference sample are heated to 300°C at a rate of 10°C / min and the resulting heat flow response is monitored. Prior to analysis, the instrument is calibrated for temperature and heat flow using an indium reference standard. Sample analysis is performed with the aid of TA Universal Analysis 2000 software, where the temperature of the thermal event is quoted as the starting temperature and peak temperature, measured according to the manufacturer's instructions. Method gas: N2, at 60.00mL / min.

[0194] 1 H-NMR spectroscopy ( 1 H-NMR)

[0195] Proton NMR was performed on a Bruker Avance 300 MHz spectrometer. The solid was dissolved in 0.75 mL of deuterated solvent in a 4 mL vial and transferred to an NMR tube (Wilmad 5 mm thin wall 8" 200 MHz, 506-PP-8). A typical measurement was usually 16 scans. Typical parameters for NMR are listed below.

[0196]

[0197]

[0198] Dynamic Vapor Sorption (DVS)

[0199] Dynamic Vapor Sorption (DVS) was performed using the DVS Intrinsic 1. The sample was loaded into a sample pan and suspended from a microbalance. The typical sample mass for DVS measurements was 25 mg. Nitrogen bubbled through distilled water provided the required relative humidity. The sample was kept at each level for at least 5 minutes and was only raised to the next humidity level if the weight change was < 0.002% between two measurements (interval: 60 seconds) or after 240 minutes had passed. A typical measurement consists of the following steps:

[0200] 1- Equilibrated at 50% RH

[0201] 2-50% to 2% (50%, 40%, 30%, 20%, 10% and 2%)

[0202] a. Maintain at each humidity for at least 5 minutes and at most 60 minutes. The passing standard is a change of less than 0.002%

[0203] 3-2% to 95% (2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%)

[0204] a. Maintain at each humidity for at least 5 minutes and at most 60 minutes. The passing standard is a change of less than 0.002%

[0205] 4-95% to 2% (95%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 2%)

[0206] a. Maintain at each humidity for at least 5 minutes and at most 60 minutes. The passing standard is a change of less than 0.002%

[0207] 5-2% to 50% (2%, 10%, 20%, 30%, 40%, 50%)

[0208] a. Maintain at each humidity for at least 5 minutes and at most 60 minutes. The passing standard is a change of less than 0.002%

[0209] High performance liquid chromatography (HPLC)

[0210] 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.

[0211]

[0212] Karl Fischer titration

[0213] Karl Fischer titration for water determination was performed using a Mettler Toledo C20S Coulometric KF titrator equipped with a current generator unit with diaphragm and a double platinum needle electrode. Aquastar TM CombiCoulomat non-melting reagent. About 0.03-0.10 g of sample is dissolved in the anode compartment and titrated until the solution potential drops below 100 mV. Prior to sample analysis, a Hywtnal 1 wt% water standard is used for verification.

[0214] Microscopic examination

[0215] Optical microscopy was performed using a Zeiss AxioScope A1 equipped with 2.5x, 10x, 20x, and 40x objectives and a polarizer. Images were captured with a built-in Axiocam 105 digital camera and processed using the ZEN 2 (blue version) software provided by Zeiss.

[0216] Example 1: Combined salt screening

[0217] 1.1 Salt screening

[0218] According to Marvin Sketch software prediction, the free base of compound (I) has multiple pKa. The compound has three basic nitrogens, and its theoretical pKa values ​​are 8.95, 3.57 and 2.86. The theoretical logarithm P is 2.98.

[0219] 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. Table 1 provides a list of counterions.

[0220] A stock solution of Compound (I) was prepared in anhydrous EtOH (20 wt%, density 0.8547 g / mL). Stock solutions of all counterions were also prepared in EtOH. Counterion stock solutions of solid counterions were prepared at 0.02 g / mL and liquid counterions were prepared at 10 vol%.

[0221] Salt formation was performed 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 equivalents and 1.1 equivalents of counterion were added. In the case of HCl, 1.1 equivalents and 2.2 equivalents of counterion were added. The solvent was evaporated at 30° C. while stirring overnight, followed by thorough drying under vacuum at 50° C. for 4 hours.

[0222] About 25 volumes of solvent (0.625 mL) were added to each vial for screening. The three selected solvents were EtOH, EtOAc, and IPA:water (9:1 volume ratio). Once the solvent was added, the mixture (or solution) was heated to 45 ° C, maintained for 1.5 hours, cooled to room temperature and stirred overnight. When a slurry was formed, the solid was filtered for XRPD analysis.

