RU2791189C2 - Crystal forms of free base of benzofuran derivative and production method - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a crystal form D of a compound of the formula I, which has a range of X-ray powder diffraction, obtained using Cu-Kα radiation and represented by a diffraction angle 2θ, which has characteristic peaks at diffraction angles 2θ: 8.41, 8.85, 11.38, 12.18, 13.45, 15.15, 16.73, 17.59, 17.68, 20.45, 21.51, 22.72, 24.53, 24.91, and 27.11, where a range of error of the angle 2θ of each characteristic peak is ±0.2. The crystal form D is obtained by dissolution of a compound of the formula I in a solvent for crystallization, filtration, and drying of the resulting crystal to obtain a desired crystal form D, where the solvent is selected from a group including methanol, isopropanol, simple ether solvent, mixed solvent of methanol and water, and mixed solvent of ethanol and water, wherein the simple ether solvent is 1,4-dioxane. A crystallization method is crystallization with evaporation of the solvent. The invention also relates to options of a method for the production of a crystal form D of a compound of the formula I. The crystal form D of the compound of the formula I, according to the invention, is intended for preparation of a drug for inhibition of EZH2.

EFFECT: crystal form D of a compound of the formula I, having a stability property.

5 cl, 17 dwg, 6 tbl, 24 ex

Description

Technical field

The present invention relates to crystalline forms A, B, C and D N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro -2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboximide, as well as methods for their preparation.

Prior Art

Lymphoma is a malignant tumor originating from the lymphoid hematopoietic system. It is subdivided into non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL) depending on the tumor cells. In Asia, 90% of lymphoma patients are NHL patients with lymphocytes, histiocytes, or reticular cells with varying degrees of pathological differentiation. According to the natural course of NHL, it can be divided into three main clinical types, namely: highly invasive, invasive, and indolent lymphomas. According to the different origin of lymphocytes, it can be divided into B-cell lymphoma, T-cell lymphoma, and natural killer lymphoma (NK lymphoma). The main function of B cells is to secrete various antibodies to protect the body from various external intrusions.

Histone methyltransferase, encoded by the EZH2 gene, is the catalytic component of the polycomb repressive complex 2 (PRC2). EZH2 levels are abnormally elevated in cancerous tissues compared to normal tissues, and EZH2 is most highly expressed in advanced cancers or poor prognosis. In some types of cancer, EZH2 overexpression occurs simultaneously with amplification of the EZH2 gene. A number of experimental studies of si/shRNA (small interfering or short interfering RNA) show that a decrease in the expression of EZH2 in tumor cell lines can inhibit tumor cell proliferation, migration and invasion or angiogenesis and lead to apoptosis. WO 2017084494 (PCT/CN2016/104318, filed November 2, 2016) discloses an EZH2 inhibitor having the following structure:

Applications for patents in which the selective inhibitor EZH2 is disclosed, include WO 2012005805, Wo 2012050532, Wo 2012111812, Wo 2012142513, Wo 20132504, Wo 2013049770, Wo 20139988, Wo 2013067300, Wo 2015141616, Wo 2011140325 and so on.

The crystal structure of a pharmaceutically active ingredient often affects the chemical stability of the drug. Different crystallization conditions and storage conditions can lead to a change in the crystal structure of the compound, and sometimes to the concomitant production of other crystalline forms. In general, an amorphous drug product does not have a regular crystalline structure and often has other disadvantages such as poor product stability, excessively small crystals, difficult filtration, easy agglomeration, and poor flowability. Polymorphic forms of a drug have different requirements for storage, production and distribution. Therefore, it is necessary to develop a crystalline form of the compound of formula (I) and a method for its preparation in order to improve various properties of the compound of formula (I).

The essence of the invention

The technical problem solved by the present invention is to develop crystalline forms A, B, C and D of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl- 6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboximide, as well as methods for their preparation. The crystalline forms obtained according to the present invention have good stability.

The technical solution according to the present invention is as follows:

The present invention provides crystalline form A of the compound of formula (I), characterized in that crystalline form A has an X-ray powder diffraction spectrum, which is obtained using Cu-Kα radiation and is represented by a diffraction angle of 26, which has characteristic peaks at diffraction angles of 2θ: 7.60, 8.51, 11.80, 12.38, 13.52, 13.73, 14.48, 15.23, 15.99, 16.10, 16.82, 16.99, 17.35, 18.24, 20.82, 21.57, 21.91, 22.57, 22.76, 22.88, 24.29, 24.47, 25.24, 25.90, 27.23 and 27.23 and 25.90, 27.23 and 27.23 and 22 27.74 where the error range of the 2θ angle of each characteristic peak is ±0.2,

There are preferably characteristic peaks at the angles of diffraction 2θ: 7.60, 8.51, 10.37, 11.16, 11.80, 12.38, 12.89, 13.52, 13.73, 14.03, 14.48, 15.23, 15.99, 16.10, 16.43, 16.82, 16.99, 17.35, 18.24, 18.92, 19.17, 19.17, 19.17, 19.17, 19.17, 19.17, 19.92, 19.17, 19.17, 19.17, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17, 19.92, 19.17. 20.68, 20.82, 21.57, 21.91, 22.57, 22.76, 22.88, 23.53, 23.68, 24.00, 24.29, 24.47, 24.91, 25.24, 25.72, 25.90, 27.23, 27.74 and 35.63, where the angle of the angle of each characteristic peak is ± 0 ± 0 ,2.

α=β=γ=90,0°; и объем элементарной ячейки составляет

The present invention also provides crystal form A single crystal data, characterized in that crystal form A is an orthorhombic crystal system; the space group is Pbca; unit cell parameters:

α=β=γ=90.0°; and the unit cell volume is

The present invention also provides a method for obtaining crystalline form A, characterized in that the method is selected from the group consisting of:

method I comprising dissolving a compound of formula (I) in a crystallization solvent, filtering, washing and drying the resulting crystal to obtain the desired crystalline form A, wherein the solvent is selected from the group consisting of an amide solvent, a mixed solvent of an amide solvent and water, a mixed solvent halocarbon and nitrile, wherein the amide solvent is selected from the group consisting of N,N-dimethylformamide and N,N-dimethylacetamide, the halocarbon solvent is dichloromethane and the nitrile solvent is acetonitrile, and the crystallization method is selected from the group consisting of crystallization at room temperature temperature, crystallization upon cooling, crystallization upon evaporation of the solvent, and crystallization caused by the addition of a seed crystal;

method II, comprising dissolving a compound of formula (I) in a good solvent, adding an anti-solvent for crystallization, filtering and drying the resulting crystal to obtain the desired crystalline form A, where a good solvent is an alcohol solvent, and the alcohol solvent is selected from the group consisting of methanol, ethanol and isopropanol, and the anti-solvent is water, and the crystallization method is selected from the group consisting of room temperature crystallization, cooling crystallization, solvent evaporation crystallization, and seed crystallization; And

method III, comprising adding a compound of formula (I) to a solvent, suspending this mixture, filtering and drying the resulting crystal to obtain the desired crystalline form A, where the solvent is selected from the group consisting of a mixed solvent of an amide solvent and water, and a mixed solvent of a halocarbon and nitrile, wherein the amide solvent is selected from the group consisting of N,N-dimethylformamide and N,N-dimethylacetamide, the halocarbon solvent is dichloromethane, and the nitrile solvent is acetonitrile.

