EA043785B1 - CRYSTAL FORMS 7-CHLORO-2-(4-(3-METHOXYAZETIDIN-1-YL)CYCLOHEXYL)-2,4-DIMETHYL-N-((6-METHYL-4-(METHYLTHIO)-2-OXO-1,2 -DIHYDROPYRIDIN-3-YL)METHYL)BENZO[D][1,3]DIOXOL-5-CARBOXAMIDE - Google Patents

Description

Related applications

This application claims benefit from U.S. Provisional Patent Application No. 62/878012, filed July 24, 2019, the entire contents of which are incorporated herein by reference.

State of the art

Eukaryotic chromatin consists of macromolecular complexes called nucleosomes. The nucleosome contains 147 base pairs of DNA wrapped around a protein octamer having two subunits each of the histone proteins H2A, H2B, H3 and H4. Histone proteins undergo post-translational modifications, which in turn affect chromatin structure and gene expression. One type of post-translational modification found in histones is the methylation of lysine and arginine residues. Histone methylation plays a critical role in the regulation of gene expression in eukaryotes. Methylation disrupts chromatin structure and is associated with both activation and repression of transcription (Zhang and Reinberg, Genes Dev. 15:2343-2360, 2001). Enzymes that catalyze the addition and removal of methyl groups from histones are involved in gene silencing, embryonic development, cell proliferation, and other processes.

One class of histone methylases is characterized by the presence of a Zeste Trithorax mosaic enhancer suppressor (SET) domain containing about 130 amino acids. Enhancer of Zeste homolog 2 (EZH2) is an example of a SET domain-containing methylase in humans. EZH2 associates with EED (embryonic ectoderm development) protein and SUZ12 (suppressor of Zeste homologue 12) to form a complex known as PRC2 (Polycomb group repressive complex 2), which has the ability to trimethylate histone H3 at lysine 27 (Cao and Zhang, Mol. Cell 15 :57-67, 2004). PRC2 complexes may also contain RBAP46 and RBAP48 subunits. Another example is the related methylase EZH1.

Many studies have demonstrated the oncogenic activity of EZH2. In cell line experiments, overexpression of EZH2 induces cell invasion, soft agar growth, and motility, whereas knockdown of EZH2 inhibits cell proliferation and cell invasion (Kleer et al., 2003, Proc. Nat. Acad. Sci. USA 100:11606-11611 ; Varambally et al., (2002), The polycomb group protein EZH2 is involved in the progression of prostate cancer, Nature 419, 624-629). EZH2 has been shown to repress the expression of several tumor suppressors, including E-cadherin, DAB2IP, and RUNX3, among others. In xenograft models, knockdown of EZH2 slows tumor growth and metastasis. Recently, down-modulation of EZH2 in mouse models was shown to block prostate cancer metastasis (Min et al., An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB, Nat Med. 2010 Mar; 16( 3):286-94). Overexpression of EZH2 is associated with the aggressiveness of some cancers, such as breast cancer (Kleer et al., Proc. Nat. Acad. Sci. USA 100:11606-11611, 2003). Recent studies also suggest that the oncogenic prostate cancer-specific TMPRSS2-ERG fusion protein induces repressive epigenetic programs through direct activation of EZH2 (Yu et al., An Integrated Network of Androgen Receptor, Polycomb, and TMPRSS2-ERG Gene Fusions in Prostate Cancer Progression, Cancer Cell. 2010 May 18; 17(5):443-454). Compound 1, 7-chloro-2-(4-(3-methoxyazetidin-1yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2- Dihydropyridin-3yl)methyl)benzo[d][1,3]dioxol-5-carboxamide, is a small molecule EZH2 inhibitor showing high therapeutic potential for the treatment of various pathological conditions associated with methyl-modifying enzyme, such as the treatment of proliferative disorders , such as cancer. Compound 1 is disclosed in US Provisional Patent Application 62/659408, the contents of which are incorporated herein by reference, and has the structure

The development of alternative forms of compound 1 represents a promising area for further treatment of diseases or disorders susceptible to EZH2 inhibition.

The essence of the invention

Provided herein are crystalline forms of Compound 1. Also provided herein are pharmaceutical compositions containing one or more of the disclosed crystalline forms of Compound 1. Additionally provided is the use of one or more of the disclosed crystalline forms of Compound 1 for the treatment of diseases or disorders responsive to EZH2 inhibition , such as cancer.

Brief description of graphic materials

In fig. Figure 1 shows the X-ray powder diffraction (XPD) profile of crystalline form 1 of compound 1.

In fig. Figure 2 shows an exemplary differential scanning calorimetry (DSC) thermogram for Form 1 of Compound 1.

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In fig. Figure 3 shows the X-ray powder diffraction (XPD) profile of crystal form 2 of compound 1.

In fig. Figure 4 shows a combined thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) thermogram for Form 2 of Compound 1.

In fig. Figure 5 shows the X-ray powder diffraction (XPD) profile of crystal form 3 of compound 1.

In fig. Figure 6 shows a combined thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) thermogram for form 3 of compound 1.

In fig. 7 shows an X-ray powder diffraction (XPD) profile of an amorphous form of Compound 1 prepared by the methods described in Example 17 of US Provisional Application No. 62/659,408.

Detailed Description of the Invention Definitions

In this context, crystalline refers to the solid form of a compound in which there is long-range atomic order in the arrangement of atoms. The crystalline nature of the solid can be confirmed, for example, by studying the X-ray powder diffraction profile.

Unless otherwise stated, the crystalline forms (Form 1, Form 2 and Form 3) of Compound 1 are individual crystalline forms. A separate crystalline form means that the specified compound, i.e. compound 1 exists in the form of a single crystal or multiple crystals, where each crystal has the same crystalline form (eg, form 1, 2 or 3). The weight percent of a particular crystalline form is defined as the mass of the particular crystalline form divided by the sum of the mass of the particular crystal, the mass of other crystalline forms present, and the mass of the amorphous form present, multiplied by 100%.

Form 1, crystalline form 1, or single crystalline form 1 are used interchangeably. Form 2, crystalline form 2, or separate crystalline form 2 are used interchangeably. Form 3, crystalline form 3, or separate crystalline form 3 are used interchangeably.

Chemical purity refers to the extent to which the form described is free from materials having other chemical structures. The chemical purity of a compound in the described crystalline forms means the mass of the compound divided by the sum of the mass of the said compound and materials/impurities having other chemical structures multiplied by 100%, i.e. mass percent. The term amorphous refers to a solid that is in a non-crystalline state or form. Amorphous solids have a disordered arrangement of molecules and therefore do not have a defined crystal lattice or unit cell and, accordingly, do not have a specific long-range order. The order of solids in the solid state can be determined by standard techniques known in the art, such as X-ray powder diffraction (XPD) or differential scanning calorimetry (DSC).