[0223] XRPD analysis was performed in three stages. XRPD of wet cake was performed on all samples (when solids were observed). The unique solids were then left on the XRPD plate and dried under vacuum at 50°C for at least 3 hours. XRPD of the unique dried solids was then performed. The solids were then exposed to>90% relative humidity for 1 day and the resulting solids were subjected to XRPD processing. A humid environment was created by placing a saturated potassium sulfate beaker in water in a sealed container. All XRPD patterns were compared with the counter-ion XRPD patterns and the known free molecule patterns.

[0224] If solids were not formed with the first three screening solvents (EtOH, EtOAc, IPA:water), the lid was opened and the solvents were evaporated at 30°C with stirring. The solids were evaporated to dryness by placing them at 50°C in vacuum for 3-4 hours and a second round of solvents (IPOAc, MBK, MtBE) was added. If solids were not formed with the second round of solvents, the solvents were evaporated to dryness again and DEE was added.

[0225] Table 1 - Counterions and associated pKa values ​​used for initial salt screening.

[0226] ID Counter ions pKa(minimum) Equivalents for screening 1 Acetic acid 4.75 1.1 2 Benzenesulfonic acid -2.8 1.1 3 benzoic acid 4.19 1.1 4 Citric Acid 3.08 1.1 5 Fumaric Acid 3.03 1.1 6 hydrochloric acid -7 1.1、2.2 7 Malic Acid 3.4 1.1 8 Maleic acid 1.9 1.1 9 Phosphoric acid 2.15 1.1 10 Salicylic acid 2.97 1.1 11 sulfuric acid -3 0.55、1.1 12 Succinic acid 4.2 1.1 13 tartaric acid 2.89 1.1

[0227] Crystalline solids were observed when screening with benzenesulfonic acid (BSA), benzoic acid, fumaric acid, HCl (1 eq. and 2 eq.), maleic acid, salicylic acid, and succinic acid. A unique XRPD pattern was observed with BSA, benzoic acid, HCl (2 eq.), salicylic acid, and succinic acid. Multiple patterns were observed with HCl (1 eq.) and fumaric acid. Two patterns were observed with maleic acid, and both deliquesced under humidity exposure. Among the crystalline solids, the solids obtained by screening with benzoic acid, fumaric acid, HCl (1 eq.), salicylic acid, and succinic acid did not deliquesce under humidity exposure.

[0228] The crystalline salts were characterized and their activity evaluated based on melting point, crystallinity, stability under drying and humidity exposure, aqueous solubility, polymorphism, and counterion acceptability.

[0229] Given the acceptable physicochemical properties, the mono-hydrochloride, succinate, and fumarate salts were selected for further development. The free base was also included in further characterization for comparison.

[0230] Benzoate was not selected due to poor water solubility and high mass loss on melting. Salicylate was not selected due to poor water solubility, high mass loss on melting and possible polymorphism. Benzenesulfonate, maleate and dihydrochloride were not selected due to low crystallinity and instability in humid environments (deliquesce).

[0231] The free base sample showed a melting onset at 116.19° C. in the DSC. The TGA thermogram showed a gradual mass loss of 0.16 wt % before melting and a stepwise mass loss of 0.05 wt % while melting. The solid was a fine powder by microscopic inspection. Karl Fischer titration of the free base showed 0.37 wt % water.

[0232] The free alkali shows high solubility (>200 mg / mL at room temperature in most of the tested organic solvents) in many organic solvent systems, high solubility in simulated fluids (0.08 mg / mL water, about 17 mg / mL simulated gastric fluid in the fasting state, about 7 mg / mL simulated intestinal fluid in the fasting state), acceptable melting temperature (starting at 116°C) and low residual solvent (<0.20 weight % by thermogravimetric analysis). The disadvantage of the free alkali is that it has polymorphs (4 figures were observed during the limited screening period), and is physically unstable in a humid environment (>90% relative humidity), and becomes a viscous glue within 4 days, and then forms a glue in water. The results of the laboratory scale also show that on a production scale, the free alkali will be difficult to separate into a crystalline solid.

[0233] The monohydrochloride salt exhibits a high melting point (starting at 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 water and fasting state simulated gastric fluid, about 7 mg / mL fasting state simulated intestinal fluid). The disadvantages of the monohydrochloride salt include sensitivity to the equivalent added (dihydrochloride is formed at as low as 1.3 molar equivalents of HCl) and sensitivity to drying.

[0234] The succinate salt showed only one pattern during screening, was stable upon drying and humidity exposure, was less hygroscopic than the mono-hydrochloride salt and free base, exhibited high solubility in water and simulated fluids (>22 m / mL in all fluids), a high melting point (onset 173°C), and an acceptable mass loss upon melting by thermogravimetric analysis (0.27 wt %).

[0235] The fumarate salt exhibits high solubility in water and simulated fluids (>15 m / mL in all fluids), and the putative hydrate (referred to as Form B) is stable upon drying and moisture exposure. Form A (anhydrous) exhibits a high melting point (onset 221°C).

