EA050257B1 - CRYSTALLINE FORMS OF 3-(5-(2-HYDROXY-2-METHYLPROPOXY)-6-METHYLPYRAZINE-2-YL)-1H-INDOL-7-CARBONITRIL - Google Patents

Abstract

The invention provides crystalline forms of an androgen receptor modulator, a pharmaceutical composition containing said crystalline forms, methods for treating neurodegenerative disorders and cancer, a method for modulating androgen receptor (AR) activity using these crystalline forms, as well as methods for obtaining these forms.

Description

Related applications

This application claims priority to U.S. Provisional Application No. 63/299,678, filed January 14, 2022, the entire contents of which are incorporated by reference.

State of the art

Prostate cancer is the second leading cause of cancer-related mortality in Western men (Damber, J. E. and Aus, G. Lancet (2008) 371:1710–1721). Numerous studies have shown that the androgen receptor (AR) is critical not only for the development of prostate cancer but also for the progression of the disease to a castration-resistant state (Taplin, M. E. et al. J. Clin. Oncol. (2003) 21:2673–8; and Tilley, W. D. et al. Cancer Res. (1994) 54:4096–4102). Thus, effective inhibition of human AR remains one of the most effective therapeutic approaches for the treatment of advanced, metastatic prostate cancer.

Spinal and bulbar muscular atrophy (SBMA) or Kennedy disease is an X-linked recessively inherited neuromuscular disorder. Its main symptoms are weakness and atrophy of the bulbar and limb muscles due to degeneration of the lower motor neurons of the brainstem and spinal cord along with primary muscle damage. At the onset of the disease, patients often experience weakness of the limbs, seizures, tremors and fasciculations, especially noticeable on the face and tongue. Dysarthria with hypernasality, laryngospasm and swallowing disorders are also common, which often lead to aspiration pneumonia as the disease progresses. More than half of patients die from respiratory infections. The disease is caused by a CAG expansion in exon 1 of the androgen receptor (AR), and androgen is required for its occurrence. There is currently no treatment for Kennedy disease.

Brief description of the essence of the invention

Provided herein are crystalline forms useful for treating diseases such as Kennedy's disease in a subject in need thereof. In a particular aspect, provided herein are crystalline forms of the free base of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile having the formula:

In another aspect, the present document provides a method of treating Kennedy's disease in a subject in need thereof, comprising administering to the subject a crystalline form of the free base of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-1-indole-7-carbonitrile.

Brief description of graphic materials

Fig. 1 shows the XRD pattern of the crystalline anhydrate form of C3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 2 shows the DSC thermogram of the crystalline anhydrate form of C3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 3 shows the TGA thermogram of the anhydrate form of C 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-III-indole-7-carbonitrile.

In fig. Figure 4 shows the ¹ H NMR spectrum of the anhydrate form of C 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 5 shows a schematic diagram of the interconversion of polymorphs of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 6 shows the XRD pattern of form O of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 7 shows the XRD pattern of the anhydrate form A of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 8 shows the XRD pattern of the hydrated form B of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 9 shows the XRD pattern of the solvate form D of 3-(5-(2-hydroxy-2-methylpropoxy)6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile.

Fig. 10 shows the XRD pattern of the solvate form E of 3-(5-(2-hydroxy-2-methylpropoxy)6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile.

Fig. 11 shows the XRD pattern of the hydrated form F of 3-(5-(2-hydroxy-2-methylpropoxy)6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile.

Fig. 12 shows the XRD pattern of Form G of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 13 shows the XRD pattern of the anhydrate form of H 3-(5-(2-hydroxy-2

- 1 050257 methylpropoxy)-6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile.

Fig. 14 shows the XRD pattern of the solvate form I of 3-(5-(2-hydroxy-2-methylpropoxy)6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 15 shows the XRD pattern of the anhydrate form J of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-ivdol-7-carbonitrile.

Fig. 16 shows the XRD pattern of the anhydrate form of K 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 17 shows the XRD pattern of the anhydrate form of L 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 18 shows the XRD pattern of form M of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Fig. 19 shows the XRD pattern of the N-hydrate form of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Detailed description

The solid state of a compound may be important when the compound is used for pharmaceutical purposes. The physical properties of a compound may vary from one solid form to another, which may affect the suitability of that form for pharmaceutical use. For example, a particular crystalline solid compound may compensate for the disadvantages of other solid forms of the compound, such as instability and/or reduced purity.

This document provides solid crystalline forms of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile:

This compound is useful for the treatment of a number of indications, including Kennedy's disease, in the subject.

In particular, this document provides crystalline forms of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile or a solvate or hydrate thereof.

The compound 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-1-indole-7-carbonitrile is described in PCT Application No. PCT/US2021/042355 and U.S. Patent Application No. 17/380,736, the entire contents of which are incorporated herein by reference.

The crystalline forms proposed in this paper can be characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).

Definitions

Listed below are definitions of various terms used to describe the crystalline forms disclosed in this document. These definitions apply to the terms as used in the text of the description and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

Unless otherwise defined, all technical and scientific terms used herein generally have the meaning commonly understood by one of ordinary skill in the art to which the compound and its crystalline forms belong. In general, the nomenclature used herein and laboratory procedures in the fields of cell culture, molecular genetics, organic chemistry, and peptide chemistry are well known and widely applicable in the art.

In the context of this document, singular forms refer to one or more than one (i.e. at least one) grammatical entity. As an example, element refers to one element or more than one element. In addition, the use of the term including, as well as other forms such as include, includes and included, is not limiting.

In the context of this document, the term EC ₅₀ refers to the concentration of a compound required to achieve an effect that corresponds to 50% of the maximum observed effect of the compound.

As used herein, the term pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in carrying or transporting a compound useful in the present invention in or into a patient's body so that it can perform its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation, including the compound provided herein, and not deleterious to the patient.

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Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic solution; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations.

As used herein, a pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like that are compatible with the activity of a compound as provided herein and are physiologically acceptable to the patient. Additional active compounds may also be included in the compositions. Other additional ingredients that may be included in pharmaceutical compositions useful for practicing the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

In the context of this document, the expressions therapeutically effective dose and therapeutically effective amount refer to the amount of a compound that prevents the onset, alleviates symptoms, stops the progression of a disease, or results in another desired biological result, such as, for example, improved clinical parameters.

The term treat, treat, treating or treating includes reducing or alleviating at least one symptom associated with or caused by the condition, disorder or disease being treated. In certain embodiments, treating includes contacting an opioid receptor with an effective amount of a compound provided herein for androgen receptor-related conditions.

In the context of this document, the term prevent or preventability means not developing a disorder or disease if it has not already occurred, or not further developing a disorder or disease if the disorder or disease has already occurred. It also includes the ability to prevent some or all of the symptoms associated with a disorder or disease.

As used herein, the term patient, individual, or subject refers to a human or non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, bovines, pigs, dogs, cats, and mice. In one embodiment, the patient, subject, or individual is a human.

The term "administering" and similar terms refer to providing a therapeutic agent, such as a crystalline form described herein, to a subject in need of treatment. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

In the context of this document, the term about will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. In the context of this document, the term about, when used in relation to a measurable value such as a quantity, time duration, etc., covers variations of up to ±10%, including ±5%, ±1% and ±0.1% of the stated value, if such deviations are suitable for performing the described methods.

Solvents can be broadly classified as polar (hydrophilic) and non-polar (lipophilic). Polarity can be measured as the permittivity or dipole moment of a compound.

Non-polar solvents include alkanes such as pentane, hexane, heptane, and cyclohexane. Additional examples of non-polar solvents include benzene, toluene, chloroform, diethyl ether, and petroleum ether.

Examples of polar solvents include pyridine, isopropyl acetate, dichloromethane (DCM), acetone, dimethylformamide (DMF), tert-butyl alcohol, dimethyl sulfoxide (DMSO), acetonitrile, isopropanol, benzyl alcohol, acetic acid, ethanol, methanol, and water.

An aprotic solvent is an organic solvent that does not contain an O-H or N-H bond or exchange protons with the substance dissolved in it. Examples of aprotic solvents include dimethyl sulfoxide (DMSO), isopropyl acetate, dimethylformamide (DMF), dichloromethane (DCM), acetonitrile, acetone, methyl ethyl ketone (MEK), methyl tert-butyl ether (MTBE), 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, tetrahydrofuran (THF), heptane,

- 3 050257 methylcyclohexane and toluene. Additional non-limiting examples include N-methylpyrrolidone, pyridine, piperidine, dimethyl ether, and methyl dodecyl sulfoxide.