The present invention also provides a method for producing a single crystal of crystalline form A, characterized in that the method consists of the following steps:

dissolving the compound of formula (I) in a crystallization solvent, filtering and drying the resulting crystal to obtain a desired single crystal of Form A, wherein the solvent is a mixed solvent of halocarbon and nitrile, the halocarbon solvent is dichloromethane, and the nitrile solvent is acetonitrile; the ratio of halocarbon to nitrile is 20:1 to 1:20 and preferably 1:10, the crystallization method is selected from the group consisting of room temperature crystallization, cooling crystallization, solvent evaporation crystallization and seed crystallization.

The present invention also relates to crystalline form B of the compound of formula (I), characterized in that crystalline form B has an X-ray powder diffraction spectrum, which is obtained using Cu-Kα radiation and is represented by a 2θ diffraction angle, which has characteristic peaks at diffraction angles 2θ: 6.31, 7.24, 10.99, 12.07, 14.56, 17.94, 19.13, 19.71, 21.48, 24.15, 27.10 and 28.83, where the error range of the 2θ angle of each characteristic peak is ±0.2.

Предпочтительно характеристические пики имеются при углах дифракции 2θ: 6.31, 7.24, 8.92, 10.38, 10.99, 12.07, 14.56, 15.10, 16.36, 17.29, 17.94, 19.13, 19.71, 21.12, 21.48, 22.59, 24.15, 25.45, 26.28, 26.52, 27.10 , 28.83, 30.07, 31.37, 32.56, 33.65, 34.64, 36.09, 37.13 and 40.04, where the error range of the 2θ angle of each characteristic peak is ±0.2.

The present invention also provides a method for obtaining crystalline form B, characterized in that the method is selected from the group consisting of:

method I, comprising dissolving a compound of formula (I) in a crystallization solvent, filtering and drying the resulting crystal to obtain the desired crystalline form B, where the solvent is ethanol, and the crystallization method is selected from the group consisting of crystallization at room temperature, crystallization with cooling , crystallization by evaporation of the solvent and crystallization caused by the addition of a seed crystal; And

method II, comprising dissolving a compound of formula (I) in a good solvent, adding an anti-solvent for crystallization, filtering and drying the resulting crystal to obtain the desired crystalline form B, where the good solvent is ethanol, the anti-solvent is water, and the crystallization method is selected from the group consisting of from room temperature crystallization, cooling crystallization, solvent evaporation crystallization, and seed crystallization.

The present invention also relates to crystalline form C of the compound of formula (I), characterized in that crystalline form C has an X-ray powder diffraction spectrum, which is obtained using Cu-Kα radiation and is represented by a 2θ diffraction angle, which has characteristic peaks at diffraction angles 2θ: 7.40, 8.62, 9.49, 12.32, 13.39, 15.52, 19.15, 19.17, 21.42 and 22.78, where the 2θ angle error range of each characteristic peak is ±0.2.

Preferably characteristic peaks are available at the angles of diffraction 2θ: 7.40, 8.62, 9.49, 9.96, 11.12, 12.32, 13.39, 14.21, 14.85, 15.52, 16.50, 17.67, 18.28, 19.15, 19.17, 20.06, 20.80, 21.42, 21.89, 22.20, 22.78, 22.78, 22.78, 22.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.78.7L , 23.41, 24.74, 25.34, 26.70, 27.38, 28.64, 29.63, 30.20 and 31.15, where the error range of the 2θ angle of each characteristic peak is ±0.2.

The present invention also provides a method for obtaining crystalline form C, characterized in that it includes the following steps:

dissolving the compound of formula (I) in a good solvent, adding an anti-solvent to crystallize, filtering and drying the resulting crystal to obtain the desired crystalline form C, where the good solvent is an ether solvent, the ether solvent is 1,4-dioxane, the anti-solvent is selected from the group consisting of an aliphatic hydrocarbon solvent and an alicyclic hydrocarbon solvent, the aliphatic hydrocarbon solvent is n-heptane and the alicyclic hydrocarbon solvent is cyclohexane, and the crystallization method is selected from the group consisting of room temperature crystallization, cooling crystallization, evaporation crystallization solvent and crystallization caused by the addition of a seed crystal.

The present invention also relates to crystalline form D of the compound of formula (I), characterized in that crystalline form D has an X-ray powder diffraction spectrum, which is obtained using Cu-Kα radiation and is represented by a diffraction angle 2θ, which has characteristic peaks at diffraction angles 2θ: 8.41, 8.85, 11.38, 12.18, 13.45, 15.15, 16.73, 17.59, 17.68, 20.45, 21.51, 22.72, 24.53, 24.91 and 27.11, where the error range of the 2θ angle of each characteristic peak is ±0.2.

Preferably characteristic peaks are available at the angles of diffraction 2θ: 8.41, 8.85, 10.15, 11.38, 12.18, 13.45, 14.40, 15.15, 16.73, 17.59, 17.68, 18.42, 18.91, 19.22, 20.45, 21.15.51, 22.11, 22.72, 24.03, 24.53, 24.53, 24.53.53.53, 24.53.53, 24.53.53, 24.5 , 24.91, 25.54, 26.54, 27.11, 27.61, 29.04, 30.49, 31.31, 33.00, 33.88, 35.52, 37.53, 40.46, 41.36, 42.40 and 44.02, where the error range of the 2θ angle of each characteristic is ±0.