[0236] A summary of the physicochemical properties of the free base and selected salts is given in Table 2 below.

[0237] Table 2 - Physicochemical properties of free base and selected salts

[0238]

[0239] 1.2 Humidity exposure of free base

[0240] The crystalline form of the free base was exposed to high humidity (>90% RH) overnight.A humid environment was created by placing a beaker saturated with potassium sulfate in water in a sealed container.

[0241] After overnight humidity exposure, the solid remained in the same crystalline form, but lost some crystallinity. After XRPD analysis, the same sample was placed back in a humid environment. After one week, it was noted that the sample had deliquesced on the XRPD plate. A second experiment was started under the same conditions. The solid darkened in color and became stickier. XRPD of the sample was collected on the 6th day. The peak intensity was lower and a change in the baseline was observed, indicating an increase in amorphous content.

[0242] Example 2: Preparation and characterization of the crystalline form (Form A) of 1.5:1 Compound (I) sesquisuccinate

[0243] 2.1 Preparation

[0244] Method A:

[0245] Compound (I) as a free base was weighed into a 4 mL vial and 1.1 equivalents of succinic acid were 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 kept for two hours while stirring, then cooled to room temperature naturally. The solid remained in solution, so the solution was inoculated with a succinate sample obtained from the screening. The seeds were retained and a white slurry was quickly formed. The slurry was stirred at room temperature overnight. Before filtering, the slurry was a beige / off-white slurry of medium consistency. The slurry was filtered and washed twice with 2 volumes of EtOH, then dried overnight in a vacuum at 50 ° C. The purity obtained by HPLC was 99.79 area%. The solid obtained was obtained by XRPD (see Figure 1 and Table 3), TGA-DSC ( Figure 2 ), 1 H-NMR( Figure 3 ) and single crystal X-ray crystallography.

[0246] The initial mass loss by TGA was 0.19 wt % and then 0.30 wt % after melting, see Figure 2 The DSC thermogram shows that the melting onset is at 172.9 °C, and then the sample decomposes above 200 °C.

[0247] Table 3. XRPD of Compound (I) Sesquisuccinate (Form A)

[0248]

[0249]

[0250] Method B:

[0251] Using compound (I) free alkali and 1.6 equivalents of succinic acid, salification was carried out under several different solvent conditions. About 30 mg of free alkali was weighed into a 2 mL vial, and 10 volumes of solvent were added. In all solvents except MtBE, the free alkali was dissolved at room temperature. Then succinic acid was added as a stock solution in EtOH, making each solvent composition about 40% by volume EtOH. The solution / slurry was stirred at room temperature until precipitation was observed, and then solids were collected for XRPD analysis. An overview of the solids obtained from the salification experiment is given in Table 4.

[0252] Table 4 - Summary of solids obtained from salt formation experiments

[0253]

[0254] Method C: Amorphous Slurry

[0255] About 30 mg of compound (I) sesquisuccinate was melted in a 2 mL vial to produce an amorphous glassy solid. Solvent (450 μL) was added to each vial along with a stirring bar at room temperature. In all cases, the glassy 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 was formed immediately after loosening the solid. After precipitation was observed, the slurry was sampled for XRPD analysis. The earliest sampling time point was about 30 minutes after the addition of solvent. Table 5 summarizes the results and observations of the amorphous slurry experiment.

[0256] Table 5 - Summary of XRPD patterns of solids obtained from amorphous slurry experiments

[0257]

[0258] Method D: Amorphous Vapor Diffusion

[0259] About 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. Before the solid was sampled for XRPD, the vial was kept at room temperature for a weekend. The appearance of most solids changed from light beige glass (decomposed by amorphous foam) to white / off-white powder. Amorphous solids exposed to a humid atmosphere (water as solvent) became yellow paste. Table 6 summarizes the solids obtained from amorphous vapor diffusion experiments.

[0260] Table 6 - Summary of solids obtained from amorphous vapor diffusion experiments

[0261]

[0262]

[0263] In the polymorph screening of Compound (I) sesquisuccinate, more than 10 crystallization or salt formation methods (including experiments using amorphous solids) were used to generate solids. Only Form A and amorphous solids were observed in the polymorph screening of Compound (I) sesquisuccinate.

[0264] A sample of the amorphous solid (Compound (I) sesquisuccinate) was heated to 140°C and then cooled to room temperature. The resulting solid was found to be Form A by XRPD.

[0265] The amorphous solid (Compound (I) sesquisuccinate) was exposed to 75% RH / 40°C for one week. The appearance of the solid changed from a light beige solid to a hard yellow glass. XRPD of the solid showed Form A.

