Salt of ezh2 inhibitor compound, crystal form thereof and use thereof

Abstract

A polymorph of hydrobromide or hydrochloride of 5-(6-(4-(cyclopropylmethyl)piperazin-1-yl)-2-methylpyridin-3-yl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarboxamido)-2-methylbenzamide, a pharmaceutical composition and the use thereof.

Description

A salt of an EZH2 inhibitor compound, its crystal form and applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a salt of an EZH2 inhibitor compound and its crystal form, as well as the application of the salt and crystal form. Background Technology

[0002] Chinese patent application CN108314677A discloses a class of EZH2 inhibitors and methods and uses thereof for treating cancers or precancerous conditions related to EZH2 activity.

[0003] As a type of EZH2 inhibitor compound, “5-(6-(4-(cyclopropylmethyl)piperazin-1-yl)-2-methylpyridin-3-yl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide” with the structure of Formula I has been shown to have superior inhibitory activity to the positive control group in the TR-FRET enzyme activity assay kit.

[0004] In practical applications, the study of salt forms and crystal forms of active compounds plays a crucial role in drug development. Different salt forms or crystal forms of the same drug exhibit significant differences in physicochemical and biological properties. To better control drug quality and meet the requirements of formulation, production, storage, and transportation, there is a consistent need to develop salt forms and / or crystal forms with favorable properties for active compounds.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide a polymorph of the salt of "5-(6-(4-(cyclopropylmethyl)piperazin-1-yl)-2-methylpyridin-3-yl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide" (hereinafter referred to as "Compound I"), which has improved solubility, hygroscopicity, stability, and efficacy.

[0007] One aspect of the present invention is to provide a crystal form A of the hydrobromide of compound of formula I.

[0008] In the X-ray powder diffraction patterns obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, the crystal form A of the hydrobromide of the compound of formula I has characteristic peaks at 2θ angles of 14.4±0.2°, 17.7±0.2°, 18.7±0.2°, 22.7±0.2° and 23.8±0.2°.

[0009] Preferably, in the X-ray powder diffraction pattern obtained using Cu-Kα radiation, the crystal form A of the hydrobromide of the compound of formula I also has a characteristic peak at at least one of 6.7±0.2°, 8.4±0.2°, 9.4±0.2°, 13.1±0.2°, 21.3±0.2°, 24.6±0.2°, 25.6±0.2°, 26.3±0.2° and 27.6±0.2°.

[0010] More preferably, the characteristic peak positions shown in the X-ray powder diffraction pattern of the hydrobromide crystal form A of the compound of formula I are substantially the same as the characteristic peak positions shown in Figure 6.

[0011] In the infrared spectrum, the crystal form A of the hydrobromide of compound I is at 2930±2 cm⁻¹. -1 1633±2cm -1 1455±2cm -1 1269±2cm -1 968±2cm -1 805±2cm -1 and 617±2cm -1 The characteristic absorption peak is displayed at the wavenumber.

[0012] In some embodiments, the hydrobromide of the compound of formula I, crystal form A, shows an endothermic peak at approximately 247°C in differential scanning calorimetry.

[0013] Another aspect of the present invention is to provide a crystal form A of the hydrochloride salt of compound of formula I.

[0014] In the X-ray powder diffraction patterns obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, the crystal form A of the hydrochloride salt of the compound of formula I has characteristic peaks at 2θ angles of 8.5±0.2°, 12.0±0.2°, 17.0±0.2°, 23.0±0.2° and 23.5±0.2°.

[0015] Preferably, in the X-ray powder diffraction pattern obtained using Cu-Kα radiation, the crystal form A of the hydrochloride salt of the compound of formula I also has a characteristic peak at at least one of 17.4±0.2°, 17.8±0.2°, 19.2±0.2°, 20.8±0.2° and 21.7±0.2°.

[0016] More preferably, the positions of the characteristic peaks shown in the X-ray powder diffraction pattern of crystal form A of the hydrochloride salt of Formula I are substantially the same as the positions of the characteristic peaks shown in Figure 4.

[0017] Another aspect of the present invention is to provide a pharmaceutical composition comprising the crystal form described above and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0018] Another aspect of the present invention is to provide the use of the above-described crystal form or the above-described pharmaceutical composition in the preparation of a medicament for treating cancer.

[0019] The crystal forms of the Formula I compound and its salts prepared by the method of this invention can be identified and distinguished from other crystal forms using characteristic X-ray powder diffraction (XRPD) patterns, DSC curves, and Fourier transform-infrared spectroscopy (FT-IR spectroscopy). Through comprehensive comparison of the polymorphic samples of the obtained Formula I compound and its salts from the perspectives of solubility, hygroscopicity, physicochemical stability, and pharmacodynamics, crystal form A of the hydrobromide of Formula I compound exhibits the best performance in terms of solubility, hygroscopicity, physicochemical stability, and pharmacodynamics, making this crystal form a promising candidate for drug development.

[0020] Other aspects and advantages of the invention will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the invention are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this invention enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention. Accordingly, the descriptions in the accompanying drawings and specification are merely exemplary and not restrictive. Attached Figure Description

[0021] The specific features of the invention are shown in the appended claims. The characteristics and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0022] Figure 1 shows the X-ray powder diffraction (XRPD) pattern of crystal form A of the compound of formula I obtained in Example 1;

[0023] Figure 2 shows the infrared spectrum of crystal form A of the compound of formula I obtained in Example 1;

[0024] Figure 3 shows the differential scanning calorimetry (DSC) spectrum of crystal form A of the compound of formula I obtained in Example 1;

[0025] Figure 4 shows the X-ray powder diffraction (XRPD) pattern of crystal form A of the hydrochloride salt of compound I obtained in Example 3;

[0026] Figure 5 shows the X-ray powder diffraction (XRPD) pattern of crystal form A of the sulfate of compound I obtained in Example 4;

[0027] Figure 6 shows the X-ray powder diffraction (XRPD) pattern of the hydrobromide of the compound of formula I obtained in Example 5, crystal form A.