α=β=γ=90.0° и объем элементарной ячейки составляет

The present invention also provides crystal form D single crystal data, characterized in that crystal form D is an orthorhombic crystal system; the space group is Pbca; unit cell parameters:

α=β=γ=90.0° and the unit cell volume is

The present invention also provides a method for obtaining crystalline form D, characterized in that the method is selected from the group consisting of:

method I comprising dissolving a compound of formula (I) in a crystallization solvent, filtering and drying the resulting crystal to obtain the desired crystalline form D, wherein the solvent is selected from the group consisting of an alcoholic solvent, an ethereal solvent, a mixed solvent of alcohol and water, mixed a solvent of ether and water, a mixed solvent of alcohol and an aliphatic hydrocarbon, and a mixed solvent of ether and an aliphatic hydrocarbon, wherein the alcohol solvent is selected from the group consisting of methanol, ethanol and isopropanol, the ethereal solvent is selected from the group consisting of tetrahydrofuran and 1,4-dioxane, and the aliphatic hydrocarbon solvent is n-heptane, and the crystallization method is selected from the group consisting of room temperature crystallization, cooling crystallization, solvent evaporation crystallization, and seeded crystallization. crystal;

method II, comprising dissolving a compound of formula (I) in a good solvent, adding an anti-solvent for crystallization, filtering and drying the resulting crystal to obtain the desired crystalline form D, where the good solvent is selected from the group consisting of an alcoholic solvent and a simple ether solvent, the alcoholic solvent is selected from the group consisting of methanol and isopropanol, the ethereal solvent is tetrahydrofuran and the antisolvent is water; or a good solvent is ether, the ether solvent is tetrahydrofuran, the anti-solvent is selected from the group consisting of an aliphatic hydrocarbon solvent and an alicyclic hydrocarbon solvent, the aliphatic hydrocarbon solvent is n-heptane, and the alicyclic hydrocarbon solvent is cyclohexane, and the crystallization method is selected from the group consisting of room temperature crystallization, cooling crystallization, solvent evaporation crystallization, and seed crystallization;

method III, comprising adding a compound of formula (I) to a solvent, suspending this mixture, filtering and drying the resulting crystal to obtain the desired crystalline form D, where the solvent is selected from the group consisting of water, ester, ether, aliphatic hydrocarbon, alicyclic hydrocarbon , a nitroalkane solvent, an arene, an alcohol, a nitrile, a halocarbon, a ketone, a sulfoxide, an amide, a mixed solvent of an alcohol and an ether, a mixed solvent of an alcohol and water, and a mixed solvent of one or more alcohols, the ester solvent is selected from the group consisting of ethyl acetate, isopropyl acetate and butyl acetate, the ether solvent is selected from the group consisting of tetrahydrofuran, 1,4-dioxane, propylene glycol methyl ether and methyl t-butyl ether, the aliphatic hydrocarbon is n-heptane, the alicyclic hydrocarbon is cyclohexane, the nitroalkane solvent before is nitromethane, the arene solvent is selected from the group consisting of xylene and cumene, the alcohol solvent is selected from the group consisting of methanol, ethanol and isopropanol, the nitrile solvent is acetonitrile, the halocarbon solvent is dichloromethane, the ketone solvent is acetone, the sulfoxide is dimethyl sulfoxide, mixed the solvent of one or more alcohols is selected from the group consisting of a mixed solvent of methanol and ethanol, a mixed solvent of methanol and isopropanol, and a mixed solvent of ethanol and isopropanol.

The present invention also provides a method for obtaining a single crystal of crystalline form D, characterized in that the method consists of the following steps:

dissolving a compound of formula (I) in a crystallization solvent, filtering and drying the resulting crystal to obtain a desired single crystal of crystal Form D, wherein the solvent is a mixed solvent of alcohol and water, the alcohol solvent is selected from the group consisting of methanol and ethanol; the ratio of alcohol to water is 20:1 to 1:20 and preferably 6:1, the crystallization method is selected from the group consisting of room temperature crystallization, cooling crystallization, solvent evaporation crystallization and seed crystallization.

The present invention also relates to a pharmaceutical composition in crystalline form A, B, C or D, characterized in that it further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention also relates to the use of a crystalline Form A, B, C or D or a pharmaceutical composition for the preparation of a medicament for the treatment of a disease associated with an EZH2 inhibitor.

According to the use of the present invention, the disease associated with the EZH2 inhibitor is selected from the group consisting of lymphoma, leukemia, breast cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, melanoma, rhabdoid tumor, synovial sarcoma, mesothelioma, cervical cancer, colon cancer, rectal cancer, stomach cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, bone cancer, kidney cancer, bladder cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, glioma , glioblastoma, head and neck tumors, and myeloma; preferably from lymphoma, leukemia, breast cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, melanoma, rhabdoid tumor, synovial sarcoma and mesothelioma; the leukemia is preferably chronic myeloid leukemia, acute myeloid leukemia or leukemia of mixed origin; and the lymphoma is preferably non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, or follicular lymphoma.

The resulting crystalline forms of the compound of formula (I) are determined by X-ray powder diffraction (XRPD) spectrum and differential scanning calorimetry (DSC).

The recrystallization method for the crystal form is not particularly limited, and can be carried out by a conventional recrystallization method. For example, a material, ie a compound of formula (I), may be dissolved in an organic solvent, followed by the addition of an anti-solvent for crystallization. After crystallization is completed, the desired crystal can be obtained by filtration and drying.

The crystallization method of the present invention includes room temperature crystallization, cooling crystallization, solvent evaporation crystallization, seed crystal addition-induced crystallization, and the like; and the cooling temperature is less than 40°C, preferably -10°C to 40°C.

The starting material used in the method for obtaining a crystalline form according to the present invention may be a compound of formula (I) in any form, and specific forms include, but are not limited to, amorphous form, arbitrary crystalline forms, and the like.

Definitions

In the description and claims of the present application, unless otherwise indicated, the scientific and technical terms used herein have the meanings generally understood by a person skilled in the art. However, in order to better understand the present invention, definitions and explanations of some relevant terms are given. In addition, when the definitions and explanations of terms provided in this application do not correspond to the meanings commonly understood by a person skilled in the art, the definitions and explanations of terms provided in this application shall prevail.

The term "C _1-6 alkyl" as used in the present invention refers to a linear or branched alkyl containing from 1 to 6 carbon atoms. Specific examples thereof include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2, 3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl and the like.

The term "ethereal solvent" used in the present invention refers to a chain compound or a cyclic compound having an ether bond -O- and having from 1 to 10 carbon atoms. Specific examples thereof include, but are not limited to: tetrahydrofuran, diethyl ether, propylene glycol methyl ether, methyl t-butyl ether, and 1,4-dioxane.

The term "alcohol solvent" as used in the present invention refers to the group resulting from the replacement of one or more hydrogen atoms on "C _1-6 alkyl" with one or more "hydroxy", where "hydroxy" and "C _1-6 alkyl ' are as defined above. Specific examples thereof include, but are not limited to, methanol, ethanol, isopropanol, n-propanol, isopentanol, and trifluoroethanol.

The term "ester solvent" as used in the present invention refers to a combination of a lower organic acid with 1-4 carbon atoms and a lower alcohol with 1-6 carbon atoms. Specific examples thereof include, but are not limited to, ethyl acetate, isopropyl acetate, or butyl acetate.