Protic solvents are solvents that contain an O-H or N-H bond. Typical protic solvents that may be used in this document include various types of glycols, such as tripropylene glycol methyl ether, dipropylene glycol, and propylene glycol. Examples of other protic solvents include water, ammonia, acetic acid, formic acid, and alcohols such as methanol, ethanol, isopropanol, propanol, and butanol.

Determination of characteristics of crystalline forms.

In certain embodiments, the crystalline forms described herein can be identified based on characteristic peaks in an X-ray powder diffraction analysis. X-ray powder diffraction (XPD) is a scientific technique using the diffraction of X-rays, neutrons, or electrons by powder, microcrystals, or other solid materials to determine structural characteristics of solid materials. A description of the methods used to obtain specific X-ray powder diffraction patterns in connection with the crystalline forms provided herein can be found in the examples below. In one embodiment, the X-ray powder diffraction data provided herein is obtained by a method that utilizes Cu Κα radiation.

In one aspect, the document provides a crystalline form of 3-(5-(2-hydroxy-2methylpropoxy)-6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile.

In one embodiment, the crystalline form is anhydrous.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, and 15.8.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, 15.8, 11.5, 12.2, 12.5, 16.3, 22.5, 22.9, and 30.0.

In one embodiment, the crystalline form is characterized by a XRD pattern having peaks selected from Table 1 (Form C).

Table 1

Corner d-value Rel. Intensity 8,142 ° 10.84978 A 2.2% 10,018 ° 8,82193 A 0.3% 11,495 ° 7,69214 A 2.1% 11,694 ° 7,56146 A 30.4% 12,237 ° 7,22731 A 3.0% 12,502 ° 7,07472 A 3.0% 12,843 ° 6,88737 A 6.2% 14,191 ° 6,23609 A 0.5% 15,819 ° 5,59789 A 24.6% 16,303 ° 5,43251 A 3.0% 17,623 ° 5,02854 A 0.6% 19,122 ° 4.63774 A 0.7% 19,747 ° 4,49214 A 1.3% 20,099 ° 4,41425 A 1.4% 20,714 ° 4,28467 A 33.3% 22,505 ° 3.94764 A 5.0% 22,876 ° 3,88442 A 2.8% 23,822 ° 3.73227 A 100.0% 24,584 ° 3.61828 A 31.8% 25,024 ° 3,55557 A 0.5% 25,444 ° 3,49787 A 0.6% 26,556 ° 3.35384 A 0.8% 27,134 ° 3,28366 A 0.3% 27,889 ° 3,19655 A 0.6% 28,337 ° 3,14701 A 1.7% 29,725 ° 3,00312 A 0.6%

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30,029 ° 2,94776 A 7.1% 30,989 ° 2,88345 A 0.3% 32,066 ° 2,78902 A 0.9% 32,273 ° 2,77163 A 1.6% 32,842 ° 2,72489 A 1.5% 34,830 ° 2,57378 A 0.3% 35,240 ° 2,54472 A 0.9% 35,956 ° 2,49572 A 0.6% 37,378 ° 2,40394 A 0.4%

In another embodiment, the crystalline form has a XRD pattern substantially as depicted in Fig. 1.

In another embodiment, the crystalline form has a DSC thermogram characterized by an endotherm with an onset temperature of about 179°C. In another embodiment, the crystalline form has a DSC thermogram characterized by an endotherm with an onset temperature of 179.3°C.

Treatment methods

This document provides methods for treating a disease comprising administering a crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile or a pharmaceutical composition comprising the crystalline form and a pharmaceutically acceptable carrier.

In one aspect, the document provides a method of treating a neurodegenerative disorder in a subject in need thereof, comprising administering to the subject a crystalline form or a pharmaceutical composition comprising a crystalline form and a pharmaceutically acceptable carrier.

In one embodiment, the neurodegenerative disorder is spinal muscular atrophy (SBMA).

In one embodiment of the methods, the crystalline form is anhydrous.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, and 15.8.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, 15.8, 11.5, 12.2, 12.5, 16.3, 22.5, 22.9, and 30.0.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks selected from Table 1.

In another aspect, the document provides a method of modulating androgen receptor (AR) activity in a subject in need thereof, comprising administering to the subject a crystalline form or a pharmaceutical composition comprising a crystalline form and a pharmaceutically acceptable carrier.

In one embodiment, the androgen receptor (AR) is allosterically modulated. In another embodiment, modulation of androgen receptor (AR) activity provides treatment for spinal muscular atrophy (SBMA) in a subject.

In another embodiment, the crystalline form selectively binds to the binding function domain 3 (BF3) of the androgen receptor.

In another embodiment of the method of modulating AR activity, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, and 15.8.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks selected from Table 1.

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In another aspect, the present document provides a method of treating spinal muscular atrophy (SBMA) in a subject in need thereof, comprising administering to the subject a crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-11-indole-7-carbonitrile.

In another aspect, the document provides a method for treating spinal muscular atrophy (SBMA) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin2-yl)-11-indole-7-carbonitrile and a pharmaceutically acceptable carrier.

In one embodiment of the methods, the crystalline form is anhydrous.

In one aspect, the present document provides a method of treating spinal muscular atrophy (SBMA) in a subject in need thereof, comprising administering to the subject a crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile, wherein the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In one embodiment of the methods, the crystalline form is anhydrous.

In one embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, and 15.8.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks selected from Table 1.

In another aspect, the present document provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a crystalline form or a pharmaceutical composition comprising a crystalline form and a pharmaceutically acceptable carrier.

In one embodiment of the methods, the crystalline form is anhydrous.

In another embodiment of the method of treating cancer, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment of the method, the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, and 15.8.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks selected from Table 1.

In one embodiment, the cancer is selected from hematological malignancies, sarcomas, lung cancer, gastrointestinal cancer, genitourinary cancer, liver cancer, bone cancer, nervous system cancer, gynecological cancer, and skin cancer.

In another embodiment, the cancer is selected from non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma, squamous cell bronchogenic carcinoma, undifferentiated small cell bronchogenic carcinoma, undifferentiated large cell bronchogenic carcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellular carcinoma, bronchial adenoma, and pleuropulmonary blastoma.

In another embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In another embodiment, the lung cancer is adenocarcinoma.

In one embodiment, the gastrointestinal tract is selected from esophageal squamous cell carcinoma, esophageal adenocarcinoma, esophageal leiomyosarcoma, esophageal lymphoma, gastric carcinoma, gastric lymphoma, gastric leiomyosarcoma, pancreatic exocrine carcinoma, pancreatic ductal adenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma, pancreatic gastrinoma, pancreatic carcinoid tumors, pancreatic vipoma, small intestinal adenocarcinoma, small intestinal lymphoma, small intestinal carcinoid tumors, Kaposi's sarcoma, small intestinal leiomyoma, small intestinal hemangioma, small intestinal lipoma, small intestinal neurofibroma, small intestinal fibroma, colon adenocarcinoma, colon tubular adenoma, colon villous adenoma, colon hamartoma, colon leiomyoma, colorectal cancer, gallbladder cancer and anal cancer.

In one embodiment, the gastrointestinal tract is

- 6 050257 colorectal cancer.

In another embodiment, the cancer is a carcinoma. In another embodiment, the carcinoma is selected from pancreatic carcinoma, colorectal carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical carcinoma, skin carcinoma, and thyroid carcinoma.

In another embodiment, the cancer is a hematological malignancy. In one embodiment, the hematological malignancy is selected from multiple myeloma, acute myelogenous leukemia, and myeloproliferative neoplasms.

In another embodiment, the cancer is a neoplasm. In another embodiment, the neoplasm is a glioblastoma or sarcoma.

In one embodiment, the cancer is selected from the group consisting of hematological malignancies, sarcomas, lung cancers, gastrointestinal cancers, genitourinary cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.

In another embodiment, the cancer is selected from the group consisting of pancreatic cancer, cervical cancer, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, carcinoma, and adenocarcinoma.

In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is a solid tumor.

In one embodiment of the methods described herein, the subject is a human.

Methods for obtaining form C.

In one aspect, this document provides a method for preparing a crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile, comprising:

a) combining 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile with a first solvent to form a mixture;

b) adding 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin2-yl)-1H-indole-7-carbonitrile crystal seed to the mixture;

c) stirring the mixture;

d) collecting the solid 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile;

e) washing the solid with a second solvent; and

f) collecting and drying the crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)1H-indole-7-carbonitrile.

In one embodiment, the first solvent is a polar solvent and the second solvent is a non-polar solvent.