The terms anhydrous and anhydrate are used interchangeably and mean that the crystalline form is substantially free of water in the crystal lattice, eg less than 1% by weight as determined by Karl Fischer analysis.

2Θ values in X-ray powder diffraction profiles for the crystalline forms described herein may vary slightly between instruments and are also affected by variability in sample receipt and batch-to-batch composition variations due to factors such as temperature variation, sample drift, and the presence or lack of internal standard. Therefore, unless otherwise noted, the RAP/signal assignment profiles presented in this document should not be interpreted as absolute and may vary by ±0.2°. It is known in the art that such variability takes into account the above factors without interfering with the undisputed identification of the crystalline form. Unless otherwise stated, the 2Θ values presented in this document are obtained using Cu Ka1 radiation. Temperature values, such as those for DSC peaks herein, may vary slightly between instrument recordings and are also affected by variability in sample receipt, batch-to-batch compositional variations, and environmental factors. Therefore, unless otherwise stated, temperatures given herein should not be interpreted as absolute and may vary by ±5° or ±2°.

Substantially the same RPD profile or X-ray powder diffraction profile substantially similar to a particular image means that at least 90% of the represented peaks are present for comparison purposes. In addition, it should be understood that, for comparison purposes, slight variability in peak intensities around the stated values, such as ±0.2°, is acceptable.

The amount of one crystalline form relative to another crystalline form in a sample can be estimated by preparing a series of mixtures of the two crystalline forms with known mass ratios and recording the RPD spectrum for each. For example, the relative amount

- 2 043785 crystalline form 1 and form 2 in a sample can be assessed by selecting one or more characteristic peaks of crystalline form 1 and form 2 presented in FIG. 1 and 3, respectively, and the correlation of their relative intensities in the RPD of the sample by their relative intensities in the RPD of the mixture.

As used herein, the terms subject and patient may be used interchangeably and mean a mammal in need of treatment, e.g., domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, pigs, horses, sheep , goats, etc.) and laboratory animals (for example, rats, mice, guinea pigs, etc.). Typically the subject is a person in need of treatment.

The term pharmaceutically acceptable carrier refers to a non-toxic carrier, adjuvant or vehicle that does not adversely affect the pharmacological activity of the compound with which it is formulated and that is also safe for human use. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate , polyvinylpyrrolidone, polyvinylpyrrolidone vinyl acetate, cellulose based substances (e.g. microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate), starch, lactose monohydrate, mannitol, sodium lauryl sulfate and croscarmellose sodium, polyethylene glycol, sodium carboxymethylcellulose, polyac rylates, polymethacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. The terms treat, treat, and treatment refer to reversing, alleviating, reducing the likelihood of developing, or slowing the progression of a disease or disorder or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed, i.e. therapeutic treatment. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be provided to a predisposed individual prior to the onset of symptoms (eg, based on a history of symptoms and/or genetic or other predisposing factors), i.e. preventive treatment. Treatment can also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

The term effective amount or therapeutically effective amount includes that amount of a compound described herein that produces a biological or medical response in a subject, for example, a dose of 0.001 to 100 mg of Compound 1 per kg of body weight per day.

Illustrative Forms

In a first aspect, this document provides crystalline Form 1 of a compound having the structural formula

In a second aspect, crystalline Form 1 is characterized by at least three X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2° and 22.5°. Alternatively, as part of a second aspect, crystalline form 1 is characterized by at least four X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2° and 22.5°. As another alternative, as part of the second aspect, crystalline form 1 is characterized by at least five X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20. 8°, 22.2° and 22.5°. As another alternative, as part of the second aspect, crystalline form 1 is characterized by at least six X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20. 8°, 22.2° and 22.5°. As another alternative, as part of the second aspect, crystalline form 1 is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22 ,2° and 22.5°. As another alternative, as part of the second aspect, crystalline form 1 is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 10.2°, 12.3°, 12.7°, 13.3°, 14 ,9°, 15.3°, 20.2°, 20.8°, 21.3°, 22.2°, 22.5° and 23.8°. As another alternative, in a second aspect, crystalline form 1 is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 10.2°, 11.0°, 11.4°, 11.8°, 12. 3°, 12.7°,

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13.3°, 14.9°, 15.3°, 16.1°, 17.4°, 20.2°, 20.8°, 21.3°, 22.2°, 22.5° and 23.8°. As another alternative, in a second aspect, crystalline Form 1 is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 14.9°, 20.2° and 20.8°. As another alternative, as part of the second aspect, crystalline Form 1 is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 14.9°, 20.2° and 20.8°. As another alternative, in a second aspect, crystalline Form 1 is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 14.9°, 20.2°, 20.8° and 22.2°. As another alternative, in a second aspect, crystalline Form 1 is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8° and 22. 2°. As another alternative, in a second aspect, crystalline form 1 is characterized by substantially the same X-ray powder diffraction profile as in FIG. 1.

In a third aspect, crystalline Form 1 is characterized by a differential scanning calorimetry (DSC) profile with a sharp endotherm at 179.5°C (onset temperature) or a thermogravimetric analysis (TGA) profile with a mass loss of 1.0% between 36 and 179°C, or both profiles, wherein crystal form 1 may also contain RPD peaks at 2Θ angles selected from any of the values described in the second aspect. In other aspects, crystalline Form 1 is characterized by substantially the same differential scanning calorimetry (DSC) profile as in FIG. 2, wherein crystal form 1 may also contain RPD peaks at 2Θ angles selected from any of the values described in the second aspect.

In a fourth aspect, crystalline form 1 is anhydrous, wherein crystalline form 1 may also contain RPD peaks at 2Θ angles selected from any of the values described in the second aspect, and/or the TGA or DSC values or profiles specified in the third aspect.

In a fifth aspect, crystalline form 1 described herein (e.g., in the first, second, third, or fourth aspects) is at least 60% distinct crystalline form, at least 70% distinct crystalline form, at least 80% distinct. % individual crystalline form, at least 90% individual crystalline form, at least 95% individual crystalline form, or at least 99% individual crystalline form by weight.

In a sixth aspect, crystalline Form 1 described herein (e.g., in the first, second, third, fourth or fifth aspects) has a chemical purity of at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% by weight.

In a seventh aspect, there is provided a crystalline form 2 of a compound having the structural formula

In an eighth aspect, crystalline form 2 is characterized by substantially the same X-ray powder diffraction (XRD) profile as in FIG. 3. In a ninth aspect, crystalline form 2 is characterized by a thermogravimetric analysis (TGA) profile with a mass loss of 3.48% between 46 and 114°C, and also with a mass loss of 0.97% between 114 and 156°C, or a differential scanning profile calorimetry (DSC) with two endotherms at 34.2 and 122.6°C (onset temperatures), or both profiles, and crystalline form 2 may also have RPD peaks at essentially the same 2Θ angles as in FIG. 3. Alternatively, as part of a ninth aspect, crystalline form 2 is characterized by substantially the same thermogravimetric analysis (TGA) or differential scanning calorimetry (DSC) profile as in FIG. 4, wherein crystal form 2 may also contain RPD peaks at substantially the same 2Θ angles as in FIG. 3. In a tenth aspect, crystalline form 2 described herein (e.g., in the seventh, eighth, or ninth aspects) is at least 60% distinct crystalline form, at least 70% distinct crystalline form, at least 80% distinct crystalline form, % individual crystalline form, at least 90% individual crystalline form, at least 95% individual crystalline form, or at least 99% individual crystalline form by weight.