[0028] Figure 7 shows the infrared spectrum of crystal form A of the hydrobromide of compound I obtained in Example 5;

[0029] Figure 8 shows the differential scanning calorimetry (DSC) spectrum of the hydrobromide of the compound of formula I obtained in Example 5, crystal form A.

[0030] Figure 9 shows the crystal structure and unit cell packing diagram of the hydrobromide of Formula I obtained in Example 5.

[0031] Figure 10 shows the X-ray powder diffraction (XRPD) pattern of crystal form B of the hydrobromide of the compound of formula I obtained in Example 6.

[0032] Figure 11 shows the differential scanning calorimetry (DSC) spectrum of crystal form B of the hydrobromide of compound I obtained in Example 6. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0034] Terminology Definition

[0035] In this invention, the term "5-(6-(4-(cyclopropylmethyl)piperazin-1-yl)-2-methylpyridin-3-yl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide", with the chemical structure shown in Formula I, is an oral inhibitor of wild-type and Y641F mutant forms of the human histone methyltransferase EZH2.

[0036] The terms “polymorph,” “polymorphs,” “crystal modification,” “crystal form,” “crystalline modification,” “polymorphic form,” and “crystalline form” as used in this invention are understood to be synonymous and refer to the solid crystalline form of a compound or complex, including, but not limited to, single-component or multi-component crystals, and / or polymorphs, solvates, hydrates, inclusion compounds, eutectics, salts, solvates of salts, and hydrates of salts.

[0037] Polymorphs can be detected, identified, classified, and characterized using techniques well known to those skilled in the art, including, but not limited to: differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD), single-crystal X-ray diffraction, vibrational spectroscopy, solution calorimetry, solid-state nuclear magnetic resonance (SSNMR), Fourier transform-infrared spectroscopy (FT-IR), Raman spectroscopy, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography, as well as quantitative analysis, particle size analysis (PSA), surface area analysis, solubility, and dissolution rate. A polymorph can be described as the ability of a specific compound to crystallize in different crystalline forms while maintaining the same chemical structure. Polymorphs of a given substance are chemically equivalent, containing the same atoms bonded together in the same manner, but differing in their crystalline forms, which can affect one or more physical properties, such as dissolution rate, melting point, bulk density, stability, flow properties, etc. The graphical representation of such data can vary slightly (e.g., relative peak intensities and peak positions) due to factors such as changes in instrument response and sample concentration and purity, as is well known to those skilled in the art. Nevertheless, those skilled in the art can compare the graphical data in the figures of this invention with graphical data generated for unknown crystal forms and can confirm whether the two sets of graphical data characterize the same crystal form.

[0038] Unless otherwise stated, when this invention refers to spectra or data presented in graphical form (e.g., XRPD, IR, Raman, and NMR spectra), the term "peak" refers to a peak or other special feature that is not caused by background noise and can be identified by a person skilled in the art.

[0039] As is well known in the field of X-ray powder diffraction (XRPD), for any given crystal form, the apparatus used to obtain the X-ray powder diffraction pattern, humidity, temperature, powder crystal orientation, and other parameters can all cause some variability in the appearance, intensity, and position of peaks in the diffraction pattern. In the present case, a variability of ±0.2°2θ peak positions takes into account these possible variations without hindering the clear identification of the indicated crystal form. Crystal form identification can be based on any unique difference peaks (in °2θ units) or combinations thereof, typically more prominent peaks. Therefore, in some embodiments, the crystalline compounds of the present invention are characterized by XRPD patterns with certain peak positions that have substantially the same characteristics as the XRPD patterns provided in the accompanying drawings. With the instrumentation used according to the present invention, an error tolerance of ±0.2° may exist for the diffraction peak positions. For example, an X-ray powder diffraction pattern “substantially consistent” with the pattern provided in the present invention may be identical to the XRPD pattern in the accompanying drawings, or more likely it may be slightly different. Such XRPD plots may not necessarily show every peak in the diffraction pattern presented in this invention, and / or may show slight variations in the appearance, intensity, or shift of said peaks due to differences in the conditions involved in obtaining the data. Those skilled in the art can determine whether a sample of the crystalline compound has the same or different crystal form as that of this invention by comparing their XRPD plots. Similarly, those skilled in the art can determine whether the given diffraction peak positions (expressed in °2θ) derived from the XRPD plot are at approximately the same positions as the values ​​presented in this invention. In the context of this invention, 2θ values ​​in X-ray powder diffraction patterns are all in degrees (°).

[0040] Similarly, as is well known in the field of differential scanning calorimetry (DSC), the melting peak height of a DSC curve depends on many related factors, such as sample preparation and instrument conditions, while the peak position is relatively insensitive to experimental details. Therefore, in some embodiments, the crystalline compounds of the present invention are characterized by DSC plots with characteristic peak positions, possessing substantially the same properties as the DSC plots provided in the accompanying drawings. Depending on the instrument used in this experiment, the melting temperature has an error tolerance of ±3°C, ±4°C, or ±5°C.