[0266] Form A was found to be crystalline with a melting onset of 173 °C, stable under drying and humidity exposure, and exhibited high solubility in water and simulated fluids (>22 mg / mL in all fluids.

[0267] Method E:

[0268] 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. Patent No. 10,233,186 was dissolved in isopropyl acetate (IPAc, 2.75 volumes) containing (R)-tetrahydrofuran-3-yl 1H-imidazole-1-carboxylate (1.2 equiv). The mixture was heated and stirred until complete conversion. When the reaction was quenched with ammonia (2 volumes), additional IPAc (4 volumes) was added. The phases were separated, washed with water and distilled to give a dry IPAc solution of compound (I) (about 3.5 kg, 3 volumes). While heating at 40-60°C, succinic acid (1.45 equiv, 10 volumes) in ethanol was added. 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 give Compound (I) sesquisuccinate Form A.

[0269] 2.2 Humidity exposure

[0270] The sesquisuccinate obtained in Example 2.1 was exposed to 75% relative humidity at 40°C for one week. The 4 mL vial was covered and then placed in a 20 mL vial containing 3-4 mL of saturated NaCl aqueous solution. The 20 mL vial was sealed and kept at 40 ° C. After one week, the solid was collected for XRPD analysis. After one week under humid conditions, Form A was determined to be physically stable by XRPD.

[0271] 2.3DVS

[0272] DVS showed a mass change of 0.59-0.60 wt% ( Figure 4 ). When the relative humidity exceeds 80%, a mass change of 0.34-0.35 wt% occurs. The XRPD after DVS measurement remains Form A ( Figure 5 ).

[0273] DVS was also performed on the free base of compound (I) ( Fig.24), at 25°C, between 2% and 95% relative humidity, it exhibits a reversible mass change of 0.88-0.92% by weight. Among them, a mass change of 0.46-0.53% by weight occurs when the relative humidity exceeds 70%.

[0274] 2.4VT-XRPD and VH-XRPD

[0275] Variable humidity experiments using XRPD on Compound (I) sesquisuccinate Form A showed that no changes in the crystal structure were observed under humidity (see Figure 6 ).

[0276] Variable temperature experiments using XRPD on Compound (I) sesquisuccinate Form A showed that no changes in the crystalline structure were observed below 160°C (i.e., the melting point) (see Figure 7 ).

[0277] Example 3: Preparation and characterization of 1:1 crystalline HCl salt monohydrate of compound (I)

[0278] 3.1 Preparation

[0279] Method A:

[0280] First, 25-35 mg of Compound (I) free base was weighed into a 2 mL vial. Solvent was then added to the vial (25 volumes or 5 volumes), followed by 0.9 mol equivalents, 1.1 mol equivalents, 1.5 mol equivalents, 2.2 mol equivalents, and 3.5 mol equivalents of HCl stock solution in IPA.

[0281] Initially, IPA: water (9: 1 volume) was added to give a total of 25 volumes (including the volume of the HCl stock solution). Initially, all formed solutions. The 1.1 equivalent experiment showed precipitation overnight, but all other precipitations remained in solution. This may be due to differences in solvent composition, so the remaining solution (0.9 equivalents, 1.5 equivalents, 2.2 equivalents, and 3.5 equivalents) was evaporated to dryness at 50°C at atmospheric pressure, followed by evaporation at 50°C in an active vacuum for about 3 hours. Another experiment of 1.1 equivalents was prepared in a similar manner by the following steps: 5 volumes of IPA and an appropriate amount of HCl stock solution were added, then evaporated to dryness at 50°C in a weak vacuum, and then evaporated to dryness at 50°C in an active vacuum for about 3 hours.

[0282] To the evaporated solid, 25 volumes of EtOAc were added and stirred at room temperature overnight. In all cases a slurry was formed. The slurry color changed from white slurry (0.9 eq.) to bright / dark yellow slurry (1.5 eq. and above).

[0283] The slurry was then filtered and the solids recovered for XRPD analysis. The salt formed with 0.9 eq. and 1.1 eq. was shown by XRPD to be Form A (monohydrochloride salt) ( Figure 8 ). A mixture of Form A (monohydrochloride) and Form B (dihydrochloride) was obtained using 1.3 equivalents and 1.5 equivalents. Form B (dihydrochloride) was obtained using 2.2 equivalents and 3.5 equivalents.

[0284] By TGA-DSC ( Fig. 9 ), 1 H NMR ( Fig.10 ) and single crystal X-ray crystallography (Table 7) further characterized Compound (I) crystalline HCl salt Form A. The sample showed a melting onset at 202.86° C. in DSC ( Fig. 9 ). The TGA thermogram showed a mass loss of 2.81 wt% before melting (related to the very broad endotherm in the DSC) and a stepwise mass loss of 0.44 wt% upon melting. Karl Fischer titration of the HCl salt showed 3.17 wt% water, which supports that the crystalline HCl salt obtained is a monohydrate. The theoretical water content in the monohydrate of the HCl salt is 3.0 wt%.