The term "ketone solvent" used in the present invention refers to a compound in which a carbonyl group (-C(O)) is bonded to two hydrocarbon groups. Ketones can be classified into aliphatic ketones, alicyclic ketones, aromatic ketones, saturated ketones and unsaturated ketones, depending on the hydrocarbon group in the molecule. Specific examples thereof include, but are not limited to, acetone, acetophenone, methyl isobutyl ketone, or methyl pyrrolidone.

The term "nitrile solvent" as used in the present invention refers to a group resulting from the replacement of one or more hydrogen atoms on "C _1-6 alkyl" with one or more "cyano", where "cyano" and "C _1-6 alkyl ' are as defined above. Specific examples thereof include, but are not limited to: acetonitrile or propionitrile.

The term "aliphatic hydrocarbon solvent" as used in the present invention refers to a hydrocarbon compound having the basic properties of an aliphatic compound and having 1 to 10 carbon atoms, where the carbon atoms in the molecule are bonded to a chain carbon skeleton in which the two ends are open and do not form ring, for example, a saturated aliphatic hydrocarbon, including an alkane solvent. Specific examples thereof include, but are not limited to, n-butane, n-pentane, n-hexane, or n-heptane.

The term "alicyclic hydrocarbon solvent" as used in the present invention refers to a hydrocarbon compound having properties similar to an aliphatic hydrocarbon and having a cyclic carbon skeleton with 1 to 8 ring atoms. Its specific examples include, but are not limited to: cyclopentane and cyclohexane.

The term "amide solvent" used in the present invention refers to a compound containing carbonylamino (-C(O)N) and having from 1 to 10 carbon atoms. Specific examples thereof include, but are not limited to, N,N-dimethylformamide and N,N-dimethylacetamide.

The term "arene solvent" as used in the present invention refers to a generic term for a carbon ring compound and its derivative, where the molecule has a conjugated closed ring system and the number of π electrons follows Haeckel's rule. Its specific examples include, but are not limited to: isopropylbenzene and xylene.

The term "halohydrocarbon solvent" as used in the present invention refers to a group derived from "C _1-6 alkyl" in which one or more hydrogen atoms are replaced by one or more "halogen atoms", where "halogen atom" and "C _{1 -6} alkyl" are as defined above. Specific examples thereof include, but are not limited to, methyl chloride, dichloromethane, chloroform, and carbon tetrachloride.

The term "nitroalkane solvent" as used in the present invention refers to a group derived from "C _1-6 alkyl" in which one or more hydrogen atoms are replaced by one or more "nitro", where "C _1-6 alkyl" is such , as defined above. Specific examples thereof include, but are not limited to: nitromethane.

The term "mixed solvent" used in the present invention refers to a solvent obtained by mixing one or more different kinds of organic solvents in a certain ratio, or a solvent obtained by mixing an organic solvent and water in a certain ratio. The mixed solvent is preferably a mixed solvent of one or more alcohols, a mixed solvent of an alcohol and an ether, a mixed solvent of an alcohol and an aliphatic hydrocarbon, a mixed solvent of an ether and an aliphatic hydrocarbon, a mixed solvent of an alcohol and water, a mixed solvent of a halocarbon, and nitrile, a mixed solvent of amide and water, or a mixed solvent of ether and water, wherein the alcohol, ether, aliphatic hydrocarbon, halocarbon, amide, and nitrile are as defined above.

порядок дифракции n - любое положительное целое число, обычно принимающее значение дифракционного максимума первого порядка, n=1), когда рентгеновское излучение падает на определенную атомную плоскость кристалла или частичного образца кристалла, имеющего расстояние d между плоскостями, под углом скользящего падения θ (комплементарный угол падения, также называемый углом Брэгга), удовлетворяющему уравнению Брэгга.The term "X-ray powder diffraction spectrum" or "XRPD" as used in the present invention refers to an X-ray powder diffraction spectrum, which is obtained in accordance with the Bragg formula 2d sinθ=nλ (where λ is the wavelength of X-rays,

diffraction order n is any positive integer, usually taking the value of the first order diffraction maximum, n=1), when X-rays are incident on a specific atomic plane of a crystal or partial crystal sample having a distance d between the planes, at a grazing incidence angle θ (complementary angle incidence, also called the Bragg angle) that satisfies the Bragg equation.

The term "differential scanning calorimetry" or "DSC" as used in the present invention means the measurement of the temperature difference and the difference in heat fluxes between the sample and the reference during the heating process or constant temperature of the sample, in order to characterize all the physical and chemical changes associated with this thermal effect. , and to obtain information about the phase change of the sample.

The term "2θ" or "angle 2θ" used in the present invention refers to the angle of diffraction, θ is the Bragg angle, and the unit is ° or degree. The 2θ error range is ±0.1 to ±0.5, preferably ±0.1 to ±0.3, and more preferably ±0.2.

The term "interplane distance" or "interplanar distance (d value)" as used in the present invention means that a spatial lattice is characterized by three non-parallel unit vectors a, b, c, each of which connects two adjacent points of the lattice and these three vectors divide the lattice into adjacent parallel juxtagonal elements, called the interplanar spacing. The spatial grid is divided according to certain lines of parallelepiped elements to obtain a set of linear grids, which is called a spatial grid or lattice. The lattice reflects the periodicity of the crystal structure with geometric points and lines. Different crystal planes have different interplanar spacings (that is, distances between two adjacent parallel crystal planes); the unit of measure is A or angstrom.

The present invention also relates to a pharmaceutical composition containing the crystalline form A, B, C or D of the compounds of formula (I) and, optionally, one or more pharmaceutically acceptable carriers and/or diluents. The pharmaceutical composition may be formulated into any of the pharmaceutically acceptable dosage forms. For example, a crystalline form A, B, C or D of a compound of formula (I) or a pharmaceutical composition according to the present invention may be formulated as a tablet, capsule, pill, granule, solution, suspension, syrup, injection (including injectable solution, sterile powder for injection and concentrated solution for injection), suppository, inhaler or spray.

In addition, the pharmaceutical composition of the present invention may also be administered to a patient or subject in need of such treatment by any suitable route of administration such as oral, parenteral, rectal, intrapulmonary or topical administration. For oral administration, the pharmaceutical composition may be formulated as an oral composition, for example an oral solid composition such as a tablet, capsule, pill, granule, and the like; or an oral liquid composition such as an oral solution, oral suspension, syrup, and the like. When preparing an oral composition, the pharmaceutical composition may further contain a suitable excipient, binder, disintegrant, lubricant, and the like. For parenteral administration, the pharmaceutical composition may be formulated as an injectable preparation comprising an injection solution, a sterile injection powder and a concentrated injection solution. When formulating a composition for injection, the pharmaceutical composition can be prepared in the usual way of the modern pharmaceutical industry. When a composition is prepared for injection, an additional agent may be omitted, or a suitable additional agent may be added, depending on the nature of the drug. For rectal administration, the pharmaceutical composition may be formulated as a suppository and the like. For intrapulmonary administration, the pharmaceutical composition may be formulated as an inhaler or spray, and the like. In certain preferred embodiments, the implementation of the crystalline form A, B, C or D of the compounds of formula (I) according to the present invention is present in a pharmaceutical composition or drug in a therapeutically and/or prophylactically effective amount. In some preferred embodiments, the crystalline form A, B, C or D of the compound of formula (I) according to the present invention is present in a pharmaceutical composition or medicament in a unit dose.

