EA042455B1 - Crystalline Form of an Inhibitor Targeting CDK4/6 Kinase - Google Patents

Description

This application claims the priority of Chinese Patent Application No. 201810084351.3, filed January 29, 2018, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Technical field

The present invention relates to a crystalline form of an inhibitor targeting CDK4/6 kinase and a method for producing the same, and also relates to a pharmaceutical composition containing said crystalline form and the use of such a compound for reducing or inhibiting CDK4/6 kinase activity in cells and for treating and/or prevention of diseases associated with cancer mediated by CDK4/6 kinase.

Prior Art

Chemical name of the compound of formula (I) -5-((4-ethylpiperazin-1-yl)methyl)-N-(5-fluoro-4-(4fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazole -6-yl)pyridin-2-yl)pyrimidin-2-amine (hereinafter referred to as the compound of formula (I), which is described in patent application PCT/CN 2014/095615), and it acts as an inhibitor targeting cyclin-dependent kinase 4/ 6 (CDK4/6). Studies have shown that among CDK isoforms involved in the cell cycle, CDK4/6 plays an indispensable role. Cancer-associated cell cycle mutations are mainly present in the G1 phase and during the G1/S phase transition. The complex formed from CDK4/6 and cyclin D results in the phosphorylation of the Rb anti-oncogene to form pRb and the release of the associated transcription factor E2F, causing transcription of genes associated with S-phase initiation, which promotes cell checkpoint progression from G1 to S phase. Approximately 80% of human tumors have abnormalities in the DCDK4/6-INK4-Rb cyclin pathway. Altering this pathway leads to an acceleration of the G1 phase so that tumor cells proliferate at an accelerated rate and thus gain a survival advantage. Therefore, intervention in this pathway has become a treatment strategy, and CDK4/6 has become a new antitumor target. CDK4/6 has advantages as an antitumor target in the following two aspects:

(1) the proliferation of most proliferative cells depends on CDK2 or CDK4/6; however, CDK4/6 inhibitors do not exhibit the cytotoxicity of pan-CDK inhibitors such as bone marrow suppression and intestinal reactions;

(2) Preclinical experiments have shown that if the level of cyclin D is increased or P16INK4a is inactivated in cells, drug sensitivity of the cells can be increased, and since tumor cells have the above-mentioned qualities compared to normal cells, the targeting ability of drugs is increased to some extent.

Formula (I)

The study of crystal forms plays an important role in the drug development process, and there are significant differences in solubility, stability, bioavailability, etc. between different crystal forms of the same drug. In order to better control the quality of the drug to meet the requirements for obtaining, manufacturing, transporting, storing, etc., the authors studied the crystalline form of the compound of formula (I) to find crystalline forms with good properties.

Brief description of the invention

The present invention relates to crystalline Form A 5-((4-ethylpiperazin-1-yl)methyl)-N(5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazole -6-yl)pyridin-2-yl)pyrimidin-2-amine represented by formula (I) as an inhibitor targeting CDK4/6 kinase. The present invention also relates to a method for preparing crystalline form A, a pharmaceutical composition containing crystalline form A, and the use of such a compound for reducing or inhibiting CDK4/6 kinase activity in cells and for treating and/or preventing diseases associated with cancer mediated by CDK4 kinase. /6.

In some embodiments, the present invention relates to crystalline form A of the compound of formula (I)

Formula (I) wherein crystalline form A of the compound of formula (I) is characterized by an X-ray powder diffraction pattern containing characteristic peaks at 6.6±0.2, 10.0±0.2, 13.2±0.2, 17.4± 0.2

- 1 042455

20.1±0.2 and 20.6±0.2° expressed as 2θ (°) angles using Cu-Ka radiation.

In some embodiments, crystalline form A of the compound of formula (I) further has characteristic peaks at 8.7±0.2, 10.9±0.2, 15.7±0.2, 16.4±0.2, and 30 .4±0.2° in addition to the above characteristic peaks.

In some embodiments, crystalline form A of the compound of formula (I) further has characteristic peaks at 16.7±0.2, 19.3±0.2, 22.2±0.2, 23.3±0.2, 24 .0±0.2, 25.9±0.2 and 28.1±0.2° in addition to the above characteristic peaks.

In some embodiments, crystalline form A of the compound of formula (I) is characterized by an X-ray powder diffraction pattern containing characteristic peaks at 6.6±0.2, 8.7±0.2, 10.0±0.2, 10.9±0 .2, 13.2±0.2, 15.7±0.2, 16.4±0.2, 17.4±0.2, 20.1±0.2, 20.6±0.2 and 30.4±0.2° expressed as 2θ angles using Cu-Ka radiation.