[0041] As is well known in the field of Fourier transform-infrared spectroscopy (FT-IR), the position and shape of the absorption peaks in an infrared spectrum depend on the transitions of the covalent bond energy levels in the sample molecule. Therefore, in some embodiments, the crystalline compounds of the present invention are characterized by Fourier transform-infrared spectra with characteristic peak positions and shapes, possessing substantially the same properties as the Fourier transform-infrared spectra provided in the accompanying drawings of the present invention. According to Appendix IV C of the Chinese Pharmacopoeia (2010 edition) – Infrared Spectrophotometry, and based on the instrumentation used in this experiment, the absorption peak is at 3000 cm⁻¹. -1 There is ±5cm nearby-1 The error tolerance is within 1000cm. -1 There is ±2cm nearby -1 Error tolerance.

[0042] The phrase "substantially identical" or "essentially identical" in X-ray powder diffraction patterns, DSC curves, and Fourier transform-infrared spectra generally means that at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% of the peaks are shown in the X-ray powder diffraction pattern, DSC curve, and infrared spectrum.

[0043] The terms “about” and “approximately” as used in this invention generally mean within ±10% of a given value or range, appropriately within ±5%, and particularly within ±1%. Alternatively, to those skilled in the art, the terms “about” and “approximately” mean within an acceptable standard error of the average value.

[0044] As used in this invention, the term "solution" generally refers to a mixture containing at least one solvent and at least one compound, which is at least partially dissolved in the solvent.

[0045] Hygroscopicity is an important physical property of active pharmaceutical ingredients (APIs), directly affecting their storage stability, processability, and manufacturing process. Table A defines the hygroscopicity of drugs after equilibration at 25°C and 80% RH, according to the 2020 edition of the Chinese Pharmacopoeia.

[0046] Table A

[0047] The term "pharmaceutical composition" generally refers to a mixture of one or more compounds described in this invention, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, with other chemical components such as physiologically / pharmaceutical acceptable excipients, excipients, diluents, adjuvants, carriers, and additional therapeutic agents. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a living organism.

[0048] Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffering substances, 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, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene-blocking polymers; lanolin; sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethyl cellulose. Sodium cellulose, ethyl cellulose, and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and suitable lubricants such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; coatings; sweeteners; flavorings; fragrances; preservatives and antioxidants.

[0049] The pharmaceutical compositions of the present invention can be administered orally, by injection, topically, sublingually, or via an implantable cartridge. The term "injection" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intramembranous, intraocular, intrahepatic, intralesional, and intracranial injection or infusion techniques. For example, the pharmaceutical compositions of the present invention can be administered orally in any acceptable oral dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. As another example, the aseptic injection method of the pharmaceutical compositions of the present invention can be an aqueous or oil-based suspension, which can be formulated according to known techniques using suitable dispersants, wetting agents, and suspending agents.

[0050] The term "cancer" refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancers are classified according to the type of cells that resemble tumor cells and are therefore presumed to be the origin of the tumor. These types include carcinomas, sarcomas, lymphomas and leukemias, germ cell tumors, and blastomas. The term "carcinoma" is used to refer to cancers that originate from epithelial cells. This group includes many of the most common cancers and includes almost all cancers that develop in the breast, prostate, lung, pancreas, and colon.

[0051] For example, the term "cancer" includes, but is not limited to, solid tumors, hematologic malignancies (e.g., leukemia, lymphoma, myeloma (such as multiple myeloma)), and metastatic lesions. In one embodiment, cancer is a solid tumor. Examples of solid tumors include malignant tumors such as sarcomas and carcinomas, adenocarcinomas affecting multiple organ systems such as those affecting the lungs, breasts, ovaries, lymph nodes, gastrointestinal tract (e.g., colon), anus, genitals and genitourinary tract (e.g., kidneys, urothelial cells, bladder cells, prostate), pharynx, CNS (e.g., brain cells, nerve cells, or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas that include malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Cancer can be in the early, intermediate, or late stages, or it can be metastatic.

[0052] As used in this invention, the term "treatment" refers to any disease or condition, and in some embodiments, it means improving the disease or condition (i.e., slowing down or stopping or alleviating the development of the disease or at least one of its clinical symptoms). In other embodiments, "treatment" means alleviating and / or improving at least one bodily parameter, including bodily parameters that may not be perceived by the patient. In still other embodiments, "treatment" means regulating the disease or condition from a physical (e.g., stabilizing perceptible symptoms), physiological (e.g., stabilizing bodily parameters), or both. In still other embodiments, "treatment" means preventing or delaying the onset, occurrence, or worsening of the disease or condition.

[0053] The term "combination" refers to a fixed combination, or combination administration, of a dosage unit, in which the crystal form of a compound having Formula I and its combination partner (i.e., an immunotherapeutic agent) can be administered independently at the same time or separately at time intervals, particularly where these time intervals allow the combination partner to exhibit synergy, such as a co-existing effect. Individual components may be packaged in a single box or separately. One or both components (e.g., powder or liquid) may be reconstituted or diluted to the desired dose prior to administration.

[0054] The term "drug combination" refers to a fixed combination or a non-fixed combination or kit for combined administration in the form of a single dose unit, wherein two or more therapeutic agents may be administered independently at the same time or separately at time intervals, particularly where these time intervals allow the combination couple to exhibit synergistic effects, such as a co-occurrence. The term "fixed combination" means a crystalline form of a compound having Formula I and a combination couple (i.e., an immunotherapeutic agent), administered simultaneously to a patient as a single entity or dose. The term "non-fixed combination" means a crystalline form of a compound having Formula I and a combination couple (i.e., an immunotherapeutic agent), administered simultaneously, in parallel, or sequentially to a patient as separate entities (without a specific time limit), wherein such administration provides a therapeutically effective level of the two compounds in the patient's body. The latter also applies to cocktail therapies, such as the administration of three or more therapeutic agents. In a non-limiting example, the drug combination is a non-fixed combination.