The crystalline form A, B, C or D of the compound of formula (I) according to the present invention can be used for the preparation of a medicament for the treatment of a disease associated with an EZH2 inhibitor. Therefore, the present application also relates to the use of crystalline form A, B, C or D of the compound of formula (I) according to the present invention for the preparation of a medicament for the treatment of an EZH2 inhibitor related disease. In addition, the present application also relates to a method for inhibiting an EZH2 inhibitor-associated disease comprising administering a therapeutically and/or prophylactically effective amount of a crystalline form A, B, C or D of a compound of formula (I) of the present invention or a pharmaceutical composition of the present invention to a subject, in need of it.

In certain preferred embodiments, the disease is an EZH2 inhibitor-associated disease selected from pain.

Advantageous Effects of the Present Invention

Compared with the prior art, the technical solution according to the present invention has the following advantages.

Studies have shown that the crystalline forms A, B, C and D of the compound of formula (I) obtained according to the present invention have good stability and high purity. Single crystals of crystalline forms A and D are obtained. The crystalline forms A, B, C and D of the compound of formula (I), obtained using the technical solution according to the present invention, can meet the requirements for the production, transportation and storage of drugs. The method of their production is stable, reproducible and controllable, and can be adapted to industrial production.

Brief description of graphic materials

In FIG. 1 shows the XRPD spectrum of the crystalline form A of the compound of formula (I).

In FIG. 2 shows the XRPD spectrum of crystalline form B of the compound of formula (I).

In FIG. 3 shows the XRPD spectrum of crystalline form B of the compound of formula (I).

In FIG. 4 shows the XRPD spectrum of the crystalline form C of the compound of formula (I).

In FIG. 5 shows the XRPD spectrum of crystalline Form C of the compound of formula (I).

In FIG. 6 shows the XRPD spectrum of the crystalline form D of the compound of formula (I).

In FIG. 7 shows the XRPD spectrum of the crystalline form D of the compound of formula (I).

In FIG. 8 shows a Dynamic Vapor Sorption (DVS) Cycle 1 plot of crystalline form D of a compound of formula (I).

In FIG. 9 shows a Cycle 2 DVS plot of the crystalline form D of the compound of formula (I).

In FIG. 10 shows the XRPD spectrum of crystalline form B of the compound of formula (I) at day 0.

In FIG. 11 shows the XRPD spectrum of crystalline form B of the compound of formula (I) after 20 days at 25° C. and 65% relative humidity.

In FIG. 12 shows the XRPD spectrum of the crystalline form D of the compound of formula (I) at day 0.

In FIG. 13 shows the XRPD spectrum of crystalline form D of the compound of formula (I) after 20 days at 40° C. and 75% relative humidity.

In FIG. 14 shows the XRPD spectrum of crystalline form D of the compound of formula (I) after 20 days at 25° C. and 65% relative humidity.

In FIG. 15 is a schematic X-ray single crystal diffraction molecular stereostructure of the crystalline form A of the compound of formula (I).

In FIG. 16 shows a schematic x-ray single crystal diffraction molecular stereostructure of the crystalline form D of the compound of formula (I).

In FIG. 17 shows the XRPD spectrum of the amorphous form of the compound of formula (I).

Detailed description of the invention

The present invention will be illustrated in detail by the following examples. The examples of the present invention are only intended to describe the technical solution of the present invention and should not be construed as limiting the essence and scope of the present invention.

Test conditions for instruments used in experiments:

1. Differential Scanning Calorimeter, DSC

Instrument type: Mettler Toledo DSC3 + STARe system

Purge gas: nitrogen (50 ml/min)

Heating rate: 10.0°C / min

Temperature range: 20-250°C

2. X-ray powder diffraction, XRPD

Instrument type: powder diffractometer BRUKER D8 Discover A25

)Radiation: Cu-Kα monochromatic radiation (

)

Scan mode: 9/29, scan range: 10-48°

Voltage: 40 kV, Electric current: 40 mA

3. Dynamic vapor sorption, DVS

Device type: DVS advantage

Temperature: 25°C

Solvent: water

Humidity change: 0-95-0-95-0% RH, dm/dt=0.002

Comparative example 1 Production method in example 2 of WO 2017084494 (PCT/CN2016/104318)

Preparation of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)- 2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (compounds of formula (I))

Stage 1

3-Bromo-2-ethyl-5-nitrobenzoic acid 1b

2-Ethylbenzoic acid 1a (20.0 g, 133 mmol, prepared according to the method described in "Journal of American Chemical Society, 1991, 113 (13), 4931-6") was added to 150 ml of sulfuric acid, then sodium nitrate (11.3 g, 133 mmol) was added portionwise in an ice bath. The reaction solution was stirred for 3 hours, then N-bromosuccinimide (2.6 g, 14.5 mmol) was added portionwise. The reaction system was stirred for 1 hour at 60°C. After completion of the reaction, the reaction solution was poured into ice water, stirred well, and filtered. The filtrate was washed with water and concentrated under reduced pressure to give the crude title product 3-bromo-2-ethyl-5-nitrobenzoic acid 1b (35 g) as a white solid, which was used directly in the next step without purification.

Stage 2

Methyl 3-bromo-2-ethyl-5-nitrobenzoate 1s

The crude 3-bromo-2-ethyl-5-nitrobenzoic acid 1b (35 g, 128 mmol) was dissolved in 200 ml of N,N-dimethylformamide, then iodomethane (21.8 g, 153 mmol) and potassium carbonate (35.3 g, 255 mmol). The reaction system was stirred for 2 hours at room temperature. After completion of the reaction, the reaction solution was concentrated under reduced pressure. An excess of water was added to the reaction solution, and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give crude title product methyl 3-bromo-2-ethyl-5-nitrobenzoate 1c (36 g) as a yellow oil which was used directly in the next step without purification.

Stage 3

Methyl 5-amino-3-bromo-2-ethylbenzoate 1d

Crude methyl 3-bromo-2-ethyl-5-nitrobenzoate 1c (35.0 g, 121 mmol) was added to 250 ml of ethanol and 150 ml of water. The reaction solution was heated to 70° C., ammonium chloride (52.8 g, 969 mmol) was added, then iron powder (34 g, 606 mmol) was added in portions. The reaction system was stirred for 2 hours at 70°C. After completion of the reaction, the reaction solution was filtered through celite while hot. The filter cake was washed with hot ethanol, then the filtrate was combined and concentrated under reduced pressure. Ethyl acetate and saturated sodium bicarbonate solution were added. The two phases were separated and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl 5-amino-3-bromo-2-ethylbenzoate 1d (22.0 g, yield 70%) as a yellow solid.