In another embodiment, the first solvent is a non-polar solvent and the second solvent is a non-polar solvent.

In another embodiment, the first solvent is an aprotic solvent and the second solvent is an aprotic solvent.

In another embodiment, the first solvent is a protic solvent and the second solvent is an aprotic solvent.

In one embodiment, the first solvent is selected from the group consisting of acetone, 2-methyltetrahydrofuran, tetrahydrofuran, ethyl acetate, methyl ethyl ketone, isopropanol, methyl isobutyl ketone, ethanol, isopropyl acetate, or a combination thereof.

In another embodiment, the second solvent is selected from the group consisting of heptane, methylcyclohexane, and toluene, or a combination thereof.

In another embodiment, the first solvent is 2-methyltetrahydrofuran and the second solvent is heptane.

In another aspect, this document provides a method for producing a crystalline form of 3-(5(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile, comprising:

a) combining 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile with a polar aprotic solvent to form a mixture;

b) adding 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin2-yl)-1H-indole-7-carbonitrile crystal seed to the mixture;

c) stirring the mixture;

d) collecting the solid 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile;

(e) re-equilibrating the solids in a polar aprotic solvent by temperature cycling from about 5°C to about 50°C; and

f) collecting and drying the crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-

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1H-INDOLE-7-CARBONITRIL.

In one embodiment, the polar aprotic solvent in step a) is acetone. In another embodiment, the polar aprotic solvent in step e) is acetone.

In one embodiment of the above methods, the starting material is amorphous.

In another embodiment, the seed for the crystals of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-11-indole-7-carbonitrile in step b) is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In another embodiment, the seed for the crystals of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-11-indole-7-carbonitrile in step b) is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment, the seed for the crystals of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-11-indole-7-carbonitrile in step b) is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6 and 15.8.

In another embodiment, the seed for the crystals of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-11-indole-7-carbonitrile in step b) is characterized by a XRD pattern having peaks, expressed in degrees-2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, 15.8, 11.5, 12.2, 12.5, 16.3, 22.5, 22.9 and 30.0.

In one embodiment, the seed for the 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-1H-indole-7-carbonitrile crystals in step b) is characterized by a XRD pattern having peaks selected from Table 1 (Form C).

In another embodiment, the mixture in step c) is stirred at room temperature. In another embodiment, the mixture in step c) is stirred at 25°C. In another embodiment, the mixture in step c) is stirred for 10 days.

In another embodiment, the temperature cycle of step e) is a cycle from 5°C to 50°C. In one embodiment, the temperature cycle of step e) is carried out in three cycles.

In another embodiment, the crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6methylpyrazin-2-yl)-11-indole-7-carbonitrile in step f) is dried at about 50°C under vacuum.

In another aspect, the document provides a method for preparing the crystalline form described herein, comprising dissolving 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile in a first solvent, adding a second solvent to form a slurry, and filtering the slurry to isolate the crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile.

In one embodiment, the first solvent is selected from the group consisting of acetone, ethyl acetate, methyl tert-butyl ether (MTBE), and 2-methyltetrahydrofuran (MeTHF), or a combination thereof. In another embodiment, the first solvent is ethyl acetate. In another embodiment, the first solvent is acetone. In another embodiment, the first solvent is 2-methyltetrahydrofuran (MeTHF). In one embodiment, the second solvent is heptane. In one embodiment, the second solvent is methyl tert-butyl ether (MTBE).

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, and 15.8.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, 15.8, 11.5, 12.2, 12.5, 16.3, 22.5, 22.9, and 30.0.

In one embodiment, the crystalline form is characterized by a XRD pattern having peaks selected from Table 1 (Form C).

Pharmaceutical compositions.

In one aspect, the present document provides a pharmaceutical composition comprising the crystalline form provided herein and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition comprises a crystalline form that is substantially free of other crystalline forms.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, and 11.7.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6.

In another embodiment, the crystalline form is characterized by a XRD pattern,

- 8 050257 having peaks, expressed in degrees 2-theta, at angles (±0.2 degrees) 23.8, 20.7, 11.7, 24.6 and 15.8.

In another embodiment, the crystalline form is characterized by a XRD pattern having peaks selected from Table 1.

The pharmaceutical compositions can be formulated for oral, intravenous, intramuscular, subcutaneous or parenteral administration for the therapeutic or prophylactic treatment of Kennedy's disease.

The pharmaceutical preparations described herein can be prepared according to standard procedures and administered in dosages selected to ameliorate, prevent, or eliminate disease. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, NY, the contents of which are incorporated herein by reference for a general description of routes of administration of various agents for human therapy.

The pharmaceutical compositions described herein may comprise the crystalline form described herein in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants and/or excipients.

For oral or parenteral administration, the crystalline form described herein can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. Compositions containing the crystalline form described herein can contain from about 0.1% to about 99% by weight of the active compound, such as from about 10% to about 30%.

For oral use, solid formulations such as tablets and capsules are suitable. It is also possible to obtain sustained-release or enteric-coated preparations. For pediatric and geriatric use, in one embodiment, suspensions, syrups and chewable tablets are proposed. For oral use, the pharmaceutical compositions have the form of, for example, tablets, capsules, suspensions or liquids.

Pharmaceutical compositions can be prepared in the form of a dosage unit containing a therapeutically effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, tablets and capsules can contain, in addition to the active ingredient, conventional carriers such as binding agents, fillers, lubricants, disintegrants or acceptable wetting agents. Liquid oral preparations generally take the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs.

The pharmaceutical compositions described herein can be placed in a pharmaceutically acceptable carrier and delivered to a recipient subject (e.g., a human) according to known drug delivery techniques. In general, methods for delivering pharmaceutical compositions in vivo employ art-recognized agent delivery protocols, with the only significant procedural modification being the substitution of the crystalline form of the present invention for the drug in the art-recognized protocols.

Introduction/dosage/composition.

In another aspect, the present document provides a pharmaceutical composition comprising the crystalline form provided herein together with a pharmaceutically acceptable carrier.

The actual dosage levels of the active ingredients in the pharmaceutical compositions discussed herein can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration without toxic effects to the patient.

In particular, the dosage level selected will depend on a number of factors including the activity of the particular compound used, the time of administration, the rate of elimination of the compound, the duration of treatment, other drugs, compounds or materials used in combination with the crystalline form, the age, sex, body weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical art.

A physician, such as a physician or veterinarian, having ordinary skill in the art, can readily determine and prescribe the required effective amount of the pharmaceutical composition. For example, the physician or veterinarian can begin administration of the pharmaceutical composition so as to administer the described crystalline form at lower levels than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is particularly advantageous to formulate

- 9 050257 crystalline form in a single dosage form for ease of administration and uniformity of dosage.

As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the patients to be treated, each unit containing a predetermined quantity of the disclosed compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. Unit dosage forms of the crystalline form disclosed herein are conditioned by and directly dependent on (a) the unique characteristics of the disclosed compound and the particular therapeutic effect desired to be achieved, and (b) the limitations inherent in the art of preparing/formulating such disclosed compound for the treatment of Kennedy's disease (KD) in a patient.

In one embodiment, the crystalline form provided herein is formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions comprise a therapeutically effective amount of the described crystalline form and a pharmaceutically acceptable carrier.

In some embodiments, the dose of a disclosed compound is from about 1 mg to about 1000 mg. In some embodiments, the dose of a disclosed compound used in the compositions described herein is less than about 1000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 20 mg, or less than about 10 mg. For example, the dose is about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, 220 mg, 240, 260 mg, 280 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg or about 600 mg.

Routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual, or topical. The compound for use provided herein may be formulated for administration by any suitable route, such as oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginal), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical routes of administration. In one embodiment, the preferred route of administration is oral.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelatin capsules, troches, dispersions, suspensions, solutions, syrups, granules, spheres, transdermal patches, gels, powders, pellets, slurries, lozenges, creams, pastes, patches, lotions, disks, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol formulations for inhalation, compositions and formulations for intravesical administration, and the like. It should be understood that the formulations and compositions that may be suitable for use in the present invention are not limited to the specific formulations and compositions described herein.

For oral use, tablets, dragees, liquids, drops, suppositories or capsules, caplets and gelatin capsules are particularly suitable. Compositions intended for oral use can be prepared by any method known in the art, wherein such compositions can contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, an inert diluent such as lactose; granulating and disintegrating agents such as corn starch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets can be uncoated or they can be coated by known methods for an attractive appearance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

In the case of parenteral administration, the described compound can be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration as a bolus dose or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing or dispersing agents, can be used.