[0055] The terms “co-administration” or “combination administration” are intended to cover the administration of a selected combination of couples (i.e., immunotherapeutic agents) to a single subject (e.g., a patient) in need, and are intended to include treatment regimens in which the agents are not necessarily administered via the same route of administration or administered simultaneously.

[0056] In one aspect of the present invention, a crystal form A of the compound of formula I is provided.

[0057] In the X-ray powder diffraction patterns obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, crystal form A of the compound of Formula I shows characteristic peaks at 2θ angles of 7.9±0.2°, 14.0±0.2°, 15.0±0.2°, 18.3±0.2°, 21.3±0.2°, and 23.9±0.2°.

[0058] In some embodiments, in the X-ray powder diffraction pattern obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, the crystal form A of the compound of Formula I also has a characteristic peak at at least one of 9.2±0.2°, 11.0±0.2°, 13.3±0.2°, 16.6±0.2°, 18.9±0.2° and 22.2±0.2°.

[0059] In some embodiments, in the infrared spectrum obtained using infrared spectroscopy characterization, the crystal form A of the compound of formula I is at 3446±2 cm⁻¹. -1 3231±2cm -1 1632±2cm -1 1549±2cm -1 and 1264±2cm -1 The characteristic absorption peak is displayed at the wavenumber.

[0060] In some embodiments, the positions of the characteristic peaks shown in the X-ray powder diffraction pattern of crystal form A of the compound of Formula I are substantially the same as the positions of the characteristic peaks shown in Figure 1.

[0061] In some embodiments, the crystal form A of the compound of formula I shows an endothermic peak at about 215°C in differential scanning calorimetry.

[0062] On the other hand, the present invention provides a method for preparing crystal form A of the aforementioned compound of formula I.

[0063] In some embodiments, the method includes: adding an antisolvent dropwise to a solution of the normal solvent of compound I, stirring until a solid precipitates, to obtain crystal form A of compound I, wherein the normal solvent includes halogenated hydrocarbons, alcohols, polar aprotic solvents, or a mixture of two or more of the aforementioned solvents, and the antisolvent includes aliphatic hydrocarbons, nitriles, ketones, esters, ethers, or a mixture of two or more of the aforementioned solvents.

[0064] In a preferred embodiment, the positive solvent includes dichloromethane, methanol, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, or a mixture of two or more of the aforementioned solvents.

[0065] In a preferred embodiment, the antisolvent includes isopropyl acetate, acetonitrile, n-heptane, cyclohexane, methylcyclohexane, tetrahydrofuran, acetone, 2-butanone, methyl isobutyl ketone, ethyl acetate, methyl tert-butyl ether, or a mixture of two or more of the aforementioned solvents.

[0066] In another embodiment, the method includes: adding an appropriate amount of solvent to the compound of formula I to suspend it, filtering, and drying to obtain crystal form A of the compound of formula I, wherein the solvent includes water, nitriles, ketones, esters, ethers, alkanes, or a mixture of two or more of the aforementioned solvents.

[0067] In this embodiment, the solvent preferably includes two or more of the following solvents: acetone, 2-butanone, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, methyl tert-butyl ether, 1,4-dioxane, n-heptane, cyclohexane, methylcyclohexane, acetonitrile, and water.

[0068] On the other hand, the present invention provides a salt of a compound of formula I:

[0069] The salt comprises a free base portion and an acid portion;

[0070] The free alkali portion has the structure shown in Formula I;

[0071] The acid portion is selected from any one of hydrochloric acid, sulfuric acid, hydrobromic acid, maleic acid, or D-tartaric acid.

[0072] In some embodiments, the salt has a crystalline form.

[0073] In some embodiments, the acid portion is hydrochloric acid or hydrobromic acid.

[0074] On the other hand, the present invention provides a crystal form A of the hydrochloride salt of the compound of formula I, in which the crystal form A of the hydrochloride salt of the compound of formula I has characteristic peaks at 2θ angles of 8.5±0.2°, 12.0±0.2°, 17.0±0.2°, 23.0±0.2° and 23.5±0.2° in the X-ray powder diffraction pattern obtained by using Cu-Kα radiation.

[0075] In some embodiments, in the X-ray powder diffraction pattern obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, the crystal form A of the hydrochloride salt of Formula I also has a characteristic peak at at least one of 17.4±0.2°, 17.8±0.2°, 19.2±0.2°, 20.8±0.2° and 21.7±0.2°.

[0076] In some embodiments, the characteristic peak positions shown in the X-ray powder diffraction pattern of crystal form A of the hydrochloride salt of Formula I are substantially the same as those shown in Figure 4.

[0077] On the other hand, the present invention provides a crystal form A of the sulfate of a compound of formula I, in which the crystal form A of the sulfate of the compound of formula I has characteristic peaks at 2θ angles of 6.5±0.2°, 7.3±0.2°, 10.0±0.2°, 12.0±0.2° and 13.0±0.2° in the X-ray powder diffraction pattern obtained by using Cu-Kα radiation.

[0078] In some embodiments, the characteristic peak positions shown in the X-ray powder diffraction pattern of crystal form A of the sulfate of Formula I are substantially the same as those shown in Figure 5.

[0079] On the other hand, the present invention provides a crystal form A of the hydrobromide of a compound of formula I, in which the crystal form A of the hydrobromide of the compound of formula I has characteristic peaks at 2θ angles of 14.4±0.2°, 17.7±0.2°, 18.7±0.2°, 22.7±0.2° and 23.8±0.2° in the X-ray powder diffraction pattern obtained by using Cu-Kα radiation.