[0285] Table 7-1: Peak list of XRPD pattern of Compound (I) crystalline HCl salt monohydrate (Form A)

[0286] 2θ(degrees) d spacing (angstroms) Relative strength (%) 5.69 15.53 8 10.14 8.72 15 12.63 7.00 11 12.91 6.85 42 13.79 6.42 16 15.14 5.85 16 17.02 5.21 100 18.98 4.67 33 19.59 4.53 21 20.32 4.37 14 21.12 4.20 28 22.17 4.01 14 22.76 3.90 35 23.35 3.81 17 23.98 3.71 36 24.80 3.59 11 25.47 3.49 17 26.12 3.41 5 28.58 3.12 6

[0287] Method B:

[0288] The free base of compound (I) (4.3 kg) was dissolved in isopropyl acetate (5.5 volumes) and isopropanol (2.5 volumes). The mixture was heated to reflux and fed with HCl (16.5%-w / w in water, 0.95 equiv.) over 0.75 hours. After reflux for 1 hour, the solution was cooled to 20-25° C. over 2 hours and maintained for 0.5 hours. The crystalline product was isolated by filtration and washed with a mixture of IPAc, IPA and water to give 1:1 Compound (I) crystalline HCl salt monohydrate Form A.

[0289] 3.2 Humidity exposure

[0290] The HCl salt monohydrate Form A was placed at 40°C / 75% relative humidity for one week. Approximately 10 mg of the sample was placed in a The vial was then placed in a 20 mL sealed vial containing a saturated sodium chloride solution. After one week of humidity exposure, some minor peak shifts were observed in the XRPD. Peak shifts were also observed in the XRPD patterns of some long-term slurries, indicating that the HCl salt monohydrate Form A is a channel hydrate and that the peak shifts may be due to changes in water content.

[0291] In the laboratory, the sampling day was low humidity (<25% RH) from a week of humidity exposure (75% RH and 40°C).

[0292] After 14 days in a sealed vial (ambient conditions), the solid was sampled again. The solid was identified by XRPD as 1:1 Compound (I) crystalline HCl salt monohydrate (Form A).

[0293] 3.3 DVS of HCl salt monohydrate form A

[0294] DVS was performed on the HCl salt monohydrate Form A ( Fig.11 ). At 25°C, between 2% and 95% relative humidity, it exhibits a mass change of 1.30-1.43% by weight. Of these, a mass change of 0.99-1.11% by weight occurs below 20% relative humidity.

[0295] After DVS measurement, the XRPD ( Fig.12 ). All peaks of the HCl salt monohydrate Form A were present in the XRPD, but additional peaks were observed.

[0296] 3.4VT-XRPD and VH-XRPD

[0297] Variable humidity experiments performed using XRPD on the HCl salt monohydrate Form A are shown in Fig.13 The small shifts observed at 0% RH towards higher angles (ie lower d-spacings) in the peaks around 10° and 13° (2θ) are consistent with contraction of the crystal structure upon water loss and are therefore consistent with channel hydrates.

[0298] Variable temperature experiments using XRPD are shown in Fig.14 This confirms that the changes observed in the diffraction patterns at 100 °C or higher are essentially due to thermal expansion and unit cell contraction due to the removal of water molecules. The crystalline structure does not appear to collapse and / or rearrange as it does in the presence of bound / crystallization water, which is again consistent with channel hydrates.

[0299] 3.5 Exposure to dry conditions and rehumidification

[0300] 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.

[0301] The conditions were: (1) room temperature in a vial containing P2O5 at 50°C, (2) 60°C in vacuum, and (3) heated to 140°C in a DSC.

[0302] 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 was below 20%.

[0303] On day 7, the HCl salt of P2O5 exposed to 50°C was sampled for XRPD. XRPD performed 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. The same sample was analyzed by XRPD after being left on the bench overnight and was determined by XRPD to remain Form A. The XRPD diagram is shown in Fig.12 middle.

[0304] Example 4: Preparation and Characterization of Anhydrous 1:1 Compound (I) Crystalline HCl Salt (Form D)

[0305] 4.1 Preparation

[0306] The anhydrous 1:1 crystalline HCl salt of compound (I) (Form D) was prepared by drying Form A (monohydrate) for a long time 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. Before sampling, a 4 mL open vial containing the sample was placed in a 20 mL sealed vial containing P2O5 at 50°C for two days. XRPD identified it as a new crystalline form (Form D) ( Fig.15 ).