Numerous equivalents to the specific procedures, embodiments, claims, and examples described herein will become apparent to those skilled in the art, or can be determined using no more than routine experimentation. Such equivalents are considered to be within the scope of the present invention and are described herein.

- 10 050257 with the following claims. For example, it should be understood that variations in reaction conditions, including but not limited to reaction time, reaction size/volume, and experimental reagents such as solvents, catalysts, pressure, atmospheric conditions such as nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It should be understood that wherever values and ranges are presented in this document, all values and ranges encompassed by these values and ranges are to be included within the scope of this disclosure. Moreover, all values falling within these ranges, as well as the upper or lower limits of the range of values, are also considered by this application.

The following examples further illustrate aspects of the present invention. However, they are in no way limiting of the stated principles of the present invention.

Examples

The invention is further illustrated by the following examples, which should not be considered as further limiting. In the practical implementation of the present invention, unless otherwise indicated, conventional methods of organic synthesis, cell biology, cell culture and molecular biology, which are within the competence of specialists in this field of technology, will be used.

Synthesis of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

Step 1. To a degassed mixture of 5-iodo-3-methyl-2-[2-methyl-2-(oxan-2-yloxy)propoxy]pyrazine (150 mg, 0.38 mmol) and tert-butyl 7-cyano-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indole-1-carboxylate (140 mg, 0.38 mmol) in dioxane (2 mL) and water (0.2 mL) were added potassium carbonate (105 mg, 0.76 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (27 mg, 0.04 mmol) at room temperature. The mixture was stirred at 100 °C for 2 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography with 0-35% ethyl acetate in petroleum ether to give 3-[6-methyl-5-[2-methyl-2-(oxan-2-yloxy)propoxy]pyrazin-2-yl]-1H-indole-7-carbonitrile (67 mg, 43%) as a white solid. MS m/z 407.1 [M+1]+.

Step 2. To a solution of 3-[6-methyl-5-[2-methyl-2-(oxan-2-yloxy)propoxy]pyrazin-2-yl]-1H-indole-7-carbonitrile (62 mg, 0.15 mmol) in dichloromethane (2.1 mL) was added trifluoroacetic acid (0.7 mL) at room temperature. The mixture was stirred at room temperature for 3 h. The mixture was concentrated in vacuo. The residue was purified by reverse phase flash column chromatography with 10~80% acetonitrile in water (10 mmol/L ammonium bicarbonate) to give 3-[5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl]-1H-indole-7-carbonitrile (21.3 mg, 43%) as a white solid. ^1H NMR (400 MHz, DMSO^) δ 12.35 (s, 1H), 8.73 (d, J=8.0 Hz, 1H), 8.56 (s, 1H), 8.19 (d, J=2.4 Hz, 1H), 7.69-7.67 (m, 1H), 7.28 (t, J=8.0 Hz, 1H), 4.70 (br, 1H), 4.10 (s, 2H), 2.54 (s, 3H), 1.24 (s, 6H); MS (IER) calc. for C18H18N4O2 [M+1]+, 323.1; received, 323.0.

Biological analysis.

LNCaP cells expressing ARR2PB-FireflyLuc and CMV-RenillaLuc were treated with the indicated concentrations of test compound, enzalutamide (negative control), or DHT (positive control) +/- 0.5 nM DHT for 48 h at 37°C. Fluorescent signals were recorded using the ImageXpress microconfocal system. Residual activity (antagonist mode) was calculated as % residual activity = 100X [( _sample counts - LC _cp .)/(HC _cp . LC _cp .)], where HC represents cells treated with 0.5 nM DHT alone and LC represents cells treated with 10 μM enzalutamide + 0.5 nM DHT. Activation (agonist mode) was calculated as % activation = 100X [(sample counts - LCcp.)/ (HCcp. - LCcp.)], where HC represents cells treated with 1 μM DHT and LC represents cells treated with DMSO. Dose response curves and _IC50 values were calculated using nonlinear regression analysis in XLfit. 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-N-indole-7-carbonitrile was found to have an _IC50 value of 50 nM.

Analytical methods for studying polymorphs.

Unless otherwise stated, powder X-ray diffraction (PXRD) was performed on a Bruker D8 Advance diffractometer in reflection mode using collimated Cu Κα radiation with an operating characteristic of 40 kV, 40 mA. Scans were performed over the range of 2-40 degrees 2-theta with a step size of 0.02 degrees and a scan time of 0.3 s per step.

Differential scanning calorimetry was performed on a TA Discovery 2500 with a Tzero crucible and a Tzero sealed lid with a 0.7 mm diameter opening. DSC analysis was performed by stepping the sample at 10°C/min or 20°C/min from 30 to 250°C or from 0 to 250°C.

Thermal gravimetric analysis (TGA) was performed on a Discovery 5500 or Q5000 with an initial temperature at ambient (below 35°C) and a final temperature of 300°C (or cancellation of the next segment if mass < 80% (w/w)) with heating at 10°C/min.

Nuclear magnetic resonance (NMR) analysis was performed on a Bruker Avance-AV 400M at

- 11 050257 frequency 400 MHz and eight scans.

Dynamic vapor sorption (DVA) was performed on an Intrinsic, Advantage or Adventure with an oven temperature of 25°C using water as the solvent. The sample weight was about 5-10 kg using the following method:

Cycle: 40-0-95-0-40% RH

Stage step: 10%

Equilibrium: 0.002 dm/dt (%/min)

Minimum stability duration dm/dt: 60 min

Maximum time of the dm/dt stage: 360 min.

Polymorph analysis.

The free form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile exhibits complex polymorphic behavior. A total of 15 forms were identified as polymorphs or pseudopolymorphs of the free form, including seven anhydrates, forms A, C, H, J, K, L, and O; four hydrates, forms B, F, M, and N; and three solvates, forms D, E, and I. In addition, a metastable form, form G, and an amorphous form were obtained.

Form A is an anhydrate. It has been prepared from many solvent systems by equilibration, slow cooling, rapid cooling, evaporation, vapor diffusion, and antisolvent addition experiments. Form A has a high degree of crystallinity. DSC shows a melting point peak at a T _{onset of} 170.2°C, followed by another endothermic peak at a T _{onset of} 178.9°C. TGA shows a mass loss of about 0.2% at about 160°C. ¹ H NMR shows no detectable residual solvent. Form A converts to Form C at both 25°C and 50°C, suggesting that Form A is thermodynamically less stable than Form C. The XRD pattern of Form A is shown in Fig. 7.

Form B is a hydrate. It was prepared from methanol only system by temperature cycling, slow cooling, slow evaporation and fast evaporation. Form B has high crystallinity. DSC shows a dehydration peak at T _onset of 16.7 °C with an enthalpy of about 22 J/g and a small exothermic peak at T _{onset of} 130.5 °C. This is followed by 2 unresolved endothermic peaks at T _{onset of} 170.2 °C and T _{onset of} 173.1 °C, respectively. After that, it shows two small unresolved endothermic peaks at T _{onset of} 177.8 °C and T _{onset of} 179.0 °C, respectively. TGA shows a mass loss of about 2.1% at about 150 °C. CF shows that it contains about 3.8% water by weight, which is equivalent to 0.7 water molecules. ¹ H-NMR shows no detectable residual solvent. Form B exhibits reversible dehydration-hydration behavior. It converts to Form K upon dehydration, and Form K converts back to Form B upon exposure to 25°C at 40-70% RH for 6 days. Form B is a metastable hydrate and converts to the anhydrate form C over the entire range of water activity (0 < a. w. < 1). The XRD pattern of Form B is shown in Fig. 8.

Form C is an anhydrate. It was prepared from several solvent systems by equilibration, slow cooling, rapid cooling, slow evaporation, and vapor diffusion. Form C has a high degree of crystallinity. DSC shows a melting peak at a T _{onset of} 179.3 °C with an enthalpy of about 114 J/g (Figure 2). TGA shows a mass loss of about 0.8% at about 150 °C (Figure 3). 1H NMR shows no detectable residual solvent (Figure 4). Form C is the most stable polymorph compared to the other polymorphs described herein. Competitive equilibration experiments show that Form C is a thermodynamically stable anhydrate at both 25 °C and 50 °C. Water activity studies show that Form C is stable over the entire range of water activities (0 < a. w. < 1). The XRD pattern of Form C is shown in Figure 1. 1.