In an eleventh aspect, crystalline Form 2 described herein (e.g., in the seventh, eighth, ninth, or tenth aspects) has a chemical purity of at least 60%, at least 70%, at least 80%, at least 90% , at least 95% or at least 99% by weight. A twelfth aspect provides crystalline form 3 of a compound having the structural formula

- 4 043785

In a thirteenth aspect, crystalline form 3 is characterized by substantially the same X-ray powder diffraction (XRD) profile as in FIG. 5. In a fourteenth aspect, crystalline form 3 is characterized by a thermogravimetric analysis (TGA) profile with a mass loss of 5.93% between 43 and 143 °C or a differential scanning calorimetry (DSC) profile with two endotherms at 34.5 and 107.0 °C ( onset temperature) and one exotherm at 249.0° C. (onset temperature), or both profiles, wherein crystal form 3 may also contain RPD profiles at substantially the same 2θ angles as in FIG. 5. Alternatively, as part of the fourteenth aspect, crystalline form 3 is characterized by substantially the same thermogravimetric analysis (TGA) or differential scanning calorimetry (DSC) profile as in FIG. 6, wherein crystal form 3 may also contain RPD profiles at substantially the same 2θ angles as in FIG. 5.

In a fifteenth aspect, crystalline form 3 described herein (e.g., in the twelfth, thirteenth, or fourteenth aspects) is at least 60% distinct crystalline form, at least 70% distinct crystalline form, at least 80% distinct crystalline form, at least 90% distinct crystalline form, at least 95% distinct crystalline form, or at least 99% distinct crystalline form by weight.

In a sixteenth aspect, crystalline Form 3 described herein (e.g., in the twelfth, thirteenth, fourteenth, or fifteenth aspects) has a chemical purity of at least 60%, at least 70%, at least 80%, at least 90 %, at least 95% or at least 99% by weight.

In a seventeenth aspect, crystal form 1 described herein (for example, in the first, second, third, fourth or fifth aspects), crystal form 2 described herein (for example, in the seventh, eighth, ninth, tenth or eleventh aspects) , or crystalline form 3 (for example, described in the twelfth, thirteenth, fourteenth, fifteenth or sixteenth aspects) is represented by the following structural formula:

^{0 0} ।

Alternatively, as part of the seventeenth aspect, crystal form 1 described herein (e.g., in the first, second, third, fourth or fifth aspects), crystal form 2 described herein (e.g., in the seventh, eighth, ninth, tenth or eleventh aspects), or crystalline form 3 (for example, described in the twelfth, thirteenth, fourteenth, fifteenth or sixteenth aspects) is represented by the following structural formula:

Alternatively, as part of the seventeenth aspect, crystal form 1 described herein (e.g., in the first, second, third, fourth or fifth aspects), crystal form 2 described herein (e.g., in the seventh, eighth, ninth, tenth or eleventh aspects), or crystalline form 3 (for example, described in the twelfth, thirteenth, fourteenth, fifteenth or sixteenth aspects) is represented by the following structural formula:

having the chemical name (2R)-7-chloro-2-(trans-4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4dimethyl-N-((6-methyl-4-(methylthio)-2- oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxol-5carboxamide.

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Application, formulation and administration

The crystalline forms described herein and compositions thereof are useful for the treatment of diseases or disorders responsive to EZH2 inhibition. Such diseases and disorders include those associated with cellular proliferation. In some embodiments, the crystalline forms and compositions thereof described herein are useful for the treatment of diseases and/or disorders associated with dysregulated cell cycle or DNA repair. In some embodiments, the crystalline forms and compositions thereof described herein are useful for the treatment of cancer. Exemplary types of cancer include, for example, adrenal cancer, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia goats, adenocarcinoma , adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenal cortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK cell leukemia, AIDS-associated lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblast new fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal -cell carcinoma, cancer biliary tract, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear cell renal sarcoma, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland tumor, tumor endodermal sinus, T-cell lymphoma associated with enteropathy, esophageal cancer, included fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of bone , glial tumor, moltiform glioblastoma, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancies, hepatoblastoma, T-cell liver and spleen lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, malignant midline carcinoma of the face, leukemia, Leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma , acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant newt tumor, mantle cell lymphoma, B-cell marginal zone cell lymphoma, mast cell leukemia, mediastinal germinal cell tumor, medullary breast carcinoma, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic urothelial carcinoma, mixed müllerian tumor, mucinous tumor, multiple myeloma , neoplasm of muscle tissue, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal cancer, neuroma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, eye cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer , osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytum, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor of T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, schwannomatosis, seminoma, Sertoli cell tumor, tumor ovarian sex cord stroma, signet ring cell carcinoma, small round cell blue cell tumor, small cell carcinoma, soft tissue sarcoma, somatostatinoma, chimney sweep cancer, spinal cord cancer, marginal zone lymphoma of the spleen, squamous cell carcinoma, synovial sarcoma, Sézary's disease, small cell intestinal cancer, squamous cell cancer, gastric cancer, T-cell lymphoma, testicular cancer, tecoma, thyroid cancer, transitional cell cancer, throat cancer, urinary duct cancer, genitourinary cancer, urothelial carcinoma, uveal melanoma, metastatic castration-resistant prostate cancer, clear cell ovarian carcinoma, uterine cancer, verrucous carcinoma, optic pathway glioma, vulvar cancer, vaginal cancer, macroglobules

- 6 043785 Waldenström's disease, Warthin's tumor and Wilms' tumor.

In one aspect, the cancers that can be treated with the crystalline forms and compositions thereof described herein are selected from breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme, bladder cancer, melanoma, bronchial cancer, lymphoma, liver cancer, multiple myeloma, lymphoma, ovarian cancer, NSCL, pancreatic cancer, malignant rhabdoid tumor, synovial sarcoma and glioma.

Another aspect of this disclosure provides the use of one or more of the crystalline forms described herein for the manufacture of a medicament for use in treating a disorder or disease described herein. Another aspect of this disclosure is one or more crystalline forms or compositions described herein for use in treating a disorder or disease described herein.

Also provided are pharmaceutical compositions containing one or more of the described crystalline forms and a pharmaceutically acceptable carrier.

The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or through an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, interstitial, and intracranial injection or infusion routes.