[0080] In some embodiments, in the X-ray powder diffraction pattern obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, the crystal form A of the hydrobromide of Formula I compound also has a characteristic peak at at least one of 6.7±0.2°, 8.4±0.2°, 9.4±0.2°, 13.1±0.2°, 21.3±0.2°, 24.6±0.2°, 25.6±0.2°, 26.3±0.2°, and 27.6±0.2°.

[0081] On the other hand, the present invention provides a crystal form A of the hydrobromide of compound I, in which, in the infrared spectrum obtained by characterization using infrared spectroscopy, crystal form A of the hydrobromide of compound I is located at 2930±2 cm⁻¹. -1 1633±2cm -1 1455±2cm -1 1269±2cm -1 968±2cm -1 805±2cm -1 and 617±2cm -1 The characteristic absorption peak is displayed at the wavenumber.

[0082] In some embodiments, the characteristic peak positions shown in the X-ray powder diffraction pattern of the hydrobromide crystal form A of the compound of formula I are substantially the same as those shown in Figure 6.

[0083] In some embodiments, the hydrobromide of the compound of formula I, crystal form A, shows an endothermic peak at approximately 247°C in differential scanning calorimetry.

[0084] On the other hand, the present invention provides a crystal form B of the hydrobromide of a compound of formula I, in which the crystal form B of the hydrobromide of the compound of formula I has characteristic peaks at 2θ angles of 13.1±0.2°, 14.2±0.2°, 21.3±0.2°, 21.8±0.2°, 22.7±0.2° and 25.3±0.2° in the X-ray powder diffraction pattern obtained by using Cu-Kα radiation.

[0085] In some embodiments, the characteristic peak positions shown in the X-ray powder diffraction pattern of crystal form B of the hydrobromide of compound of formula I are substantially the same as those shown in Figure 10.

[0086] In some embodiments, the hydrobromide of the compound of formula I, crystal form B, shows an endothermic peak at approximately 233°C in differential scanning calorimetry.

[0087] On the other hand, the present invention provides a method for preparing crystal forms A of the hydrochloride salt, A of the sulfate salt, and A and B of the hydrobromide salt of the aforementioned compound I, the method comprising: weighing a certain amount of free base and adding an appropriate amount of solvent; adding a certain amount of acid solution, directly precipitating a solid, filtering, and drying to obtain the polymorph of the salt of compound I, or adding a certain amount of acid solution and then adding an appropriate solvent (or adding the entire system to an appropriate solvent), precipitating a solid, filtering, and drying to obtain the polymorph of the salt of compound I.

[0088] On the other hand, the present invention provides a pharmaceutical composition comprising a crystal form of the aforementioned compound of formula I, or a salt of the aforementioned compound of formula I or a crystal form of a salt thereof, and a pharmaceutically acceptable excipient.

[0089] In some embodiments, the pharmaceutical composition comprises crystal form A of the hydrobromide of formula I, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0090] In some embodiments, the pharmaceutical composition comprises crystal form A of the hydrochloride salt of compound of formula I, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0091] On the other hand, the present invention provides the use of the crystal form of the aforementioned Formula I compound, or the crystal form of a salt of the aforementioned Formula I compound or a salt thereof, in the preparation of a medicament for treating a disease, wherein the disease is cancer.

[0092] In some embodiments, the present invention provides the use of the hydrobromide of Formula I, crystal form A, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease, said disease being cancer.

[0093] In some embodiments, the present invention provides the use of crystal form A of the hydrochloride salt of compound of formula I or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease, said disease being cancer.

[0094] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following detailed description of the technical solutions of the present invention using embodiments will help to further understand the advantages and effects of the technical solutions of the present invention. The embodiments do not limit the scope of protection of the present invention, which is determined by the claims.

[0095] The abbreviations used in this invention are explained as follows:

[0096] XRPD: X-ray powder diffraction;

[0097] IR: Infrared spectrum;

[0098] DSC: Differential Scanning Calorimetry;

[0099] HPLC: High Performance Liquid Chromatography;

[0100] RH: Relative humidity.

[0101] Instruments and methods

[0102] X-ray powder diffraction (XRPD)

[0103] XRPD images were acquired using a DX-2700BH X-ray diffractometer, with the following scanning parameters:

[0104] Equipment parameters: Cu Kα rays, scattering slit #2: 1°, anti-scattering slit #3: 1°, receiving slit #4: 0.2mm;

[0105] Detection parameters: tube current: 40mA, tube voltage: 40kV, step rate: 0.02° / 0.5 seconds; starting angle: 3°, ending angle: 40°.

[0106] Differential scanning calorimetry (DSC)

[0107] The DSC spectrum was acquired using a TA Q2000 / Discovery 2500 differential scanning calorimeter, and the test parameters are shown in Table 1.

[0108] Table 1 DSC Test Parameters

[0109] Unless otherwise specified in the examples, the room temperature is 20℃~30℃.

[0110] Infrared spectroscopy (IR)

[0111] Samples for IR spectroscopy were prepared using a potassium bromide pellet method and analyzed using a transmission FTIR spectrometer. The infrared spectrometer was an Infrared Spectrometer Thermo Scientific Nicolet iS5+iD1 or similar instrument.

[0112] The starting materials used in the following examples can be prepared according to the prior art, for example, according to the method described in patent application CN108314677A, but the starting materials are not a limiting condition for preparing the crystal form of the present invention.