Form D is a solvate of 1,4-dioxane. It was prepared from only 1,4-dioxane system by equilibration, slow cooling, rapid cooling, slow evaporation, rapid evaporation and vapor diffusion experiments. Form D has a high degree of crystallinity. DSC shows two desolvation peaks at T _{onset of} 88.0 °C with an enthalpy of about 27 J/g and at T _{onset of} 138.6 °C with an enthalpy of about 41 J/g, suggesting that 1,4-dioxane molecules have two types of intermolecular interaction formats with the API molecule in the crystal structure of Form D. Then it melts with T _{onset of} 179.1 °C with an enthalpy of about 111 J/g. TGA shows about 9.1% mass loss at about 130°C and about 2.8% mass loss from about 130°C to 160°C. 1H-NMR shows 0.5 equiv (13.6%) of 1,4-dioxane residue by mass. Form D converts to Form C after desolvation. The XRD pattern of Form D is shown in Fig. 9.

Form E is an EtOH/water solvate. It was prepared from the ethanol system using flash evaporation experiments. Form E has a high degree of crystallinity. DSC shows a desolvation/dehydration peak at Ton at 80.4 °C with an enthalpy of about 8 J/g and unresolved melting peaks at Ton _of 165.2 °C and 170.2 °C, respectively. This unresolved melting peak is followed by a recrystallization peak at Ton _of 173.1 °C. After recrystallization, it

- 12 050257 exhibits a melting point at T _{onset of} 177.9°C, which is close to the melting point of Form C. TGA shows about 2.1% mass loss at about 120°C. ¹ H-NMR shows 1.4% EtOH by mass. CF shows that it contains about 0.6% water by mass. Form E converts to a physical mixture of Form F and Form G after heating to 100°C by TGA and cooling to room temperature. The XRD pattern of Form E is shown in Fig. 10.

Form F is a hydrate. It was prepared from THF or MEK system by flash evaporation experiments. Form F has a high degree of crystallinity. DSC shows 2 unresolved melting peaks at Tb _of 163.2 °C and 169.7 °C combined with a recrystallization peak at Tb _of 171.7 °C. After recrystallization, it shows a melting point at Tb _of 177.9 °C which is close to the melting point of Form C. TGA shows about 0.5% mass loss at about 140 °C and about 0.7% mass loss from about 140 °C to 180 °C. 1H-NMR shows 0.5% residual THF by weight. CF shows that it contains about 1.7% water by weight. Form F is a metastable hydrate and converts to Form C over the entire range of water activities (0 < a. w. < 1). The XRD pattern of form F is shown in Fig. 11.

Form G is a metastable form. It was prepared from DMSO/water using antisolvent addition experiments. Form G has a high degree of crystallinity. It is converted to anhydrated form H after equilibration in DMSO/water at 25°C for 6 days. The XRD pattern of form G is shown in Fig. 12.

Form H is an anhydrate. It was prepared from acetone or acetone/water (v:v=20:80) by slow evaporation experiments. Form H has a high degree of crystallinity. DSC shows unresolved endothermic peaks at T _{onset of} 169.3 °C and 173.2 °C, respectively. Thereafter, it shows a small endothermic peak at T _{onset of} 179.2 °C. TGA shows a mass loss of about 1.2% at about 150 °C. ¹ H NMR shows no detectable residual solvent. Form H is a metastable anhydrate. It converts to Form C at both 25 °C and 50 °C, suggesting that Form H is thermodynamically less stable than Form C. The XRD pattern of Form H is shown in Fig. 13.

Form I is a THF solvate. It was prepared from THF/heptane using antisolvent addition experiments. Form I has a high degree of crystallinity. DSC shows a desolvation peak at T _{onset of} 92.8 °C with an enthalpy of about 73 J/g. It then melts at T _{onset of} 178.5 °C with an enthalpy of about 114 J/g. TGA shows a mass loss of about 10.0% at about 130 °C. ¹ H NMR shows 0.5 equiv (10.1%) remaining THF by mass. Form I converts to a physical mixture of Form C, Form N, and an unknown peak after heating to 140 °C by TGA and cooling to room temperature. The XRD pattern of Form I is shown in Fig. 14.

Form J is an anhydrate. It was prepared by heating and cooling DSC. Form J has a high degree of crystallinity. DSC shows a small exotherm with an onset at 94.0 °C, a peak melting point at Ton of 172.1 °C, followed by another endotherm at Ton _of 177.4 °C. TGA shows about 0.6% mass loss at about 150 °C. ¹ HNMR shows no detectable residual solvent. Form J is a metastable anhydrate and converts to Form C at both 25 °C and 50 °C, suggesting that Form J is thermodynamically less stable than Form C. The XRD pattern of Form J is shown in Fig. 15.

Form K is an anhydrate. It was prepared from the methanol system from equilibration, quenching, and dehydration experiments of Form B. Form K has a high degree of crystallinity. DSC shows two unresolved melting peaks at T _{onset of} 168.7 °C and 171.9 °C combined with a recrystallization peak at T _{onset of} 173.4 °C. After recrystallization, it shows two unresolved melting peaks at T _{onset of} 177.3 °C and 178.8 °C, respectively. Form K is unstable and converts to hydrated Form B after exposure to 25 °C/92% RH for 6 days. The XRD pattern of Form K is shown in Fig. 16.

Form L is an anhydrate. It was obtained by heating Form B to 160°C and cooling to room temperature. Form L has a high degree of crystallinity. Based on the DSC thermogram and HT-XRD results, Form K converts to Form L after melting and recrystallization. Melting of Form L occurs at T _{onset of} 173.5°C with an enthalpy of about 86 J/g. Form L is a metastable anhydrate and converts to Form C at both 25°C and 50°C, suggesting that Form L is thermodynamically less stable than Form C. The XRD pattern of Form L is shown in Fig. 17.

Form M is an intermediate crystalline form. It was prepared from the methanol system by equilibration. Form M has a high degree of crystallinity. It is converted to Form B after equilibration in MeOH at 25°C for another 1 day. Therefore, complete characterization data for Form M are not available. The XRD pattern of Form M is shown in Fig. 18.

Form N is a hydrate. It was prepared by exposing the amorphous form to 25°C/92% RH in an open container for 3 days. Form N has a high degree of

- 13 050257 crystallinity. DSC shows a dehydration peak at T _{onset of} 47.1 °C, an exothermic peak at T _{onset of} 81.1 °C and an endothermic peak at T _{onset of} 128.5 °C with an enthalpy of about 11 J/g. Then it melts at T _{onset of} 171.6 °C with an enthalpy of about 76 J/g. Two small endothermic peaks at T _{onset of} 177.3 °C and 178.8 °C are present after melting. CF shows that it contains about 8.7% water by weight, which is equivalent to 1.5 water molecules. Form N is a metastable hydrate and converts to form C over the entire range of water activities (0 < a. w. < 1). The XRD pattern of form N is shown in Fig. 19.

Form O is an anhydrate. It was prepared by temperature cycling, slow evaporation, and flash evaporation in several solvent systems. Form O has a high degree of crystallinity. DSC shows unresolved melting peaks at T _{onsets of} 170.1°C and 178.5°C, respectively. TGA shows a mass loss of about 0.7% at about 180°C. 1H NMR shows no detectable residual solvent. The XRD pattern of Form O is shown in Fig. 6.

The amorphous form was prepared by heating and cooling TGA using the free form. The amorphous form crystallized into form N after exposure to high humidity.

Table 2 shows the screening conditions for the polymorphs discussed above.

Table 2

Summary of polymorph screening conditions

Polymorphs Screening experiments Form A, anhydrate From most solvent systems by equilibration, slow cooling, rapid cooling, slow evaporation, rapid evaporation, vapor diffusion and antisolvent addition experiments. Form B, hydrate From a methanol system using temperature cycling, slow cooling, slow evaporation and fast evaporation Form C, anhydrate From most solvent systems by equilibration, slow cooling, rapid cooling, slow evaporation and vapor diffusion. Form D, solvate From the 1,4-dioxane system by equilibration, slow cooling, rapid cooling, slow evaporation, rapid evaporation and vapor diffusion experiments. Form E, solvate From an ethanol system using fast evaporation experiments Form F, hydrate From THF or MEK system by flash evaporation experiments Form G From DMSO/water system using antisolvent experiments Form H, anhydrate From acetone or acetone/water mixture (v:v=20:80) using slow evaporation experiments Form I, solvate From THF/heptane system using antisolvent experiments for 6 days Form J, anhydrate Using DSC with heating and cooling Form K, anhydrate From the methanol system via equilibration experiments, rapid cooling and from form B after dehydration Form L, anhydrate After heating Form B to 160°C and cooling to room temperature Form M From the methanol system by equilibration Form N, hydrate Exposure of amorphous form to 25°C/92%RH in open container for 3 days Form O From water system by temperature cycle, IPAC system by slow evaporation, ACN system by fast evaporation Amorphous form Prepared by DSC with heating and cooling using the free form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)1H-indole-7-carbonitrile

Obtaining Form C.