The amount of the provided crystalline form that can be combined with carrier materials to obtain the composition in unit dosage form will vary depending on the patient being treated and the particular route of administration. The proposed compositions can be formulated such that a dose of 0.001 to 100 mg of inhibitor per kg of body weight per day can be administered to a patient taking said compositions.

It should also be understood that the specific dosage and treatment regimen for a particular patient will depend on various factors, including age, body weight, general health, gender, diet, time of administration, excretion rate, drug combination, the judgment of the treating physician, and the severity of the specific disease. to be treated. The amount of the provided crystalline form in the composition also depends on the specific compound in the composition.

Examples

As shown in the following examples, crystalline forms were prepared in accordance with the following general methods.

Preparation of amorphous compound 1

The amorphous form of compound 1 was obtained as one enantiomer, one geometric isomer using the following method described below.

The RPD profile of the amorphous product obtained in this method is presented in Fig. 7.

Intermediate 1: Methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[b][1,3]dioxol-5-carboxylate

Intermediate 1

Intermediate 1 Intermediate 1 (R-enantaomer) (S-enantiomer)

Step 1: Synthesis of methyl 5-chloro-3,4-dihydroxy-2-methylbenzoate

To a solution of methyl 3,4-dihydroxy-2-methylbenzoate (5.11 g, 27.9 mmol) in tetrahydrofuran (199 ml) at -20°C, sulfuryl chloride (2.45 ml, 30.6 mmol) was added dropwise. . The reaction mixture was stirred at -20°C for 3 hours, then quenched with saturated aqueous ammonium chloride (50 ml). The desired product was extracted with ethyl acetate (25 ml x 3). The combined organic layers were washed with brine (25 ml), dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient 0 to 60% ethyl acetate in heptane) to give the title compound (4.117 g, 68% yield) as a beige solid. LCMS [M+H]+ m/z: calc. 217.0; found 217.1 (Cl isotope profile).

Step 2: Synthesis of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-2H-1,3-benzodioxole-5carboxylate

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A mixture of methyl 5-chloro-3,4-dihydroxy-2-methylbenzoate (1.2 g, 5.53 mmol), dodecacarbonyltriruthenium (176 mg, 276 μmol) and triphenylphosphine (145 mg, 553 μmol) was degassed under vacuum and purged nitrogen (3 cycles). Toluene (8.1 ml) was added and the reaction mixture was heated to reflux for 30 minutes. A solution of 4-ethynylcyclohexan-1-one (1.34 g, 11.0 mmol) in toluene (17 ml) was then added dropwise and the reaction mixture was stirred at reflux for 23 hours. Finally, the reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient 0 to 60% ethyl acetate in heptane) to give the title compound (1.327 g, 70% yield) as a yellow oil. LCMS [M+Na]+ m/z: calc. 361.1; found 361.1 (Cl isotope profile).

Step 3: Separation of methyl (R)-7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxol-5carboxylate and methyl (S)-7-chloro- 2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxol-5-carboxylate

A racemic mixture of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxol-5carboxylate (4.4 g, 13 mmol) was separated by preparative SLC [column: ChiralPak AY from Daicel Chemical Industries (250 mm x 50 mm i.d., 10 µm). Mobile phase A: CO ₂ / Mobile phase B: 0.1% NH4OH in methanol. Isocratic elution (85% mobile phase A and 15% mobile phase B). Flow rate: 80 ml/min. Column temperature: 40°C]. Intermediate 1 (peak 1) (undesired enantiomer/distomer): Retention time = 6.2 min. Recovery = 1.4 g, 4.05 mmol, 31% yield, ee. 90%, purity 98% (yellow solid).

^1H NMR (400 MHz, chloroform-d) δ 7.48 (s, 1H), 3.78 (s, 3H), 2.44-2.36 (m, 2H), 2.35-2.25 (m, 6H), 2.19 (tdd, J=2.8, 5.6, 13.1 Hz, 2H), 1.70-1.57 (m, 5H). Intermediate 1 (Peak 2) (desired enantiomer/eutomer): Retention time = 7.0 min. Recovery = 1.1 g, 3.08 mmol, yield 23.75%, ee. 99%, 95% purity (yellow solid).

1H NMR (400 MHz, chloroform-d) δ 7.49 (s, 1H), 3.78 (s, 3H), 2.44-2.36 (m, 2H), 2.36-2.25 ( m, 6H), 2.20 (tdd, J=2.8, 5.6, 13.1 Hz, 2H), 1.72-1.59 (m, 5H). Analytical SLC method: [column: ChiralPak AY-3 (150 x 4.6 mm i.d., 3 µm). Mobile phase A: CO ₂ / Mobile phase B: 0.05% Et ₂ NH in iPrOH. Gradient: from 5 to 40% mobile phase B (in 5.5 min). Flow rate: 2.5 ml/min. Column temperature: 40°C]. Intermediate 1 (peak 1 - undesired enantiomer/distomer): Retention time = 2.853 min. Intermediate 1 (peak 2 - desired enantiomer/eutomer): Retention time = 2.979 min.

Intermediate 2: 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid

among!ici 1is 1 (single ^enantiome P; °D ^IN geometric isomer) (Peak 2) (single enantiomer) cis or trans μ.

Intermediate 2 (one enantiomer; one geometric isomer)

Step 1: Synthesis of methyl 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxol-5-carboxylate

A solution of 3-methoxyazetidine hydrochloride salt (8 g, 64.75 mmol) and N,N-diisopropylethylamine (12 ml, 68.9 mmol) in methanol (30 ml) was stirred at room temperature for 30 min, then another methyl solution was added -7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-1,3-benzodioxole-5carboxylate (intermediate 1 - peak 2) (4.1 g, 12.10 mmol) in tetrahydrofuran (30 ml ). The reaction mixture was stirred at room temperature for 1 hour, then cooled to -70°C. Lithium borohydride (500 mg, 22.96 mmol) was added and the reaction mixture was stirred at -70°C for 30 minutes [or until the starting material was completely consumed by TLC, ethyl acetate/methanol 5:1]. The two reaction mixtures were then combined and quenched with saturated aqueous ammonium chloride (120 ml) at 0°C, and the desired product was extracted with dichloromethane (200 ml x 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient 0 to 14% methanol in dichloromethane) to give the title compound (8.05 g, 67% yield, 83% purity) as a light yellow oil. The sample (50 mg) was further purified by preparative thin layer chromatography (silica gel, ethyl acetate:methanol 15:1). LCMS [M+H]+ m/z: calc. 410.2; found 410.1.

1H NMR (400 MHz, methanol-d4) δ 7.39 (s, 1H), 3.95-3.91 (m, 1H), 3.73 (s, 3H), 3.59-3.51 ( m, 2H), 3.16

- 8 043785 (s, 3H), 2.97 (shdd, J=6.4, 8.0 Hz, 2H), 2.26 (s, 3H), 2.11-2.02 (m, 1H) , 1.91-1.73 (m, 5H), 1.54 (s, 3H), 1.221.12 (m, 2H), 0.98-0.86 (m, 2H).