[0307] It was observed that after 2.25 hours of exposure to ambient conditions (22° C., 35% RH), Form D converted to Form A. Therefore, characterization of Form D was performed with minimal exposure to ambient conditions.

[0308] The DSC thermogram of the Form D sample showed an endotherm at 202.4°C ( Fig.16 ). TGA of Form D sample showed a total mass loss of 1.10 wt% ( Fig.16 ). Form A (monohydrate) also shows a DSC endotherm upon dehydration, occurring at 202-203°C.

[0309] Karl Fischer titration showed that the water content of the Form D sample was 0.72% by weight. The Form D sample exhibited a cubic morphology under a microscope. The morphology was not significantly different from the starting material (Form A). By HPLC, the purity of the Form D sample was 98.82 area %.

[0310] After humidity exposure (>90% RH overnight and 74% RH / 40°C for one week), Form D converted to Form A (monohydrate).

[0311] Compound (I) crystalline HCl salt form D is prepared by 1 H NMR further characterized ( Fig.17 ).

[0312] Table 8 - Peak listing for the XRPD pattern of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form D).

[0313] 2θ(degrees) d spacing (angstroms) Relative strength (%) 9.36 9.44 8 9.79 9.02 5 10.81 8.18 29 13.05 6.78 25 13.25 6.68 30 13.89 6.37 10 14.32 6.18 5 15.24 5.81 8 15.68 5.65 5 16.62 5.33 27 16.87 5.25 63 17.35 5.11 27 18.02 4.92 1 18.83 4.71 62 19.88 4.46 1 20.84 4.26 25 21.64 4.10 18 22.18 4.00 100 23.69 3.75 9 24.65 3.61 31 26.04 3.42 15 27.81 3.21 12

[0314] Example 5: Preparation and Characterization of Anhydrous 1:1 Compound (I) Crystalline HCl Salt (Form G)

[0315] 5.1 Preparation

[0316] Form G (low crystallinity) was observed while rapidly cooling the amorphous slurry from IPA solution and from EtOAc and MtBE. Form G was scaled up by rapidly cooling in IPA. About 200 mg of the original HCl salt was weighed in a 20 mL vial, and 60 volumes of IPA were added while stirring at 50 ° C. The solid was dissolved, and the solution was transferred to an ice water (0 ° C) beaker. The solution was inoculated with a Form G sample at 0 ° C. The seeds were retained, but no thick slurry was formed. The sample was transferred to a refrigerator at -20 ° C, and solids precipitated over the weekend.

[0317] The resulting slurry appeared fluffy. Filtration was extremely slow and the solids were somewhat sticky. The collected solids were quite wet due to poor filtration. The collected solids were dried overnight at 50 °C in vacuum. The crystallinity of the solids obtained from scale-up was lower than those observed during screening ( Fig.18 ).

[0318] The DSC thermogram of Form G showed two endothermic peaks occurring at 163.1°C and 189.6°C, respectively, followed by decomposition ( Fig.19 ). The TGA thermogram showed an initial gradual mass loss of 2.62 wt % prior to the first endothermic event, followed by smaller mass losses (0.35 wt % and 0.07 wt %) during the endothermic events. Standalone DSC agreed well with the coupled DSC-TGA data and showed a broad endotherm between 80-130°C. The Form G sample was heated in the DSC above the broad endotherm and then cooled to room temperature. No changes were observed in the XRPD.

[0319] Karl Fischer titration showed the water content of the sample to be 2.79 wt%.

[0320] Microscopic examination of the Form G sample showed large lumps of solid and some irregular / fine particles. The purity of the Form G sample was 98.89 area % by HPLC.

[0321] Form G samples partially converted to Form A overnight in a high humidity environment (>90% RH). Form G was stable (by XRPD) after one week of humidity exposure (75% RH / 40°C).

[0322] Table 9 - Peak listing for the XRPD pattern of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form G).

[0323]

[0324]

[0325] Example 6: Preparation and Characterization of Anhydrous 1:1 Compound (I) Crystalline HCl Salt (Form I)

[0326] 6.1 Preparation

[0327] Form I was observed when salting was performed in anhydrous solvent systems (MtBE: IPA and cyclohexane: IPA). Form I was scaled up by salting in cyclohexane: IPA. First, about 200 mg of the free base of compound (I) was weighed in a 4 mL vial, and 15 volumes of cyclohexane were added to form a slurry. 1.1 molar equivalents of HCl were added in the form of a 0.55 M IPA solution over 30 minutes. The HCl solution was distributed dropwise in three aliquots. After the first aliquot, a yellow slurry was formed and then in a gelled state. The gelled state was maintained after adding the last two aliquots. The vial was then heated to 45 ° C, kept for 1 hour, and inoculated with a Form I sample. After inoculation, the sample was cooled to room temperature naturally. After inoculation, a white solid was observed, and after cooling to room temperature, the sample was essentially a white slurry with some yellow colloid on the vial wall. The slurry was filtered and washed twice with two volumes of cyclohexane.