Stage 4

Methyl 3-bromo-2-ethyl-5-hydroxybenzoate 1e

Methyl 5-amino-3-bromo-2-ethylbenzoate 1d (15.0 g, 58 mmol) was dissolved in 10 ml of acetonitrile, then 200 ml of 10% sulfuric acid was added. The reaction solution was well stirred and cooled to 3°C in an ice bath with salt, then 10 ml of a pre-prepared sodium nitrite solution (4.4 g, 64 mmol) was added dropwise. The reaction solution was stirred for 4 hours at the same temperature, 200 ml of 50% sulfuric acid was added dropwise, followed by stirring for 1 hour at 90°C. After completion of the reaction, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl 3-bromo-2-ethyl-5-hydroxybenzoate 1e (5.5 g, yield 37%) as a brown solid.

Stage 5

Methyl 3-bromo-5-(2,2-diethoxyethoxy)-2-ethylbenzoate 1f

Methyl 3-bromo-2-ethyl-5-hydroxybenzoate 1e (35 g, 135 mmol) was dissolved in 200 ml of N,N-dimethylformamide, then 2-bromo-1,1-diethoxyethane (40 g, 202 mmol) was added and potassium carbonate (37 g, 269 mmol). The reaction system was stirred at 120° C. for 12 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove N,N-dimethylformamide. The reaction solution was added to water and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl 3-bromo-5-(2,2-diethoxyethoxy)-2-ethylbenzoate 1f ( 40 g, 80% yield as light yellow oil.

Stage 6

Methyl-6-bromo-5-ethylbenzofuran-4-carboxylate 1g

Polyphosphoric acid (30 g) was added to 400 ml of toluene. The reaction solution was heated to 100°C and 50 ml of a previously prepared solution of methyl 3-bromo-5-(2,2-diethoxyethoxy)-2-ethylbenzoate 1f (40 g, 107 mmol) in toluene was added with stirring. The reaction solution was stirred for 16 hours at 100°C. After completion of the reaction, the supernatant was decanted. Water and ethyl acetate were added to the residue. The two phases were separated and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium carbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl 6-bromo-5-ethylbenzofuran-4-carboxylate 1g (11.8 g, yield 39%) as a yellow solid.

Stage 7

Methyl 5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1h

Methyl 6-bromo-5-ethylbenzofuran-4-carboxylate 1g (11.0 g, 39 mmol), tetrahydro-2H-pyran-4-amine (5.89 g, 58 mmol), tris(dibenzylideneacetone)dipalladium (3 .6 g, 3.9 mmol), (.9 mmol) bis(diphenylphosphino)-1,1'-binaphthalene (4.86 g, 7.8 mmol) and cesium carbonate (38 g, 117 mmol) were dissolved in 100 ml of toluene. The reaction solution was stirred for 12 hours at 100°C. After completion of the reaction, the reaction solution was filtered through Celite, and the filter cake was washed with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino )benzofuran-4-carboxylate 1h (10.0 g, 85% yield) as a yellow solid.

Stage 8

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1i

Methyl 5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1h (10.0 g, 0.033 mmol) was dissolved in 150 ml of 1,2-dichloroethane, then acetaldehyde was added (7.2 g, 0.165 mmol) and acetic acid (9.9 g, 0.165 mmol). The reaction solution was stirred for 1 hour and sodium triacetoxyborohydride (20.8 g, 0.1 mmol) was added. The reaction solution was stirred for 12 hours at room temperature. After completion of the reaction, the reaction solution was concentrated under reduced pressure, neutralized with a saturated sodium bicarbonate solution, and extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl-5-ethyl-6-(ethyl(tetrahydro-2H-pyran)-4-yl )amino)benzofuran-4-carboxylate 1i (7.8 g, 71% yield) as a white solid.

MS m/z (LC-MS): 332.4 [M+1]

Stage 9

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-formylbenzofuran-4-carboxylate 1j

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1i (1.6 g, 4.8 mmol) was dissolved in 25 ml of tetrahydrofuran. The reaction solution was cooled to -70° C. and 2.0 M lithium diisopropylamide (3.6 ml, 7.3 mmol) was added dropwise under argon. The reaction solution was stirred for 90 minutes and N.N-dimethylformamide (536 mg, 7.3 mmol) was added. The reaction solution was stirred for 2 hours, then slowly warmed to room temperature. An excess of ammonium chloride was added to the reaction solution, stirred well, and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl-5-ethyl-6-(ethyl-(tetrahydro-2H-pyran-4-yl )amino)-2-formylbenzofuran-4-carboxylate 1j (1.3 g, 75% yield) as a yellow oil.

MS m/z (ESI):360.2 [M+1]

Stage 10

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(hydroxymethyl)benzofuran-4-carboxylate 1k

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-formylbenzofuran-4-carboxylate 1j (1.4 g, 3.9 mmol) was dissolved in 5 ml of tetrahydrofuran and 10 ml of methanol, then sodium borohydride (222 mg, 5.8 mmol) was added. The reaction solution was stirred for 30 minutes at room temperature. After completion of the reaction, the reaction solution was concentrated under reduced pressure, water and saturated sodium bicarbonate were added, and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using n-hexane and ethyl acetate as eluent to give the title product methyl-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl) amino)-2-(hydroxymethyl)-benzofuran-4-carboxylate 1k (1.4 g, 99% yield) as a yellow oil.

Stage 11

Methyl 2-(bromomethyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1l

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(hydroxymethyl)-benzofuran-4-carboxylate 1k (1.0 g, 2.8 mmol) was dissolved in 30 ml of tetrahydrofuran, then phosphorus tribromide (1.12 g, 4.2 mmol) was added dropwise. The reaction solution was stirred for 12 hours at room temperature. After completion of the reaction, the reaction solution was neutralized with a saturated sodium bicarbonate solution and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give crude title product methyl 2-(bromomethyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)benzofuran-4-carboxylate 1l (1, 15 g) as a yellow oil, which was used directly in the next step without purification.

Stage 12

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)-benzofuran-4-carboxylate 1m

Crude methyl 2-(bromomethyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-benzofuran-4-carboxylate 1l (1.15 g, 2.7 mmol) was dissolved in 15 ml of acetonitrile, then 10 ml of a previously prepared solution of piperidine (362 mg, 4.3 mmol) in acetonitrile was added dropwise. The reaction solution was stirred for 30 minutes at room temperature. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and ethyl acetate and a saturated sodium bicarbonate solution were added. The two phases were separated and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using dichloromethane and methanol as eluent to give the title product methyl-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)- 2-(piperidin-1-ylmethyl)-benzofuran-4-carboxylate 1m (1.2 g, 99% yield) as a yellow oil.