[0113] Example 1: Preparation of crystal form A of compound of formula I

[0114] 500 mg of the compound shown in Formula I was added to 15 mL of dichloromethane and stirred until dissolved. 15 mL of acetone was added dropwise, and the mixture was stirred at room temperature to induce crystallization. The crystals were filtered, the filter cake was collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified this product as crystal form A of the compound in Formula I, and the XRPD spectrum is shown in Figure 1. The infrared spectrum is shown in Figure 2. The DSC spectrum shows an endothermic peak at 215 °C, and the spectrum is shown in Figure 3.

[0115] Example 2: Preparation of crystal form A of compound of formula I

[0116] As shown in Table 2, approximately 500 mg of compound I was added to the solvents listed in Table 2 below, suspended and slurried at room temperature for 6 days, filtered, the filter cake was collected, and vacuum dried to obtain a solid. X-ray powder diffraction analysis confirmed that the solid was of crystal form A of compound I.

[0117] Table 2

[0118] Example 3: Preparation of crystal form A of the hydrochloride salt of compound I

[0119] Approximately 2 g of compound I was added to 65 mL of methanol and stirred until dissolved. Then, 1.1 eq of 37% hydrochloric acid was added, and the mixture was stirred at room temperature to induce crystallization. The crystals were filtered, the filter cake was collected, and the solid was dried under vacuum. X-ray powder diffraction analysis identified this product as crystal form A of the hydrochloride salt of compound I, specifically crystal form A of the monohydrochloride salt of compound I. The XRPD spectrum is shown in Figure 4, and the spectral analysis data of the X-ray powder diffraction pattern are shown in Table 3.

[0120] Table 3 shows the XRPD spectrum analysis data of crystal form A of the hydrochloride salt of compound I.

[0121] Example 4: Preparation of crystal form A of the sulfate of compound I

[0122] Approximately 600 mg of compound I was added to 20 mL of methanol and stirred until dissolved. Then, 1.1 eq of sulfuric acid (concentrated sulfuric acid diluted with 10 volumes of water) was added, and the mixture was stirred at room temperature to induce crystallization. The crystals were filtered, the filter cake was collected, and the solid was dried under vacuum. X-ray powder diffraction analysis identified this product as crystal form A of the sulfate of compound I, specifically crystal form A of the monosulfate of compound I. The XRPD spectrum is shown in Figure 5.

[0123] Example 5: Preparation of crystal form A of the hydrobromide of compound I

[0124] Approximately 12 g of compound I was added to 120 mL of ethanol, heated and stirred until dissolved, and 1.05 eq of hydrobromic acid (48% hydrobromic acid diluted with 1 volume of water) was added in portions. Crystallization was carried out at room temperature with stirring. 140 mL of methyl tert-butyl ether was added dropwise to the ethanol system, and the mixture was stirred for 16 hours. The mixture was filtered, the filter cake was collected, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form A of the hydrobromide of compound I, specifically crystal form A of the monohydrobromide of compound I. The XRPD spectrum is shown in Figure 6, and the spectral resolution data of the X-ray powder diffraction pattern are shown in Table 4. The infrared spectrum is shown in Figure 7. The DSC spectrum is shown in Figure 8, showing an endothermic peak at a peak temperature of 247 °C.

[0125] Table 4. XRPD spectrum analysis data of crystal form A of the hydrobromide of compound I.

[0126] Bromine ion content detection:

[0127] Test instrument: 916Ti-Touch Measurement Instrument;

[0128] Theoretical value: 11.7%;

[0129] Measured value: 11.2%.

[0130] Single crystal structure analysis:

[0131] The crystal structure and unit cell packing diagram of the hydrobromide of Formula I are shown in Figure 9.

[0132] The unit cell parameters are as follows:

[0133] It belongs to the monoclinic crystal system, space group P121 / n1.

[0134] α = 90°

[0135] β=105.4590(10)°,

[0136] γ = 90°

[0137] Unit cell volume

[0138] The number of asymmetric units within the unit cell is Z = 4.

[0139] Example 6: Preparation of crystal form B of the hydrobromide of compound I

[0140] Approximately 2 g of compound I was added to 65 mL of methanol and stirred until dissolved. Then, 1.1 eq of hydrobromic acid (48% hydrobromic acid diluted with 2 volumes of water) was added, and the mixture was stirred for approximately 16 hours. The system was concentrated to dryness to obtain a solid. 20 mL of ethyl acetate was added, and the mixture was stirred at room temperature for 5 days. The mixture was filtered, and the filter cake was collected and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified this product as crystal form B of the hydrobromide of compound I, specifically crystal form B of the monohydrobromide of compound I. The XRPD spectrum is shown in Figure 10. The DSC spectrum shows an endothermic peak at 233 °C, as shown in Figure 11.

[0141] Example 7: Study on the crystal form transformation relationship of hydrobromide of Formula I compound

[0142] Approximately 50 mg of the hydrobromide samples of Formula I obtained in Examples 5 and 6, in crystal form A and crystal form B respectively, were weighed and suspended in approximately 1 mL of solvent. The samples were magnetically stirred at room temperature (20-30°C) for 3 days, filtered, and dried to obtain solids. XRPD was tested, and the results are shown in Table 5.

[0143] Table 5 Results of the investigation on the crystal form transformation of hydrobromide

[0144] The experimental results in the table show that crystal form A of the hydrobromide of Formula I is relatively stable in most organic solvents. Slurrying at room temperature for 3 days consistently yields crystal form A of the hydrobromide of Formula I. Crystal form B of the hydrobromide of Formula I exhibits a tendency to transform into crystals in most organic solvents, with varying degrees of transformation. Crystal form B of the hydrobromide of Formula I can only be obtained in the ethyl acetate system. In summary, crystal form A of the hydrobromide of Formula I is a stable crystal form in most solvent systems.