Step 2: Synthesis of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid

To a solution of methyl 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxol-5-carboxylate (4 g, 9.75 mmol) in methanol (48 ml), a solution of lithium hydroxide hydroxide (4.03 g, 96.06 mmol) in water (12 ml) was added. The reaction mixture was stirred at 70°C for 2 hours, then the two batches were combined and concentrated under reduced pressure. Water (500 ml) was added and the pH was adjusted to 6 with saturated aqueous citric acid at 0°C. The desired product was extracted with a 3:1 mixture of dichloromethane and isopropanol (300 ml x 5). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure to give the title compound (6.1 g, crude) as an off-white solid, which was used in the next step without further purification. LCMS [M+H]+ m/z: calc. 396.2; found 396.1.

1H NMR (400 MHz, methanol^) δ 7.07 (s, 1H), 4.05-4.10 (m, 2H), 3.76-3.88 (m, 1H), 3.67 (bdd , J=10, 3.6 Hz, 2H), 3.22 (s, 3H), 2.71-2.81 (m, 1H), 2.19 (s, 3H), 1.91-1, 99 (m, 4H), 1.75-1.85 (m, 1H), 1.52 (s, 3H), 1.18-1.28 (m, 2H), 1.06-1.14 ( m, 2H).

Amorphous compound 1.

A- A cis or w _Ν Ο Η NN n _{2 n} Q

About | ZA o o , /V _HN А^Д/LokA /FT ₂ EtN, nat J N Η ϋ

R or S ^DMF f ° R or Ξ

Intermediate 2 Compound 1 (amorphous) (one enantiomer; one geometric isomer) (one enantiomer, one geometric isomer)

(5y7-chl or-2-(( 1 g/5)-4-(3-methoxyazetidine-1 and l ) cycle edema il >2,4-DI meta π-N-( (6-meta l-4 -{metalta o)-2-oxo-1,2-dihydropyridin-3-i l }m etal)benzo[<1,3]dioxol-5-carb oxamide (S)-7-x l or-2 -(( 1 h,4H)-4-(3-megox and azetadi n-1 yl)cy cl edema yl)-2,4-DI megyl-M-((6-metal-4-(m etalthio)- 2-ox o-1,2-dihydropyri di n-3-yl )meg yl)benzo[c(|[1,3]dioxol-5-carbo oke amide

(RJ-7-chloro-2-(( 1r,4 R)-4-( 3-methox iazeta din-1 (P)-7-chloro-2-((13,4E)-4-(3- megoxyazegidin-1-yl-yl)cyclohexyl)-2,4-dimegyl-L/-((6-metalL- )cycle edema yl)-2,4dimetal-L/-((6-metal^1-(meta ( metaltao)-2-oxo-1,2-dihydropyridin-3-yl) ltao)-2-oxo-1,2-dihydropyrodium n-3-yl)mehyp)ve methyl)benzo[P][1, 3]dioxol-5-carb oke amide nzo[P][1,3]dioxol-5-carb oke amide

To a solution of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5carboxylic acid (intermediate 2, one enantiomer and geometric isomer ) (5 g, 12.63 mmol) in N,N-dimethylformamide (50 ml) was added O-(7-azabenzotriazol-1-yl)N,N,N'N'-tetramethyluronium hexafluorophosphate (5.7 g, 14 .99 mmol) and N,N-diisopropylethylamine (11 ml, 63.15 mmol). Stir the mixture at 20°C for 30 minutes, then add 3-(aminomethyl)-6methyl-4-(methylthio)pyridin-2(1H)-one hydrochloride salt (intermediate 1) (4.2 g, 19. 03 mmol). The reaction mixture was stirred at room temperature for another 1.5 hours, then filtered. The filtrate was purified by preparative HPLC [column: Phenomenex Gemini C18 (250 mm x 50 mm, 10 µm). Mobile phase A: water (0.04% ammonium hydroxide v/v and 10 mM ammonium bicarbonate) / mobile phase B: acetonitrile. Gradient (from 75 to 44% mobile phase A / from 25 to 56% mobile phase B in 23 min). Column temperature: 30°C] to obtain the title compound (4.4 g, 60% yield, 96% purity) as a white solid. LCMS [M+H] ⁺ m/z: calc. 562.2; found 562.2.

1H NMR (400 MHz, methanol-d ₄ ) δ 6.91 (s, 1H), 6.29 (s, 1H), 4.50 (s, 2H), 4.01 (quin, J=6 Hz, 1H), 3.58 (dd, J=8.8, 6.4 Hz, 2H), 3.26 (s, 3H), 2.92-3.02 (m, 2H), 2.54 (s , 3H), 2.31 (s, 3H), 2.21 (s, 3H), 2.01-2.11 (m, 1H), 1.79-2.00 (m, 5H), 1, 62 (s, 3H), 1.19-1.34 (m, 2H), 0.91-1.08 (m, 2H). The RPD profile of the amorphous product obtained in this method is presented in Fig. 7.

- 9 043785

List of abbreviations

Reduction Meaning % w/w Mass percentage Ή-NMR Proton nuclear magnetic resonance DM FA dimethylformamide DMSO dimethyl sulfoxide Dec Differential scanning calorimetry Chipboard Dynamic vapor sorption EtOAc ethyl acetate EtOH ethanol SHG Gravimetric vapor sorption H ₂ O water HPLC High performance liquid chromatography NS1 Hydrochloric acid THEIR Ion chromatography IPA propan-2-ol KF Karl Fischer M.A.S. Spin at a magic angle MDSC Modulated differential scanning calorimetry IEC methyl ethyl ketone MeOH methanol N/A Not applicable PLM Polarized light microscopy OB Relative humidity room, room Room temperature MK-RD X-ray diffraction of a single crystal SAM Scanning electron microscope TFC trifluoroacetic acid Tg Glass transition temperature THF Thermogravimetric analysis THF tetrahydrofuran UV ultraviolet PT-RPD X-ray powder diffraction at variable temperature RPD X-ray powder diffraction

1. Details of instruments and methods

X-ray powder diffraction (RPD):

XRD patterns were recorded on a Bruker AXS C2 GADDS, Bruker AXS D8 Advance, or PANalytical Empyrean instrument, as described in detail below. Bruker AXS C2 GADDS