Form C was obtained using the following procedure:

200 mg of the free form was weighed into a 4 mL glass vial. 200 μL of acetone was added to the vial with stirring at 25 °C. About 3 mg of Form C seed was added to the solution. After stirring at 25 °C for 10 days, the solids were collected by suction filtration and then dried at 25 °C under vacuum for about 4 hours. The dry solids were re-equilibrated in 100 μL of acetone with a temperature cycle from 5 °C to 50 °C at a rate of

- 14 050257 heating/cooling 0.1°C/min. After 3 cycles, the solids were collected by suction filtration at 5°C and then dried at 50°C under vacuum for about 2 hours. 136 mg of Form C was obtained as an off-white solid in 67% yield.

An alternative way to obtain Form C.

Purification: A total of 11.5 kg of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-1-Nindole-7-carbonitrile was purified from crude 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-1-Nindole-7-carbonitrile (17.0 kg) in one batch using 2-MeTHF/n-heptane. The desired product was isolated as a white solid with a purity of 100% by HPLC. Purification was performed using the following procedure:

1. Loading 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile (1X (0.95-1.05X)) into the first reactor.

2. Loading 2-MeTHF (3.3-3.9 volumes) into the reactor.

3. Bringing the reactor to 45-55°C.

4. Stirring the solution in the reactor for 0.5-2 hours at 45-55°C to obtain a clear solution.

5. Decolorization of the organic layer by filtration through a carbon filter at 45-55°C for 5-15 h.

8. Taking a sample for analysis.

9. Transfer liquid to the second reactor through a cartridge filter.

10. Wash the first reactor and carbon filter with 2-MeTHF (1.5-3.0 volumes) twice.

11. Transferring the washing liquid to the second reactor through a cartridge filter.

12. Bringing the second reactor to 55-65°C.

13. Concentration of the contents of the second reactor to 2-4 volumes below 65°C in a vacuum.

14. Loading 2-MeTHF (1.0-5.0 volumes) into the second reactor via a sprayball.

15. Bringing the second reactor to 55-65°C.

16. Loading n-heptane (1.5-2.8 volumes) into the second reactor at 55-65°C for 1-5 hours.

17. Loading the seed (0.001-0.10 volumes) into the second reactor at 55-65°C.

18. Stir the second reactor for 1-4 hours at 55-65°C.

22. Taking a sample for analysis.

23. Loading n-heptane (4.1-9.0 volumes) into the second reactor at 55-65°C for 7-12 hours.

24. Stir the second reactor for 1-5 hours at 55-65°C.

25. Bringing the second reactor to 20-30°C over 7-12 hours.

26. Stir the second reactor for 5-15 hours at 20-30°C.

27. Taking a sample for analysis.

28. Filtration and washing of the precipitate with a solution of 2-MeTHF/n-heptane (1.5-2.9 volumes) twice.

29. Taking a sample of mother liquor for analysis.

30. Taking a wet sediment sample for analysis.

31. Drying the wet sediment at 40-50°C for 15-35 hours.

32. Taking a sample for analysis.

33. Sifting and packaging.

34. Preparation of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile.

35. Taking a sample for analysis.

Interrelation of polymorphs.

The mutual transformation of the identified polymorphs is shown in Fig. 5.

The relative stability of the anhydrate forms A, C, H, and J was investigated in competitive equilibration experiments. Form C was obtained in all selected solvents at both 25°C and 50°C except MeOH, which gave form B. An intermediate crystalline form, form M, was obtained from methanol as a wet cake and was readily converted to form B upon exposure to air. The presence of intermediate form M interferes with the results of the competitive equilibration experiments and may not reflect the actual thermodynamic relationships between the anhydrates in methanol. In the other solvent systems selected, form C was the most stable form.

The relative stability of the anhydrates of forms C, K and L was investigated in competitive equilibration experiments. Form C was prepared in IPAC at both 25°C and 50°C.

Therefore, form C is a thermodynamically stable anhydrate at both 25°C and 50°C.

The relative stability of the anhydrate form C, hydrate form B and hydrate form F were investigated in competitive water activity experiments. The relative stability of the anhydrate form C and hydrate form N were also investigated in water activity experiments in an acetone/water solvent mixture (a. w. = 0.95). The results show that form C is the thermodynamically stable form over the entire range of water activity (0 < a. w. < 1). Thus,

- 15 050257 thus, the anhydrate form C is the optimal polymorph of the free form of 3-(5-(2-hydroxy2-methylpropoxy)-6-methylpyrazin-2-yl)-1H-indole-7-carbonitrile.

Competitive balancing.

To determine the relative stability of the anhydrates, competitive equilibration experiments were performed in different solvent systems.

About 2 mg of each anhydrate was added to 0.3-0.4 ml of saturated solutions of the selected solvents. The resulting suspensions were stirred at 25°C for 3-10 days, respectively. The solid particles (wet sediment) were isolated by centrifugation and analyzed by XRD. The results are shown in Table 3.

Table 3

Experiments in competitive equilibration Patterns: Shape A+Shape C+Shape J+Shape H No. Solvents PRD Comments 25°C for 4 days 50°C for 4 days 1 IPAC Form C Form C // 2 2-MeTHF/heptane (v:v=1:1) Form C Form C // 3 IEC Form C Form C // 4 MeOH Form M Physical mixture of form B and form M Starting materials Forms A, C, J and H in methanol for 4 days: DRP: Form M Starting materials in methanol for 5 days: DRP: Form B DSC: Dehydration from 30°C 173.5°C, 78 J/g Unresolved melting peak: 178.2°C; 179.3°C 5 Acetonitrile Form C Form C Samples: Form A + Form C + Form J + c )form H No. Solvents PRD Comments 25°C for 10 days 50°C for 10 days 4 MeOH Form B Form K 25°C: DSC: Dehydration from 30°C; Unresolved onset: 170.2°C, 173.2°C; Recrystallization: 175.2°C; Unresolved onset of melting: 178.1°C, 179.2°C 50°C: DSC: Onset: 172.9°C, 75 J/g; Unresolved onset: 177.7°C, 179.0°C Patterns: C shape+K shape+L shape No. Solvents PRD Comments 25°C for 3 days 50°C for 3 days 6 IPAC Form C+shape A peaks Form C // 25°C for 6 days // // 7 IPAC Form C // //

//=No comments.

Physicochemical characteristics

The physicochemical characteristics of form C are summarized in Table 4.

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Table 4 Physicochemical characteristics

Polymorph Form C Parameters Way Results Purity HPLC 99.2% X-ray diffraction 3-40° (2 theta) High crystallinity - shown in Fig. 1 Onset of melting and enthalpy DSC, 10°C/min Start: 179.3°C, 114 J/g - shown in Fig. 2 Thermogravimetry TGA, 10°C/min 0.8 at 150°C, as shown in Fig. 3 Stoichiometry Ή-NMR (DMSO6) There is no detectable residual solvent, as can be seen in Fig. 4

Stability of bulk product

The stability of bulk product Form C was assessed at 25°C/92% RH in an open container, 40°C/75% RH in an open container, and 60°C in a tightly closed container for 1 week. Form C is physically and chemically stable after stress under these conditions, as shown in Table 5.

Table 5

Stability of bulk product

Exp. ID Original purity 99.2% Purity Color BS1 Solid state, 25°C/92%RH, open container, 1 week Bulk product (HPLC) 99.2% No color change Bulk product (BP) Form C BS2 Solid state, 40°C/75%RH, open container, 1 week Bulk product (HPLC) 99.1% No color change Bulk product (BP) Form C BS3 Solid state, 60°C, tightly closed container, 1 week Bulk product (HPLC) 99.2% No color change Bulk product (BP) Form C

The stability of Form C was further investigated under the following conditions and the results are summarized in Tables 5A–5F.