[0145] Example 8: Drugability Evaluation

[0146] 1. Stability evaluation

[0147] The crystal form A of the compound of formula I, the crystal form A of the hydrochloride of the compound of formula I, the crystal form A of the sulfate of the compound of formula I, and the crystal form A of the hydrobromide of the compound of formula I obtained in the above examples were taken in appropriate amounts and laid out in an open manner. The stability of the samples under light (4500 Lux), high temperature (60°C), and high humidity (RH90%) conditions was investigated. The sampling period was 30 days. The results are shown in Table 6.

[0148] Table 6 Results of the investigation of influencing factors

[0149] Experimental data show that the crystal forms of all samples remained unchanged under high temperature, high humidity, and light conditions. However, crystal form A of compound I exhibited poor chemical stability under all conditions, especially significant degradation under light. The chemical stability of each salt crystal form was significantly better than that of crystal form A of compound I, particularly under light conditions, where chemical stability improved to varying degrees. The stability of the sulfate crystal form was lower than that of the hydrochloride and hydrobromide crystal forms under all conditions, with the hydrobromide crystal form showing the best chemical stability.

[0150] 2. Hygroscopicity Study

[0151] Take appropriate amounts of each of the following samples obtained from the above examples: crystal form A of compound I, crystal form A of the hydrochloride of compound I, crystal form A of the sulfate of compound I, and crystal form A of the hydrobromide of compound I. Place them open and flat in 90% RH (room temperature). After sampling at the sampling point, weigh them and calculate the moisture absorption weight gain rate. The results are shown in Table 7.

[0152] Table 7 Results of hygroscopicity test for each sample

[0153] Experimental data show that all samples exhibit varying degrees of hygroscopicity. The crystal forms of Formula I and the sulfate crystal forms are more hygroscopic, while the crystal forms of hydrochloride and hydrobromide are less hygroscopic, with the hydrobromide crystal form exhibiting the least hygroscopicity.

[0154] 3. Solubility investigation in different media

[0155] The solubility of the crystal form A of the compound of formula I, the crystal form A of the hydrochloride salt of the compound of formula I, and the crystal form A of the hydrobromide salt of the compound of formula I obtained in the above examples in appropriate amounts in different media was investigated. The results are shown in Table 8.

[0156] Table 8. Solubility test results for each sample

[0157] The above solubility studies show that the solubility of crystal form A of hydrochloride and crystal form A of hydrobromide is significantly better than that of the free base of compound I, especially in neutral water, where crystal form A of hydrobromide has the best solubility.

[0158] 4. Pharmacokinetic Evaluation

[0159] The crystal form A of the compound of formula I, the crystal form A of the hydrochloride salt of the compound of formula I, and the crystal form A of the hydrobromide salt of the compound of formula I obtained in the above examples were taken in appropriate amounts for pharmacokinetic studies. The experimental results are shown in Table 9.

[0160] Table 9 Pharmacokinetic Evaluation Results

[0161] The results showed that, at the same dosage, the total systemic exposure (characterized by AUCall) and peak concentration of hydrobromide crystal form A were superior to those of free base crystal form A and hydrochloride crystal form A.

[0162] Example 9: Packaging stability test of crystal form A of hydrobromide of Formula I compound

[0163] A certain amount of the hydrobromide crystal form A of compound of formula I was weighed, packaged in two layers of PE bags, and then sealed in an aluminum foil bag. The packaging stability under different conditions was investigated. Samples were taken from the sampling point and the purity was tested by RP-HPLC. The test results are shown in Table 10 (RRT indicates unknown impurities).

[0164] Table 10 shows the packaging stability results of the hydrobromide of compound I in crystal form A.

[0165] Stability study data show that the hydrobromide crystal form A of compound I has good chemical stability under packaging conditions (two layers of PE bags, then sealed with an aluminum foil bag), and there is no significant increase in impurities.

[0166] Example 10: Study on key physicochemical properties of the hydrobromide of Formula I in crystal form A

[0167] 1. Powder properties

[0168] The crystal form A of the hydrobromide of the compound of formula I obtained in Example 5 was tested for its powder properties using a powder comprehensive characteristic tester (manufacturer: Dandong Better Instrument Co., Ltd., model: BT-1000). The results are shown in Table 11.

[0169] Table 11 shows the results of the analysis of the crystal form A of the hydrobromide of Formula I.

[0170] 2. Particle size distribution

[0171] The particle size distribution of the hydrobromide of the compound of formula I obtained in Example 5 was detected using a laser particle size analyzer (manufacturer: Malvern, model: Mastersizer 2000). The results are shown in Table 12.

[0172] Table 12 shows the particle size distribution of the hydrobromide crystal form A of compound formula I.

[0173] 3. Solubility

[0174] The equilibrium solubility of the hydrobromide crystal form A of compound I in different media (37℃, 24h) was investigated using a constant temperature water bath shaker method. The results are shown in Table 13.

[0175] Table 13 shows the equilibrium solubility of hydrobromide crystal form A of compound I in different media.

[0176] The dose-solubility volume (DS) is calculated as 100 mg (specification) / (sample equilibrium solubility). The dose-solubility volume (DS) of the sample is <250 ml in pH 1.0–5.5, and >250 ml in pH 6.8.

[0177] The solubility in water (37℃, 24h) is 3.97 mg / ml, and the dose-solubility volume DS = 100 mg (specification) / (3.97 mg / ml) = 25 ml < 250 ml.

[0178] In summary, the hydrobromide of Formula I, crystal form A, exhibits high solubility in the pH range of 1.0 to 5.5, and also in water.