RPD recording on a Bruker AXS C2 GADDS diffractometer was performed using Cu Ka radiation (40 kV, 40 mA), an automated XYZ platform, a laser video microscope for automatic sample placement, and a Vantec-500 2-dimensional area detector. The X-ray optics consisted of a single multilayer Gobel mirror coupled to a collimator with a 0.3 mm pinhole aperture. The beam divergence, i.e. The effective size of the X-ray beam on the sample was approximately 4 mm. A continuous θ-θ scanning mode was used with a sample-to-detector distance of 20 cm, resulting in an effective 2Θ range of 1.5°-32.5°. Typically, the sample was irradiated with an X-ray beam for 120 s. The software used for data collection and analysis was GADDS for Win7/XP and Diffrac Plus EVA, respectively. Samples tested under ambient conditions were prepared in the form of flat plate samples using the powder as received without grinding. Samples were obtained and analyzed on a glass slide or glass frit. Samples were lightly pressed onto a glass slide to provide a flat surface for analysis. A glass ceramic filter block was used to separate solids from suspensions and analyze them by adding a small amount of suspension directly onto the glass frit before filtering under low vacuum. For ex

- 10 043785 variable temperature (VT) experiments, samples were placed on an Anton Paar DHS 900 heating stage under ambient conditions. The sample was then heated to the appropriate temperature at a rate of 10°C/min and then held isothermally for 1 min before data collection began. Samples were obtained and analyzed on a silicon wafer mounted on a heating stage using a thermally conductive compound. Bruker AXS D8 Advance RPD on a Bruker D8 diffractometer was recorded using Cu Ka radiation (40 kV, 40 mA) and a θ-2θ goniometer equipped with a Ge monochromator. The incident beam was passed through a 2.0 mm divergence slit, then through a 0.2 mm anti-scattering slit and a knife prism. The diffracted beam passed through an 8.0 mm receiving slit with 2.5° Soller slits, then onto the Lynxeye detector. The software used for data acquisition and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA, respectively. The samples were tested under ambient conditions in the form of flat plate samples using the powder as received. The sample was prepared on a polished zero-background (510) silicon wafer by lightly pressing it onto a flat surface or placing it in a cut cavity. The sample was rotated in its plane. PANalytical Empyrean RPD on a PANalytical Empyrean diffractometer was recorded using Cu Ka radiation (45 kV, 40 mA) in transmission geometry. For the incident beam, a 0.5° slit, a 4 mm screen, and a 0.04 rad Soller slit with a focusing mirror were used. The PIXcel3D detector, installed for the diffracted beam, was equipped with a receiving slit and 0.04 rad Soller slits. The software used for data collection was X'Pert Data Collector with X'Pert Operator Interface. Data were analyzed and presented using Diffrac Plus EVA or HighScore Plus. Samples were obtained and analyzed on a metal plate or on a Millipore 96-well plate in transmission mode. A radiolucent film was used between metal sheets on a metal well plate and the powder (approximately 1-2 mg) was used as received. A Millipore plate was used to separate solids from suspensions and analyze them by adding a small amount of suspension directly to the plate before filtering under low vacuum.

Differential scanning calorimetry (DSC)

DSC data were recorded on a TA Instruments Q2000 instrument equipped with a 50-point autosampler. Typically, 0.5-3 mg of each sample was heated in an aluminum crucible with a pinhole at a rate of 10°C/min from 25 to 300°C. A constant flow of dry nitrogen was maintained over the sample at a rate of 50 ml/min. Temperature modulated DSC was performed using a base heating rate of 2°C/min and temperature modulation parameters of ±0.318°C (amplitude) every 60 seconds (period). Advantage for the Q Series and Thermal Advantage were used as instrument control software, and data were analyzed using Universal Analysis or TRIOS.

DSC data were also recorded on a TA Instruments Discovery DSC instrument equipped with a 50-point autosampler. Typically, 0.5-3 mg of each sample was heated in an aluminum crucible with a pinhole at a rate of 10°C/min from 25 to 300°C. A constant flow of dry nitrogen was maintained over the sample at a rate of 50 ml/min. TRIOS was used as instrument control software and data were analyzed using TRIOS or Universal Analysis.

Thermogravimetric analysis (TGA)

TGA data were recorded on a TA Instruments Q500 TGA equipped with a 16-point autosampler. Typically, 5-10 mg of each sample was loaded into a pre-weighed aluminum DSC crucible and heated at a rate of 10°C/min from room temperature to 300°C. A constant flow of nitrogen was maintained over the sample at a rate of 60 ml/min. Advantage for the Q Series and Thermal Advantage were used as instrument control software, and data were analyzed using Universal Analysis or TRIOS. TGA data were also recorded on a TA Instruments Discovery TGA equipped with a 25-point autosampler. Typically, 5-10 mg of each sample was loaded into a pre-weighed aluminum DSC crucible and heated at a rate of 10°C/min from room temperature to 300°C. A constant flow of nitrogen was maintained over the sample at a rate of 25 mL/min. TRIOS was used as instrument control software and data were analyzed using TRIOS or Universal Analysis.

2. General methods of crystallization

Selection methods for crystallization are described in the methods presented below.

Suspension maturation/aging

In the maturation chamber: The maturation suspensions were placed in a shaker platform incubator (Heidolph Titramax/Incubator 1000, Fig. 2) and subjected to a series of heating-cooling cycles from room temperature to approximately 50°C. This was ensured by turning the heating on or off every 4 hours. Shaking was continued throughout.

In the Polar Bear reactor: The suspensions were stirred (500 rpm) in the Polar Bear reactor (reactor design

- 11 043785 pa from Cambridge) for various times at 50°C.

Then the samples were cooled to 25°C at a rate of 0.1°C/min and stirred for another 4 hours. After this, the samples were again heated to 50°C at a rate of 0.1°C/min. Then the cycle was repeated.

Crystallization upon cooling

The solutions were cooled to 5°C at a rate of 0.1°C/min in a Polar Bear reactor and stirred at the indicated temperature for various times. All solids were filtered and suction dried for 10 min and initially analyzed by XRD. The solutions were further cooled to -20° C. over 16 hours and then any new solids that may have formed were processed as described above. The remaining solutions were evaporated (see method 3 below).

Controlled evaporation

Solutions placed in vials were evaporated under ambient conditions by removing the caps from the vials or by inserting a needle into the rubber diaphragm of the vial caps. Samples were allowed to slowly evaporate to dryness or until a solid appeared under ambient conditions.

Precipitation/crystallization by adding antisolvent Antisolvent was added dropwise to the solutions at 50°C until turbidity appeared. Then the samples were cooled to 5°C at a rate of 0.1°C/min and kept isothermal. If necessary, additional antisolvent was added to the suspension. The solids were filtered and suction dried for 10 minutes, and initial analysis of the residues was performed using RPD.