Table 5A

Conditions/State when placed: 25°C ± 2° C/60%RH ± 5%RH, normal Initial state 01 month 03 month 06 month Appearance Appearance Whitish powder Whitish powder Whitish powder Whitish powder Purity (% area), HPLC Purity (% area), HPLC Report 99.2 99.3 99.2 99.3 Impurities (area %), HPLC SM1 (% area) < 0.05 < 0.05 < 0.05 < 0.05 SM2 (% area) < 0.05 < 0.05 < 0.05 < 0.05 RRT 1.07 (% of area) 0.77 0.71 0.77 0.73

Table 5B

Conditions/State when placed: 25°C ± 2° C/60%RH ± 5%RH, but rma Initial state 01 month 03 month 06 month Impurities (area %), HPLC A (% of area) < 0.05 < 0.05 < 0.05 < 0.05 B (% of area) < 0.05 < 0.05 < 0.05 < 0.05 C (% area) < 0.05 < 0.05 < 0.05 < 0.05 D (% of area) < 0.05 < 0.05 < 0.05 < 0.05 Unidentified impurity < 0.05 < 0.05 < 0.05 < 0.05 PRD PRD Complies N/A N/A N/A

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form C Water content (% wt.) Water content (% wt.) < 0.05 < 0.05 < 0.05 < 0.05

Table 5C

Conditions/State when placed: 25°C ± 2° C/60%RH ± 5%RH, normal 09 month 12 month Appearance Appearance Whitish powder Whitish powder Purity (% area), HPLC Purity (% area), HPLC 99.3 99.3 Impurities (area %), HPLC SM1 (% area) < 0.05 < 0.05 SM2 (% area) < 0.05 < 0.05 RRT 1.07 (% of area) 0.72 0.74 A (% of area) < 0.05 < 0.05 B (% of area) < 0.05 < 0.05

Table 5D

Conditions/State when placed: 25°C ± 2° C/60%RH ± 5%RH, normal 09 month 12 month Impurities (area %), HPLC C (% area) < 0.05 < 0.05 D (% of area) < 0.05 < 0.05 Unidentified impurity < 0.05 < 0.05 PRD PRD N/A Corresponds to form C Water content (% wt.) Water content (% wt.) < 0.05 < 0.05 Analysis (% wt), HPLC Analysis (% wt), HPLC 99.4 99.4

Table 5E

Conditions/State when placed: 40°C ± 2° C/75%RH ± 5%RH, normal Initial state 01 month 03 month 06 month Appearance Appearance Whitish powder Whitish powder Whitish powder Whitish powder Purity (% area), HPLC Purity (% area), HPLC 99.2 99.3 99.2 99.3 Impurities (area %), HPLC SM1 (% area) < 0.05 < 0.05 < 0.05 < 0.05 SM2 (% area) < 0.05 < 0.05 < 0.05 < 0.05 RRT 1.07 (% of area) 0.77 0.70 0.77 0.73 A (% of area) < 0.05 < 0.05 < 0.05 < 0.05 B (% of area) < 0.05 < 0.05 < 0.05 < 0.05

Table 5F

Conditions/State when placed: 40°C ± 2° C/75%RH ± 5%RH, normal Initial state 01 month 03 month 06 month Impurities (area %), HPLC C (% area) < 0.05 < 0.05 < 0.05 < 0.05 D (% of area) < 0.05 < 0.05 < 0.05 < 0.05 Unidentified impurity < 0.05 < 0.05 < 0.05 < 0.05 PRD PRD Corresponds to form C N/A N/A Corresponds to form C Water content (% wt.) Water content (% wt.) < 0.05 < 0.05 < 0.05 < 0.05 Analysis (% wt), HPLC Analysis (% wt), HPLC 99.4 99.7 98.7 99.1

As can be seen above, form C is stable under conditions where temperature and relative humidity change.

Solubility.

Anhydrate form C (equivalent to 8 mg of free form) was weighed into a 20 ml glass

- 18 050257 vial. 4 ml of dissolution medium were added. The resulting suspensions were stirred at 37°C at 400 rpm. Some of the suspensions were collected after 2 h and 24 h, respectively. They were then centrifuged at 37°C at 14,000 rpm for 5 min. The supernatants were analyzed by HPLC and pH meter for solubility and pH, respectively. The residual solids (wet cake) were characterized by PDP to determine the physical form.

Table 6 Solubility

Way Solubility at 37°C, target concentration 2 mg/mL (free form), equilibration for 24 hours, LOQ: 0.35 μg/mL Exp. ID Dissolution medium 2 h. 24 h Solubility (mcg/ml) Solubility (µg/ml) (pH) PRD residual solids ES1 HCl solution with pH 1.0 (0.1 N) 17.01 14.58 (1.0) Form C ES2 Citrate buffer pH 3.0 (50 mM) 5.97 4.08 (3.1) Form C ES3 Acetate buffer pH 5.0 (50 mM) 5.44 3.85 (5.1) Form C ES4 Phosphate buffer pH 7.0 (50 mM) 5.43 11.57 (7.3) Form C

The results of additional solubility studies are presented in Table 7.

Table 7

Solvents 50°C 25°C Solubility (mg/ml solvent) PRD Solubility (mg/ml solvent) PRD Water ~ 0 Form C ~ 1 Form C Ethanol 64 27 Acetone 122 66 THF > 200 n/a 247 EA 58 Form C 32 Heptane ~ 3 ~ 2 2-MeTHF 154 117 Toluene 5 5 IEC 110 63 IPS 29 14 n-PrOH 48 21 MCG ~ 3 ~ 3 MIBK 28 21

These data indicate that form C has high solubility in acetone, THF, 2MeTHF and MEK, but poor solubility in water, n-heptane and MCH. Since potential form A/H (anhydrate) or form I (THF solvate) may exist in aqueous or THF system according to the polymorph screening report, water and THF were avoided in the subsequent study.

To achieve high yield for the crystallization process, additional solubility in acetone, 2-MeTHF and MEK in combination with antisolvents n-heptane and MCH at 25°C was measured, as shown in Table 8.

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Table 8

Solubility in a mixture of solvents

Solvent systems (v/v) 25 GO Solubility (mg/ml) PRD Adeton/MCG = 1/1 30 Form C Form C Acetone/MCG = 1/2 15 IEC/MCG = 1/1 20 IEC/MCG = 1/2 9 2-MeTHF/MCH = 1/1 1S 2-MeTHF/MCH = 1/2 6 2-MeTHF/MCH = 1/3 -4 Acetone-n-heptane = 1/1 15 Acetone/n-heptane = 1/2 10 Acetone/n-geggan = 1/3 5 MEK/'n-heptane = 1/1 16 MEK/n-heptane = 1/2 6 MEK/n-heptane = 1/3 -3 2-ME-THF/n-heptane = 1/1 13 2-ME-THF/n-heptane = 1/2 -4 2-ME-THF/n-heptane = 1/3 _ 2

These data indicate that the solubility decreased as the antisolvent was added to the MEK, 2-MeTHF, and acetone solvents.

Further solubility studies were performed in 2-MeTHF in combination with the antisolvent n-heptane at 50°C, as shown in Table 9.

Table 9 Solubility in solvent mixtures

Solvent systems (v/v) 50 (°C) Solubility (mg/ml) PRD 2-ME-THF/n-heptane=4/0.5 122 Form C 2-ME-THF/n-heptαn=4/1 90 2-ME-THF/n-heptane=4/2 49 2-ME-THF/n-heptane=4/4 21 2-ME-THF/n-heptane=4/8 10 2 - ME-TG F/n-heptane=4/12 5

The chemical stability in solution was determined in acetone, MEK, and 2-MeTHF solvent systems at 50°C and 55°C. The results are summarized in Table 10. These data indicate that the compound was stable after 48 h of storage in both acetone and 2-MeTHF systems. The purity of the compound decreased from 99.43% to 98.27% after 48 h of storage at 55°C in the MEK solvent system.

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Table 10

Study of chemical stability in solution at 50°C and 55°C

Solvent system Concentration (mg/ml solvent) T/°C Purity (a%) 0 h 6 h 24 h 48 h Acetone ~ 10 mg/ml 50 99.54% 99.50% 99.51% 99.69% IEC 55 99.43% 99.23% 98.54% 98.27% 2-MeTHF 55 99.35% 99.55% 99.62% 99.62%

2-MeTHF/n-heptane was selected as a potential solvent system for crystallization. The solution chemical stability was performed in 2MeTHF solvent systems at higher temperatures (60°C and 70°C). The data indicate that the compound was stable after 48 h in 2-MeTHF at 60°C and 70°C. The solution chemical stability study was performed in 2-MeTHF/n-heptane=1v/1v solvent system at 70°C and the data indicate that the compound was stable after 48 h in the system at 70°C.

Table 11

Study of chemical stability in solution at 60°C and 70°C

Solvent system Concentration (mg/ml solvent) T/°C Purity (a%) 0 h 24 h 48 h 2-MeTHF ~ 10 mg/ml 60 99.54% 99.69% 99.56% 70 99.53% 99.70% 99.44% 2-MeTHF/n-heptane = 1 vol./1 vol. 70 98.45% 98.44% 98.44%

Crystallization from antisolvent.