[0328] The DSC thermogram of the Form I sample showed an endotherm at 180.5°C followed by a small endotherm at 198°C ( Fig. 22 ). The TGA thermogram showed a gradual mass loss of 2.34 wt% before melting and a mass loss of 0.26 wt% upon melting.

[0329] Independent DSC is in good agreement with the coupled DSC-TGA data and also shows an endotherm between 90-120°C. The Form I sample was heated to 150°C in the DSC and then cooled to room temperature for XRPD analysis. No changes were observed in the XRPD pattern. All peaks were shifted to slightly higher 2θ, which may be due to sample displacement.

[0330] Karl Fischer titration showed that the water content of the Form I sample was 2.64 wt %.

[0331] Microscopic examination showed fine powder (needles) and agglomerates.The purity of Form I was 99.51 area % by HPLC.

[0332] The X-ray powder diffraction (XRPD) pattern of the anhydrous 1:1 crystalline HCl salt of Compound (I) (Form I) is shown in Fig.21 middle.

[0333] Form I samples partially converted to Form A overnight in a high humidity environment (>90% RH).

[0334] Table 10 - Peak listing for the XRPD pattern of the anhydrous 1:1 Compound (I) crystalline HCl salt (Form I).

[0335] 2θ(degrees) d spacing (angstroms) Relative strength (%) 5.44 16.23 52 8.22 10.75 60 10.21 8.66 37 13.06 6.77 51 14.94 5.92 23 15.40 5.75 25 16.33 5.42 100 16.51 5.37 57 17.05 5.19 30 18.35 4.83 57 18.98 4.67 22 20.05 4.43 9 20.42 4.34 28 21.49 4.13 65 21.78 4.08 43 22.49 3.95 10 23.70 3.75 11 24.10 3.69 28 25.00 3.56 7 25.55 3.48 12 26.45 3.37 10 27.30 3.26 12 30.78 2.90 5 34.03 2.63 14 35.09 2.56 4 35.87 2.50 11

[0336] Example 7: Preparation and Characterization of Anhydrous 1:1 Crystalline Fumarate of Compound (I) (Form A)

[0337] 7.1 Preparation

[0338] The scale of the fumarate salt Form A was expanded 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. Most of the solid dissolved (the slurry became very dilute), and then the solid precipitated to form a thick white slurry. Another 5 volumes of EtOAc were added to improve mixing. The slurry was heated to 45°C and kept for two hours while stirring, and then cooled to room temperature naturally. The slurry was stirred at room temperature overnight. Before filtering, the slurry was a thick white slurry. The slurry was filtered and washed twice with 2 volumes of EtOAc, and then dried in vacuo at 50°C overnight. The solid obtained was identified by XRPD (see Fig.26 and Table 11), TGA-DSC ( Fig. 27 ), 1 H-NMR( Fig.28 ) for further characterization.

[0339] Table 11 - Peak List of XRPD Pattern of Anhydrous 1:1 Compound (I) Crystalline Fumarate Salt (Form A)

[0340] 2θ(degrees) d spacing (angstroms) Relative strength (%) 5.72 15.45 100 7.54 11.72 28 9.76 9.05 24 10.29 8.59 28 11.21 7.89 15 12.25 7.22 30 14.79 5.99 21 15.25 5.80 74 16.21 5.46 13 16.86 5.25 90 17.16 5.16 28 17.52 5.06 42 18.26 4.86 23 18.78 4.72 17 19.87 4.47 24 20.66 4.30 14 21.46 4.14 5 22.39 3.97 51 23.04 3.87 50 23.49 3.78 8 25.79 3.45 29

[0341] Example 8: Preparation and Characterization of Anhydrous 1:1 Crystalline Fumarate of Compound (I) (Form C)

[0342] 8.1 Preparation

[0343] In a 4 mL vial, the free base of compound (I) was added to 1.1 equivalents of fumaric acid. IPAc (15 volumes) was then added at room temperature. Most of the solid dissolved (the slurry became very dilute), and then the solid precipitated as an off-white slurry. The slurry was heated to 45 ° C and kept for two hours while stirring, and then cooled to room temperature naturally. The slurry was stirred at room temperature overnight. Before filtering, the slurry was a thick white slurry. The slurry was filtered and washed twice with 2 volumes of IPAc, and then dried overnight in a vacuum at 50 ° C. The obtained solid was further slurried in EtOH and EtOAc and characterized by XRPD (see Fig.29 and Table 12).