[0179] 4. Stability

[0180] Forced degradation tests were performed on sample A, the crystalline form of the hydrobromide of compound I. The acid degradation conditions were: 1M hydrochloric acid, 5 days at room temperature; alkali degradation conditions: 1M sodium hydroxide solution, 5 days at room temperature; oxidative degradation conditions: 0.3wt% hydrogen peroxide, 2 days at room temperature; solid-state high-temperature degradation conditions: 130℃ for 8 hours; solution-state (acetonitrile:water mixed at a volume ratio of 1:1) high-temperature degradation conditions: 60℃ for 5 days. The light degradation conditions for both solid and solution-state samples were: illuminance of 5000 Lux ± 500 Lux, and near-UV lamp energy of 80–100 μW / cm². 2 The samples were subjected to forced degradation at a temperature of 5℃±3℃ for 3 days under light conditions to investigate the impurity profile and degradation pathway. Furthermore, data obtained from the forced degradation study can be used in formulation process design and development to prevent impurity formation. Forced degradation samples were compared with non-forced degradation samples (controls). The results of each forced degradation condition are shown in Table 14 (RRT indicates unknown impurities).

[0181] Table 14. Stability test results of hydrobromide crystal forms of Formula I compounds under various forced degradation conditions (sample A).

[0182] Note: ND indicates not detected.

[0183] The results show that the hydrobromide of Formula I, crystal form A, is essentially undegraded under high-temperature (liquid, solid), acid (solid), and alkali (solid) conditions, but degrades to varying degrees under light (liquid, solid) and oxidation (solid) conditions. In summary, the hydrobromide of Formula I, crystal form A, is sensitive to oxidation and UV photodegradation. Based on the results of Example 10, sealed, light-protected packaging conditions can be used to prevent the generation of impurities.

[0184] Industrial application

[0185] This invention provides a salt of an EZH2 inhibitor compound, its crystal form, and its applications. It improves the solubility, hygroscopicity, stability, and efficacy of "5-(6-(4-(cyclopropylmethyl)piperazin-1-yl)-2-methylpyridin-3-yl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(N-ethylcyclopropanecarbamate)-2-methylbenzamide," and is therefore suitable for industrial applications.

Claims

1. A crystal form A of the hydrobromide salt of a compound of formula I, characterized in that: In the X-ray powder diffraction patterns obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, the crystal form A of the hydrobromide of the compound of formula I has characteristic peaks at 2θ angles of 14.4±0.2°, 17.7±0.2°, 18.7±0.2°, 22.7±0.2° and 23.8±0.2°.

2. The crystal form A of the hydrobromide of the compound of formula I according to claim 1, characterized in that: In the X-ray powder diffraction pattern obtained using Cu-Kα radiation, the crystal form A of the hydrobromide of the compound of formula I also has a characteristic peak at at least one of 6.7±0.2°, 8.4±0.2°, 9.4±0.2°, 13.1±0.2°, 21.3±0.2°, 24.6±0.2°, 25.6±0.2°, 26.3±0.2° and 27.6±0.2°.

3. The crystal form A of the hydrobromide of the compound of formula I according to claim 1 or 2, characterized in that: In the X-ray powder diffraction pattern obtained using Cu-Kα radiation, the characteristic peak positions shown in the X-ray powder diffraction pattern of crystal form A of the hydrobromide of compound I are substantially the same as those shown in Figure 6.

4. The crystal form A of the hydrobromide of the compound of formula I according to any one of claims 1-3, characterized in that: In the infrared spectrum, the crystal form A of the hydrobromide of compound I is at 2930±2 cm⁻¹. -1 1633±2cm -1 1455±2cm -1 1269±2cm -1 968±2cm -1 805±2cm -1 and 617±2cm -1 The characteristic absorption peak is displayed at the wavenumber.

5. The crystal form A of the hydrobromide of the compound of formula I according to any one of claims 1-4, characterized in that: The hydrobromide of compound I, crystal form A, showed an endothermic peak at approximately 247 °C in differential scanning calorimetry.

6. A crystal form A of the hydrochloride salt of a compound of formula I, characterized in that: In the X-ray powder diffraction patterns obtained using Cu-Kα radiation and expressed in terms of diffraction angle 2θ, the crystal form A of the hydrochloride salt of the compound of formula I has characteristic peaks at 2θ angles of 8.5±0.2°, 12.0±0.2°, 17.0±0.2°, 23.0±0.2° and 23.5±0.2°.

7. The crystal form A of the hydrochloride salt of the compound of formula I according to claim 6, characterized in that: In the X-ray powder diffraction pattern obtained using Cu-Kα radiation, the crystal form A of the hydrochloride salt of the compound of formula I also has a characteristic peak at at least one of 17.4±0.2°, 17.8±0.2°, 19.2±0.2°, 20.8±0.2° and 21.7±0.2°.

8. The crystal form A of the hydrochloride salt of the compound of formula I according to claim 6 or 7, characterized in that: In the X-ray powder diffraction pattern obtained using Cu-Kα radiation, the characteristic peak positions shown in the X-ray powder diffraction pattern of crystal form A of the hydrochloride salt of compound I are substantially the same as those shown in Figure 4.

9. A pharmaceutical composition comprising a crystal form as described in any one of claims 1-8 and one or more pharmaceutically acceptable carriers, diluents or excipients.

10. Use of the crystal form as described in any one of claims 1-8 or the pharmaceutical composition as described in claim 9 in the preparation of a medicament for treating cancer.

11. The crystal form according to any one of claims 1-8, for the treatment of cancer.

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