3. Selection methods

Samples of the amorphous form of compound 1 with various impurities, one with a purity of 95.2% (30 mg), and the other with a purity of 97.6% (20 mg), were suspended or dissolved in 10-30 vol. of this solvent at room temperature. After 5 minutes of equilibration at room temperature, all samples (solutions and suspensions) were heated to 50°C for 10 minutes and the resulting samples were processed as follows: The suspensions were left to mature at a temperature of 60 to 5°C for 72 hours (4 hours at each temperature). The solutions were cooled to 50 to 5°C at a rate of 0.1°C/min and kept at 5°C for 72 hours. If no solid was obtained, the solutions were left to evaporate at room temperature. All obtained solids were analyzed using RPD and then with appropriate technologies. See table. 1-3, where sorting methods and analysis results are presented.

Based on RPD analysis, polymorph sorting using the 95.2% pure amorphous form of Compound 1 provided three crystalline forms, referred to herein as Form 1, Form 2, and Form 3 (Tables 1 and 2). Form 1 was the predominant form that was obtained from various solvent systems. Form 3 was observed only once when prepared from a 2-butanol solvent. Sorting of polymorphic forms using the amorphous form of compound 1 with a purity of 97.6% provided Form 1 and Form 2, but not Form 3 (Table 3). Form 1 was more common than form 2. Characteristics of forms 1-3 are presented below.

Table 1. Sorting of polymorphic forms using the amorphous form of compound 1 _____________________with a purity of 95.2% (30 mg)________________

Solvent 10 rev. 20 rev. 30 rev. Observation at 50 °C Treatment Evaporation at room temperature RPD n-Heptane X X X Suspension Ripening 60/5 °C N/A Form 1 Acetone - Solution Cooling at 5 °C N/A Form 1 2-Propanol - Solution Cooling at 5 °C Glassy/resinous substance N/A Ethanol - Solution Cooling at 5 °C Glassy/resinous substance N/A Ethyl acetate X - Suspension Ripening 60/5 °C N/A Form 2 Isopropyl acetate X Y X Suspension Ripening 60/5 °C N/A Form 1 tert-Butyl methyl ether X X Suspension Ripening 60/5 °C n/a Form 1 Methyl isobutyl ketone X X Suspension Ripening 60/5 °C n/a Form 1 Dimethyl sulfoxide V - Solution Cooling at 5 °C Solid Amorphous Anisole - Solution Cooling at 5 °C Glassy/resinous substance N/A Methanol - Solution Cooling at 5 °C Solid Amorphous Toluene X X Y X Suspension Ripening 60/5 °C N/A Form 2

Key: clear solution; ^x = cloudy; x = suspension; N/A = not carried out

- 12 043785

Table 2. Sorting of polymorphic forms using the amorphous form of compound 1 with a purity of 95.2% (30 mg)

Solvent 10 rev 20 rev 30 rev. Observation at 50 °C Treatment Observation after treatment (72 hours) Evaporation at room temperature RPD Tetrahydrofur an Ζ - - Solution Cooled below 5°C Solution Glassy/resinous substance n/a Dichloromethane Ζ - - Solution Cooled below 5°C Solution Glassy/resinous substance n/a Acetonitrile Ζ - - Suspension Ripening 60/5 °C Suspension I N/A Form 1 10% water/EtOH Ζ - - Solution Cooled below 5°C Solution Glassy/resinous substance N/A 10% water/IP A Ζ - - Solution Cooled below 5°C Solution Glassy/resinous substance N/A 10% water/THF Ζ - - Solution Cooled below 5°C Solution Glassy/resinous substance N/A mek X - Solution Cooled below 5°C Suspension I N/A Form 2 Water X X X Resinous suspension Ripening 60/5 °C Suspension I N/A Form 1 2-Butanol Ζ - - Solution Cooled below 5°C Suspension I n/a Form 3 Isobutyl acetate X X X Fine suspension Ripening 60/5 °C Suspension I n/a Form 1 Cyclohexane X X X Suspension Ripening 60/5 °C Suspension I n/a Form 1 + two additional flax peaks 1,4-Dioxane Ζ - - Solution Cooled below 5°C Solution Solid Amorphous

Key: clear solution; *^x = cloudy; x = suspension; N/A = not carried out

Table 3. Sorting of polymorphic forms using the amorphous form of ________________compound 1 with a purity of 97.6% (20 mg)___________

Solvent 10 rev. 20 rev. 30 rev. Observation at 50 °C Treatment Observation after treatment (72 hours) Evaporation at room temperature RPD n-Heptane X X X Suspension I Ripening 60/5 °C Suspension I N/A Form a 1 Acetone X Suspension I Ripening 60/5 °C Suspension I N/A Form a 1 2-Propanol V - - Solution Cooling at 5 °C Solution Vitreous e/ N/A

- 13 043785

resinous substance Ethanol Solution Cooling at 5 °C Solution Glassy/resinous substance N/A Ethyl acetate X X X Suspension I Ripening 60/5 °C Suspension I N/A Form a 1 Isopropylacetate X X X Suspension I Ripening 60/5 °C Suspension I N/A Form a 1 tert-Butyl methyl ether X X X Suspension I Ripening 60/5 °C Suspension I n/a Form a 1 mek - - Solution Cooling at 5 °C Suspension I n/a Form a 1 Toluene Solution Cooling at 5 °C Solution Solid Similar to the U2 form Tetrahydrofur and Solution Cooling at 5 °C Solution Solid Similar to forms 2 Acetonitrile X X X Suspension I Ripening 60/5 °C Suspension I N/A Form a 1 10% water/IPA Solution Cooling at 5 °C Solution Glassy/resinous substance N/A

Key: C= clear solution; X x = cloudy; x = suspension; N/A = not carried out

4. Selection of new crystal forms

Forms 1 and 2 were chosen for further analysis because Form 3 showed a weakly crystalline phase. For Form 1, the sample prepared from acetonitrile had the highest purity (97.4%) and contained only a trace amount of solvent. Scaling experiments with Form 1 were performed as described below. For Form 2, the sample obtained from MEK had the highest purity (97.2%) and contained only a small amount of residual MEK. Scaling experiments with Form 1 were performed as described below.

Scaling up the production of crystalline Form 1 and Form 2 Scale-up experiments for the production of Form 1 in acetone and Form 2 in MEK were carried out in accordance with the following method. The amorphous form of compound 1, which was prepared by the methods described in US patent 62/659408 (1 g), was weighed in two 20 ml scintillation tubes and dissolved in 20 volumes (20 ml) of acetone or 20 volumes (20 ml) of MEK at 50 °C with stirring. The solutions were then cooled to 5°C at a rate of 0.1°C/min and kept at this temperature for 20 hours. Both samples formed white suspensions, which were filtered and dried in a Buchner funnel under vacuum. The solids were left to air dry for an hour, then both options were described using different techniques (summarized in Tables 4 and 5 and FIGS. 1-4). Yields were also calculated for the isolated solids.