MS m/z (LC-MS): 429.2[M+1]

Stage 13

5-Ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzo-furan-4-carboxylic acid 1n

Methyl 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboxylate 1m (1.2 g, 2.7 mmol) was dissolved in 5 ml of tetrahydrofuran and 20 ml of methanol, then 5 ml of 4 M sodium hydroxide solution was added. The reaction solution was stirred for 12 hours at 60°C. After completion of the reaction, concentrated hydrochloric acid was added to adjust the pH of the reaction solution to 4. The mixture was concentrated under reduced pressure, and the residue was dissolved in a mixed solvent of dichloromethane and methanol (V:V=5:1) and filtered. The filter cake was washed with a mixed solvent of dichloromethane and methanol (V:V=5:1). The filtrates were combined and concentrated under reduced pressure to give crude title product 5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)-benzofuran-4- carboxylic acid 1n (1.1 g) as a yellow solid, which was used directly in the next step without purification.

MS m/z (LC-MS): 415.2[M+1]

Stage 14

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2 -(piperidin-1-ylmethyl)benzofuran-4-carboximide

(compound of formula (I))

5-Ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)-benzofuran-4-carboxylic acid 1n (1.0 g, 2.4 mmol) was dissolved in 30 ml of N,N-dimethylformamide, then 1-ethyl-3-(3-dimethyl-amipropyl)-carbodiimide (696 mg, 3.6 mmol), 1-hydroxybenzotriazole (490 mg, 3.6 mmol) was added and N,N-diisopropylethylamine (1.56 g, 12.1 mmol). The reaction solution was stirred for 1 hour, then 3-(aminomethyl)-4,6-dimethyl-pyridin-2(1H)-one hydrochloride 2a (593 mg, 3.0 mmol) prepared according to the method disclosed in patent application WO 2014097041). The reaction solution was stirred for 12 hours at room temperature. After completion of the reaction, an excess of water was added to the reaction solution, and extracted with a mixed solvent of dichloromethane and methanol (V:V=8:1). The organic phases were combined, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using dichloromethane and methanol as eluent to give the title product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (I) (750 mg, 57% yield) as a white solid.

MS m/z (ESI): 549.7 [M+1]

The white solid product was identified by the XRPD spectrum as an amorphous form. The XRPD spectrum of the amorphous form is shown in FIG. 17.

Example 1 (Preparation of Crystal Form A)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (20 mg, 0.036 mmol) obtained in Comparative Example 1 was added to the reaction flask, and a mixed solvent of dichloromethane-acetonitrile (v/v, 1:10, 500) was added µl). The reaction solution was stirred at 25°C for one hour and filtered. The filtrate was placed in a clean flask and slowly evaporated until the solvent was completely dry to give a white to pale yellow solid. The XRPD spectrum of the solid sample is shown in FIG. 1 and the positions of the characteristic peaks are shown in the following table:

Example 2 (Obtaining a single crystal of the crystalline form A)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)-benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in a mixed solvent of acetonitrile and dichloromethane (v/v, 10: 1.1 ml). The neck of the flask was closed with a sealing film in which two or three small holes were punched. The solvent was evaporated until a single crystal was obtained. An example of a single molecular crystal stereostructure is shown in FIG. 15 by single crystal X-ray diffraction (XRD), and the unit cell parameters are shown in the following table:

Example 3 (Preparation of Crystal Form B)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in ethanol (300 µl). The neck of the flask was closed with a sealing film in which two or three small holes were punched. The solution was left at room temperature until the solvent evaporated to dryness to give a white to pale yellow solid. The XRPD spectrum of the crystal sample is shown in FIG. 2. The DSC spectrum is shown in FIG. 3, which has one endothermic peak and one exothermic peak during the temperature rise period, the onset of the exothermic peak is about 123.69°C and the initial melting point is about 206.31°C. The crystal form was determined to be crystal form B and the positions of the characteristic peaks are shown in the following table:

Example 4 (Preparation of Crystal Form B)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in ethanol (300 µl). Water (900 µl) was added to the solution as an anti-solvent to precipitate crystals. The mixture was filtered and dried to give a pale yellow solid. The crystalline sample was identified as crystalline Form B by XRPD.

Example 5 (Preparation of Crystal Form C)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in 1,4-dioxane (1 ml). n-heptane (2 ml) was added to the solution to precipitate crystals. The mixture was filtered and dried to give a white to pale yellow powder. The XRPD spectrum of the crystal sample is shown in FIG. 4. The DSC spectrum is shown in FIG. 5, which has many endothermic peaks. The crystal form was determined to be crystal form C and the positions of the characteristic peaks are shown in the following table:

Example 6 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (50 mg, 0.091 mmol) obtained in Comparative Example 1 was added to the reaction flask, and methanol (100 μl) was added. The solution was stirred at 25° C. for five days, filtered and dried in vacuo to give a white to pale yellow powder. The XRPD spectrum of the crystal sample is shown in FIG. 6. The DSC spectrum is shown in FIG. 7, where the melting start temperature is about 205.45°C. The crystalline form was determined to be crystalline form D. From the DVS plots in FIG. 8 and 9 it can be seen that the crystalline form D does not have a clear hygroscopicity. The positions of the characteristic peaks are shown in the following table:

Example 7 (Preparation of single crystal of crystalline form D) Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H -pyran-4-yl)amino)-2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to a reaction flask and dissolved in a mixed solvent of methanol and pure water (v/v, 6:1, 1 ml). The neck of the flask was closed with a sealing film in which two or three small holes were punched. The solvent was evaporated until a single crystal was obtained. The single molecular stereostructure of the crystal sample is shown in Fig. 16 by single crystal X-ray diffraction (XRD), and the unit cell parameters are shown in the following table:

Example 8 (Preparation of single crystal of crystalline form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in a mixed solvent of ethanol and pure water (6:1 by volume). , 1 ml). The neck of the flask was closed with a sealing film in which two or three small holes were punched. The solvent was evaporated to obtain a crystal, which was identified as a single crystal of crystalline Form D by single crystal X-ray diffraction (XRD).

Example 9 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and ethyl acetate (300 µl) was added. The mixture was suspended at room temperature for two hours, filtered and dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 10 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and propylene glycol methyl ether (300 µl) was added. The mixture was suspended at room temperature for two hours, filtered and dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 11 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and cyclohexane (300 µl) was added. The mixture was suspended at 25°C for two hours, filtered and dried to give a pale yellow solid which was identified as crystalline form D by XRPD.