According to the solubility and chemical stability data, antisolvent crystallization experiments were carried out in acetone/n-heptane and 2-MeTHF/n-heptane systems as shown below.

Pre-crystallization from antisolvent in the 2-MeTHF/n-heptane system:

1. Dissolution in 8 vol. 2-MeTHF at 50°C.

2. Bring to 25°C in 0.5 h

3. Sowing (10 mg)

4. Stir for 1 hour at 25°C

5. Addition of 24 vol. n-heptane for 4 h at 25°C

6. Taking a sample for analysis of PRD-1 and MPS-1.

7. Stir for 15 hours at 25°C

8. Taking a sample for analysis of PRD-2, MPS-2 and analysis-1.

9. Stir for 8 hours at 25°C

10. Filtration and washing of precipitate 2 vol. 2-MeTHF/n-heptane=1 vol./3 vol.

11. Drying of wet precipitate in vacuum at 50°C

12. Receiving material

After addition of n-heptane, PRD showed form C even after aging at 25°C (loss of about 6% mother liquor (ML)). The dried product showed 99.41% purity with 0.46% residual 2-MeTHF and 0.07% residual n-heptane.

Pre-crystallization from antisolvent in the acetone/n-heptane system:

1. Dissolution in 9 vol. acetone at 50°C.

2. Bring to 45°C.

3. Sowing (10 mg)

4. Stirring for 0.5 h at 45°C (liquid suspension)

5. Bring to 42°C.

6. Stir for 1 hour at 42°C (high solids suspension)

7. Bring to 25°C in about 3.5 hours.

8. Selection of a sample for analysis of PRD-1 and MPS-1.

9. Addition of 27 vol. n-heptane over 4.5 h at 25°C

10. Stir for 12 hours at 25°C

11. Taking a sample for analysis of PRD-2, MPS-2 and analysis-1.

12. Stir for 6 hours at 25°C

13. Bring to 5°C in 4 hours.

14. Stir for 14 hours at 5°C

15. Taking a sample for analysis of PRD-3, MPS-3 and analysis-2.

16. Filtration and washing of sediment 2 vol. acetone/n-heptane=1 vol./3 vol.

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17. Drying of wet precipitate in vacuum at 50°C

18. Receiving material.

Before adding n-heptane, the PRD showed form C. After adding n-heptane, the PRD displayed form C even after holding at 25°C (loss of about 17% of MF). Before filtration, the MF loss was about 12%. The dried product was 99.61% pure with 0.03% residual acetone and 0.04% residual n-heptane.

Process optimization in 2-MeTHF/n-heptane.

According to the results of preliminary crystallization from antisolvent in 2-MeTHF/n-heptane and acetone/n-heptane, the 2-MeTHF/n-heptane system was determined as the main system for crystallization. Two test experiments were carried out, obtaining 11 g of crystallized product. The results can be seen in Table 12.

Table 12

Temp. Results After adding anti-solvent Before filtration dry product PRD PRD PRD Purity Residual solvent Exit 25°C Form C Form C Form C 99.46% 2-MeTHF: 0.28% n-heptane: 0.06% Loss of mammary gland: ~5% (Analysis: 0.21%) 50°C 2-MeTHF/nheptane = 4 vol./1 vol. Form C Form C Form C 99.49% 2-MeTHF: 0.07% n-heptane: 0.05% Loss of mammary gland: ~4% (Analysis: 0.17%)

Taking into account the results of the above experiment, an experiment was performed with an increase in scale to 11 g using the following procedures.

1. Dissolution in 8 vol. 2-MeTHF at 50°C.

2. Add 1.5 vol. n-heptane for 1 h at 50°C

3. Sowing (110 mg)

4. Stir for 1.5 hours at 50°C

5. Addition of 22.2 vol. n-heptane over 7 h at 50°C

6. Selecting a sample for PRD-1.

7. Stir for 15 hours at 50°C

8. Selecting a sample for PRD-2.

9. Stir for 20 hours at 50°C

10. Selecting a sample for PRD-3.

11. Add 0.3 vol. n-heptane for 1 h at 50°C

12. Stir for 1 hour at 50°C

13. Bring to 25°C in 5 hours

14. Stir for 17 hours at 25°C

15. Taking a sample for PRD-4 analysis, purity analysis and analysis-1.

16. Filtration and washing of precipitate 2 vol. 2-MeTHF/n-heptane=1 vol./3 vol.

17. Drying of wet precipitate in vacuum at 50°C

18. Receiving material

After adding n-heptane, the PRD showed form C with an additional peak. After holding at 50°C for 15 h and 35 h, the PRD again showed form C with an additional peak (loss of MF about 4%). Since the PRD showed an additional peak, the process was further optimized using the procedure below.

1. Dissolution in 8 vol. 2-MeTHF at 50°C.

2. Add 2 vol. n-heptane for 1 ~ 3 h at 50°C

3. Sowing (150 mg)

4. Stirring for 1~18h at 50°C (liquid suspension)

5. Add 2 vol. n-heptane for 2 ~ 3 h at 50°C.

6. Taking a sample for PRD analysis.

7. Stir for 2~4 hours at 50°C

8. Taking a sample for PRD analysis.

9. Add 20 vol. n-heptane for 6 ~ 8 h at 50°C.

10. Stir for 2 hours at 50°C.

11. Bring to 25°C in 5 hours

-

Claims (18)

12. Stir for 2 hours at 25°C 13. Taking a sample for PRD analysis. 14. stirring at 25°C for 6~15 h 15. Filtration and washing of precipitate 2 vol. 2-MeTHF/n-heptane=1 vol./3 vol. 16. Drying of wet precipitate in vacuum at 50°C 17. Receiving material This procedure was performed twice and both times the PRD showed form C. The measured purity was 99.24% and 97.86%. Hygroscopicity. The hygroscopicity of Form C was assessed using a dynamic vapour sorption (DVS) test at 25°C. Form C is non-hygroscopic below 90% relative humidity (RH). Form C absorbs about 0.2% water at 95% RH at 25°C. After the DVS test, the resulting sample was still in Form C. Form C assessment. Form C is the optimal polymorph identified. Its stability, solubility, hygroscopicity and formulation process feasibility were assessed as described above. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be within the scope of the appended claims. Each reference, including, without limitation, all patents, patent applications, and publications cited in this application, is hereby incorporated by reference in their entirety. CLAUSE OF THE INVENTION 1. Crystalline form of 3-(5-(2-hydroxy-2-methylpropoxy)-6-methylpyrazin-2-yl)-III-indole-7-carbonitrile. 2. The crystalline form according to claim 1, wherein the crystalline form is anhydrous. 3. The crystalline form according to claim 1 or 2, wherein the crystalline form is characterized by a XRD diffraction pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, and 11.7. 4. The crystalline form according to any one of claims 1 to 3, wherein the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, and 24.6. 5. The crystalline form according to any one of claims 1 to 4, wherein the crystalline form is characterized by a XRD pattern having peaks expressed in degrees 2-theta at angles (±0.2 degrees) of 23.8, 20.7, 11.7, 24.6, and 15.8. 6. The crystalline form of claim 1, wherein the crystalline form has a XRD pattern substantially as shown in Fig. 1. 7. A crystalline form according to any one of claims 1 to 6, having a DSC thermogram characterized by an endotherm with an onset temperature of about 179°C. 8. A crystalline form according to any one of claims 1 to 7, having a DSC thermogram characterized by an endotherm with an onset temperature of 179.3°C. 9. A pharmaceutical composition comprising a crystalline form according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 10. A method for treating a neurodegenerative disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1-8 or a pharmaceutical composition according to claim 9. 11. The method according to claim 10, characterized in that the neurodegenerative disorder is an x-linked recessive disorder. 12. The method according to claim 10 or 11, characterized in that the neurodegenerative disorder is spinal muscular atrophy (SBMA). 13. A method for modulating androgen receptor (AR) activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to claims 1-8 or a pharmaceutical composition according to claim 9. 14. The method according to claim 13, characterized in that the androgen receptor (AR) is subject to allosteric modulation. 15. The method according to claim 13 or 14, characterized in that modulation of androgen receptor (AR) activity provides treatment of spinal muscular atrophy (SBMA) in a subject. 16. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1-8 or a pharmaceutical composition according to claim 9. 17. The method according to claim 16, characterized in that the cancer is prostate cancer. 18. A method for producing a crystalline form according to any one of claims 1-8, comprising: -