For the scaled preparation of Form 1, the solid sample was confirmed to be crystalline Form 1 using XRD (Table 4 and FIG. 1). Purity determined by HPLC was 96.6%. By ¹ H NMR the sample was consistent with the reference material, an amorphous form of compound 1 with a small amount of residual acetone (0.04 eq) remaining in the sample. Thermal analysis showed a small weight loss of 1.0% w/w. (corresponding to 0.32 equiv. water), which is consistent with the KF analysis. DSC analysis showed a sharp endotherm at 179.5°C (onset) (Fig. 2). GSP analysis showed that the material is slightly hygroscopic with an absorption of 1.7% w/w. water in the RH range from 0 to 90%. The sample was stored in Form 1 after GSP analysis or storage for 5 days under elevated RH conditions. The morphology of Form 1, as determined by PLM and SEM, was agglomerates of small crystalline particles. These agglomerates can vary in size and shape (from 20 to 650 µm), and the crystalline particles are small and irregular in shape (up to 20 µm). In general, Form 1 is found to be anhydrous (See also Table 5 for a summary of the properties of Form 1).

For the scaled preparation of Form 2, the solid sample was confirmed to be crystalline Form 2 using XRD (Figure 3). However, further characterization suggested that Form 2 is a less stable crystalline form than Form 1. For example, unlike Form 1, which retains its original crystalline form, RPD analysis showed that Form 2 becomes amorphous after GSP analysis or storage for 5 days in conditions of increased RH. In contrast to Form 1 showing a cutting endotherm (Figure 2), Form 2 exhibited two endothermic events in DSC analysis (Figure 4). Form 2 also showed higher overall weight loss than Form 1 in the TGA analysis. Form 1 was more common than form 2 during sorting of polymorphic forms and in suspension competition experiments, which further underscores

- 14 043785 states that form 1 is more stable than form 2.

Table 7. RPD of crystalline form 1

Table 8. Characteristics of crystalline form 1

Analysis Scaled Form Retrieval 1 RPD Crystal Form 1 (free base) 'n-yamr Corresponds to reference material. 0.04 eq. residual acetone vezhkh 96.6% THF Weight loss 1.0% from 36 to 179 °C (corresponds to 0.32 equiv. water) DSK Endotherm at 179.5 °C (start, 61 J/g) KF 1.0% w/w (0.32 equiv. water). SHG Absorption -1.7% w/w water in the RH range from 0 to 90%. No hysteresis. RPD after GSP showed that the material retained profile 1. Exit 54.1% (part of the material passed through the filter. The actual yield should be significantly higher). Static stability at 40 °C/75% RH (5 days) RPD: Slight decrease in crystallinity, retention of profile 1, HPLC: 96.5%. Static RPD: Slight reduction in crystallinity, preservation stability at 25 °C/RH 97% (5 days) profile 1, HPLC: 96.8%. PLM Agglomerates of small crystalline particles. Agglomerates vary significantly in size and shape from 20 µm to 650 µm in length. Crystalline particles up to 20 microns long of irregular shape. SAM Agglomerates of various sizes, consisting of small crystalline particles of irregular shape.

Although many embodiments of the present invention have been described, it will be appreciated that the basic examples provided may be modified to provide other embodiments that utilize the compounds and methods described herein. Accordingly, it is to be understood that the scope of the present invention is to be defined by the appended claims and not by the specific embodiments which are presented by way of example. The entire contents of all references (including literary references, issued patents, published patent applications and pending patent applications) cited in this application are expressly incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used herein have their common meaning known to those skilled in the art.

-

Claims (20)

CLAIM 1. Crystal form 1 of the compound having the structural formula 2. Crystal form 1 according to claim 1, characterized in that said crystal form is characterized by at least three X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2 °, 20.8°, 22.2° and 22.5°. 3. Crystalline form 1 according to claim 1 or 2, characterized in that said crystalline form is characterized by at least four X-ray powder diffraction peaks at angles 2Θ selected from 10.0°, 13.3°, 14.9°, 20 ,2°, 20.8°, 22.2° and 22.5°. 4. Crystalline form 1 according to any one of claims 1 to 3, characterized in that said crystalline form is characterized by at least five X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9° , 20.2°, 20.8°, 22.2° and 22.5°. 5. Crystalline form 1 according to any one of claims 1 to 4, characterized in that said crystalline form is characterized by at least six X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9° , 20.2°, 20.8°, 22.2° and 22.5°. 6. Crystalline form 1 according to any one of claims 1 to 5, characterized in that said crystalline form is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2 °, 20.8°, 22.2° and 22.5°. 7. Crystalline form 1 according to any one of claims 1 to 6, characterized in that said crystalline form is characterized by X-ray powder diffraction peaks at angles 2Θ selected from 10.0°, 10.2°, 12.3°, 12.7 °, 13.3°, 14.9°, 15.3°, 20.2°, 20.8°, 21.3°, 22.2°, 22.5° and 23.8°. 8. Crystalline form 1 according to any one of claims 1 to 7, characterized in that said crystalline form is characterized by X-ray powder diffraction peaks at angles 2Θ selected from 10.0°, 10.2°, 11.0°, 11.4 °, 11.8°, 12.3°, 12.7°, 13.3°, 14.9°, 15.3°, 16.1°, 17.4°, 20.2°, 20.8 °, 21.3°, 22.2°, 22.5° and 23.8°. 9. Crystalline form 1 according to any one of claims 1 to 8, characterized in that said crystalline form is characterized by essentially the same RPD profile as in FIG. 1. 10. Crystal form 1 according to claim 1, characterized in that said crystal form is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 14.9°, 20.2°C and 20.8°. 11. Crystal form 1 according to claim 1 or 10, characterized in that said crystal form is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 14.9°, 20.2°C and 20.8° . 12. Crystalline form 1 according to any one of claims 1, 10 or 11, characterized in that said crystalline form is characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 14.9°, 20.2°, 20 .8° and 22.2°. 13. Crystalline form 1 according to any one of claims 1, 10-12, characterized in that said crystalline form is characterized by X-ray powder diffraction peaks at angles 2Θ selected from 10.0°, 13.3°, 14.9°, 20 ,2°, 20.8° and 22.2°. 14. Crystalline form 1 according to any one of claims 1 to 13, characterized in that said crystalline form is anhydrous. 15. Crystalline form 1 according to any one of claims 1 to 14, characterized in that crystalline form 1 is at least 90% by weight one crystalline form. 16. Crystalline form 1 according to any one of claims 1 to 15, characterized in that crystalline form 1 is at least 95% by weight one crystalline form. 17. Crystalline form 1 according to any one of claims 1 to 16, characterized in that said compound has a chemical purity of at least 90% by weight. 18. Crystalline form 1 according to any one of claims 1 to 17, characterized in that said compound has a chemical purity of at least 95% by weight. 19. Crystalline form 1 according to any one of claims 1 to 18, characterized in that said compound has a chemical purity of at least 99% by weight. 20. Crystalline form 1 according to any one of claims 1 to 19, characterized in that said compound has the structural formula -