Example 12 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide ((30mg, 0.055mmol) obtained in Comparative Example 1 was added to the reaction flask and xylene (300µl) was added. The mixture was suspended at 25°C for two hours, filtered and dried to give a pale yellow solid which was identified by XRPD as crystalline Form D.

Example 13 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and acetonitrile (300 μl) was added. The mixture was suspended at 50°C for two hours, filtered and dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 14 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and acetone (300 µl) was added. The mixture was suspended at 50°C for two hours, filtered and dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 15 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and nitromethane (300 µl) was added. The mixture was suspended at 50°C for two hours, filtered and dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 16 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and dimethylformamide (300 µl) was added. The mixture was suspended for two hours, filtered at 50°C and dried to give a pale yellow solid which was identified as crystalline form D by XRPD.

Example 17 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in isopropanol (1 ml). The solvent was evaporated and the resulting solid was then dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 18 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in a mixed solvent of methanol and water (v/v, 19:1 , 300 μl). The solvent was evaporated and the resulting solid was then filtered and dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 19 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in dioxane (1 ml). The solvent was evaporated and the resulting solid was then dried to give a pale yellow solid which was identified by XRPD as crystalline Form D.

Example 20 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask and dissolved in tetrahydrofuran (300 µl). The solvent was evaporated and the resulting solid was then filtered and dried to give a pale yellow solid which was identified as crystalline Form D by XRPD.

Example 21 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and dimethyl sulfoxide (100 µl) was added. The mixture was suspended at 50°C, filtered and dried to give a pale yellow solid which was identified by XRPD as crystalline Form D.

Example 22 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and dichloromethane (100 μl) was added. The mixture was suspended at 25°C, filtered and dried to give a pale yellow solid which was identified by XRPD as crystalline Form D.

Example 23 (Preparation of Crystal Form D)

Crude product N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-ethyl-6-(ethyl(tetrahydro-2H-pyran-4-yl)amino) -2-(piperidin-1-ylmethyl)benzofuran-4-carboximide (30 mg, 0.055 mmol) obtained in Comparative Example 1 was added to the reaction flask, and methanol (100 µl) was added. The mixture was suspended at 50°C, filtered and dried to give a pale yellow solid which was identified by XRPD as crystalline Form D.

Example 24 Long Term, Accelerated Stability Study of Crystalline Forms B and D

A sample of crystalline form B was spread over the surface in air to test the stability of the sample under conditions of 25°C and 65% relative humidity (RH), and sampling was performed on day 20. A sample of crystalline form D was spread over the surface in air to test the stability of the sample under conditions 25°C and 75% RH and 25°C and 65% RH were sampled on day 20.

Test results:

In FIG. 10 shows the XRPD spectrum of crystalline form B on day 0.

In FIG. 11 shows the XRPD spectrum of crystalline form B after 20 days at 25°C and 65% relative humidity for 20 days.

In FIG. 12 shows the XRPD spectrum of crystalline form D at day 0.

In FIG. 13 shows the XRPD spectrum of crystalline form D after 20 days at 40°C and 75% relative humidity for 20 days.

In FIG. 14 shows the XRPD spectrum of crystalline form D after 20 days at 25°C and 65% relative humidity for 20 days.

Test Conclusion:

The results of the stability study shown in FIG. 12, 13 and 14 show that the XRPD peaks of crystalline form D of the compound of formula (I) did not change significantly under conditions of 40° C. and 75% relative humidity and the crystalline form is stable. The results shown in FIG. 10 and 11 show that the XRPD peaks of crystalline form B of the compound of formula (I) changed significantly under conditions at 25° C. and 65% relative humidity. It can be seen that the physical stability of crystalline form D is better than crystalline form B under conditions of being at 25°C and 65% relative humidity.

Claims (9)

1. Crystalline form D of the compound of formula (I), characterized in that the crystalline form D has an X-ray powder diffraction spectrum obtained using Cu-Kα radiation and represented by a diffraction angle 2θ, which has characteristic peaks at diffraction angles 2θ: 8, 41, 8.85, 11.38, 12.18, 13.45, 15.15, 16.73, 17.59, 17.68, 20.45, 21.51, 22.72, 24.53, 24, 91 and 27.11, where the error range of the 2θ angle of each characteristic peak is ±0.2

2. Crystal form D according to claim 1, characterized in that it has characteristic peaks at diffraction angles 2θ: 8.41, 8.85, 10.15, 11.38, 12.18, 13.45, 14.40, 15.15, 16.73, 17.59, 17.68, 18.42, 18.91, 19.22, 20.45, 21.15, 21.51, 22.11, 22.72, 24, 03, 24.53, 24.91, 25.54, 26.54, 27.11, 27.61, 29.04, 30.49, 31.31, 33.00, 33.88, 35.52, 37, 53, 40.46, 41.36, 42.40, and 44.02, where the 2θ angle error range of each characteristic peak is ±0.2. 3. The method of obtaining a crystalline form D according to any one of paragraphs. 1 and 2, characterized in that the method is selected from the group consisting of: process I comprising dissolving a compound of formula (I) in a crystallization solvent, filtering and drying the resulting crystal to obtain the desired crystalline form D, wherein the solvent is selected from the group consisting of methanol, isopropanol, an ether solvent, a mixed solvent of methanol and water, and a mixed a solvent of ethanol and water, wherein the ethereal solvent is 1,4-dioxane and the crystallization method is solvent evaporation crystallization; method II, comprising dissolving a compound of formula (I) in a good solvent, adding an anti-solvent for crystallization, filtering and drying the resulting crystal to obtain the desired crystalline form D, where the good solvent is an ether solvent, and the ether solvent is tetrahydrofuran, and the anti-solvent is water; method III, comprising adding a compound of formula (I) to a solvent, suspending this mixture, filtering and drying the resulting crystal to obtain the desired crystalline form D, where the solvent is selected from the group consisting of an ester, a simple ether, an alicyclic hydrocarbon, a nitroalkane solvent, an arene , alcohol, nitrile, halocarbon, ketone, sulfoxide and amide, where the ester solvent is ethyl acetate, the ether solvent is propylene glycol methyl ether, the alicyclic hydrocarbon is cyclohexane, the nitroalkane solvent is nitromethane, the arene solvent is xylene, the alcohol solvent is is methanol, the nitrile solvent is acetonitrile, the halocarbon solvent is dichloromethane, the ketone solvent is acetone, the sulfoxide is dimethyl sulfoxide, the amide solvent is N,N-di methylformamide. 4. Pharmaceutical composition with inhibitory activity against EZH2, containing an effective amount of crystalline form D according to any one of paragraphs. 1 and 2, characterized in that it additionally contains one or more pharmaceutically acceptable carriers or excipients. 5. The use of a crystalline form D according to any one of claims 1 and 2, or a pharmaceutical composition according to claim 4 for the preparation of an EZH2 inhibitory medicament.

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