[0344] Table 12 - Peak Listing of XRPD Pattern of Anhydrous 1:1 Compound (I) Crystalline Fumarate Salt (Form C)

[0345] 2θ(degrees) d spacing (angstroms) Relative strength (%) 4.45 19.82 41 6.30 14.02 48 7.41 11.91 16 8.96 9.86 76 13.50 6.55 75 14.68 6.03 32 16.24 5.45 10 16.78 5.28 20 17.35 5.11 13 17.78 4.98 8 18.36 4.83 15 18.92 4.69 100 19.65 4.52 43 21.04 4.22 54 22.50 3.95 72 23.63 3.76 40 25.46 3.50 16 26.22 3.40 13 27.51 3.24 22 28.31 3.15 13

[0346] Example 9: Preparation and Characterization of Anhydrous 1:1 Crystalline Fumarate of Compound (I) (Form D)

[0347] 9.1 Preparation

[0348] In a 4 mL vial, the free base of compound (I) was added to 1.1 equivalents of fumaric acid. IPAc (15 volumes) was then added at room temperature. Most of the solid dissolved (the slurry became very dilute), and then the solid precipitated as an off-white slurry. The slurry was heated to 45°C and kept for two hours while stirring, and then cooled to room temperature naturally. The slurry was stirred at room temperature overnight. Before filtering, the slurry was a thick white slurry. The slurry was filtered and washed twice with 2 volumes of IPAc, and then dried overnight in a vacuum at 50°C. The obtained solid was further slurried in a mixture of IPA: water (95:5 volumes) and characterized by XRPD (see Fig.30 and Table 13).

[0349] Table 13 - Peak Listing of XRPD Pattern of Anhydrous 1:1 Compound (I) Crystalline Fumarate Salt (Form D)

[0350] 2θ(degrees) d spacing (angstroms) Relative strength (%) 4.62 19.10 100 10.98 8.05 26 11.94 7.41 7 14.25 6.21 5 15.08 5.87 7 18.45 4.80 23 19.40 4.57 8 20.48 4.33 9 20.98 4.23 8 22.78 3.90 6 23.62 3.76 5 24.97 3.56 7

Claims

1. A fumarate salt of compound (I), which is represented by the following structural formula: The molar ratio between compound (I) and fumaric acid is 1:1, wherein the fumarate salt is in single crystal form A, characterized in that The X-ray powder diffraction pattern includes peaks at 5.7°, 15.3°, 16.9°, 22.4°, and 23.0°±0.2 in terms of 2θ.

2. The fumarate salt of claim 1, wherein the fumarate salt is in a single crystalline form A, characterized in that The X-ray powder diffraction pattern includes 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.

3. The fumarate salt of claim 1, wherein the fumarate salt is in a single crystalline form A, characterized in that The X-ray powder diffraction pattern comprises 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 in 2θ.

4. The fumarate salt of claim 1, wherein the fumarate salt is in a single crystalline form A, characterized in that The peak phase transition temperature of the product was 224±2℃ by differential scanning calorimetry (DSC).

5. A pharmaceutical composition comprising the fumarate according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier or diluent.

6. Use of the fumarate according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating or improving fibrodysplasia ossificans progressiva in a subject.

7. The use of claim 6, wherein the subject has a mutation in the ALK2 gene, which results in the expression of an ALK2 enzyme having an amino acid modification selected from one or more of the following: L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328W, G328E, G328R, G356D and R375P.

8. The use according to claim 7, wherein the ALK2 enzyme has an amino acid modification R206H.

9. Use of the fumarate according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating or improving diffuse intrinsic pontine glioma in a subject.

10. The use of claim 9, wherein the subject has a mutation in the ALK2 gene, the mutation resulting in the expression of an ALK2 enzyme having an amino acid modification selected from one or more of: R206H, G328V, G328W, G328E, and G356D.

11. The use according to claim 10, wherein the ALK2 enzyme has an amino acid modification R206H.

12. Use of the fumarate salt of any one of claims 1 to 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for inhibiting abnormal ALK2 activity in a subject.

13. The use of claim 12, wherein the abnormal ALK2 activity is caused by a mutation in the ALK2 gene, which results in the expression of an ALK2 enzyme having an amino acid modification selected from one or more of the following: L196P, PF197-8L, R202I, R206H, Q207E, R258S, R258G, R325A, G328A, G328V, G328W, G328E, G328R, G356D and R375P.

14. The use according to claim 13, wherein the ALK2 enzyme has an amino acid modification R206H.

15. The use according to any one of claims 12 to 14, wherein the subject suffers from fibrodysplasia ossificans progressiva or diffuse intrinsic pontine glioma.

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