In some embodiments, crystalline form A of the compound of formula (I) is characterized by an X-ray powder diffraction pattern containing characteristic peaks at 6.6±0.2, 8.7±0.2, 10.0±0.2, 10.9±0 .2, 13.2±0.2, 15.7±0.2, 16.4±0.2, 16.7±0.2, 17.4±0.2, 19.3±0.2 , 20.1±0.2, 20.6±0.2, 22.2±0.2, 23.3±0.2, 24.0±0.2, 25.9±0.2, 28 ,1±0.2 and 30.4±0.2° expressed as 2θ angles using Cu-Ka radiation.

In some embodiments, crystalline form A of the compound of formula (I) has a Cu-Ka X-ray powder diffraction pattern substantially as shown in FIG. 1.

In some embodiments, crystalline form A of the compound of formula (I) is characterized by a differential scanning calorimetry (DSC) diagram containing an endothermic peak at about 195-215°C.

In some embodiments, crystalline form A of the compound of formula (I) is characterized by a differential scanning calorimetry (DSC) diagram containing an endothermic peak at 205±3°C.

In some embodiments, crystalline form A of the compound of formula (I) has a differential scanning calorimetry (DSC) pattern substantially as shown in FIG. 2.

In some embodiments, crystalline form A of the compound of formula (I) has an X-ray powder diffraction pattern substantially as shown in FIG. 1.

In some embodiments, crystalline form A of the compound of formula (I) is characterized by a differential scanning calorimetry diagram containing an endothermic peak in the range from about 195 to 215°C, preferably in the range of 205±3°C, and more preferably a differential scanning calorimetry diagram essentially, as shown in FIG. 2.

In some embodiments, the crystalline form A of the compound of formula (I) is characterized by a TGA diagram in which there is no apparent mass loss in the range from 0 to 250°C, preferably a thermogravimetric analysis diagram, essentially as shown in FIG. 2.

In some embodiments, crystalline form A of the compound of formula (I) has an H-NMR spectrum substantially as shown in FIG. 3.

In some embodiments, crystalline form A of the compound of formula (I) is characterized in that its crystalline structure is in substantially pure form.

In some embodiments, the present invention also relates to a process for preparing crystalline Form A of a compound of formula (I), comprising dissolving the compound of formula (I) in an organic solvent, then stirring and collecting the precipitated solid.

In some embodiments, a method for preparing crystalline form A of a compound of formula (I) comprises dissolving a compound of formula (I) in an organic solvent and heating it to 60-100° C. with stirring until the compound dissolves;

cooling to 0-25°C and stirring at a constant temperature for 1-24 hours; and collecting the precipitated solid.

In some embodiments, a method for preparing crystalline form A of a compound of formula (I) comprises dissolving a compound of formula (I) in an organic solvent and heating it to 60-100° C. with stirring until the compound dissolves;

cooling to 30-55° C. to precipitate a solid;

cooling to 0-25°C and stirring at a constant temperature for 1-24 hours; and collecting the precipitated solid and drying.

In some embodiments, a method for preparing crystalline form A of a compound of formula (I) comprises dissolving a compound of formula (I) in an organic solvent, heating it

- 2 042455 to 60-100°C and stirring until the compound is dissolved, cooling to 0-25°C with stirring to precipitate a solid, keeping at a constant temperature, stirring for 1-24 hours, vacuum filtration and drying to obtain a crystalline form A.

In some embodiments, a method for preparing crystalline Form A of a compound of formula (I) comprises dissolving a compound of formula (I) in an organic solvent and heating it to 70-100°C;

after dissolving the compound, cooling to 50-75°C, adding a crystalline seed and keeping at a constant temperature to precipitate a solid; and slow cooling to 0-25° C., holding at a constant temperature, collecting the precipitated solid, and drying to obtain crystalline Form A.

In some embodiments, the implementation of the crystalline seed is a crystalline Form A of the compounds of formula (I) or a solid precipitated during the preparation of crystalline Form A of the compounds of formula (I).

In some embodiments, the amount of crystal seed added is 0.1-3%, such as 0.1-0.2, 0.2-0.5, 0.5-1, 1-1.5, 1.5-2 , 2-2.5 or 2.5-3% relative to the weight of the compound of formula (I).

In some embodiments, a method for preparing crystalline Form A of a compound of formula (I) comprises dissolving a compound of formula (I) in an organic solvent and heating it to 70-100°C;

after dissolving the compound and cooling to 50-65°C optional addition of a certain amount of crystalline seed and keeping at a constant temperature;

after the appearance of a solid, slow cooling to 0-25°C and keeping at a constant temperature;

vacuum filtration and drying to obtain crystalline form A.

The seed crystal is selected from crystal form A or the solid precipitated before drying during the preparation of crystal form A, and crystal form A can be obtained by another method for obtaining crystal form A in which no crystal seed is added, as described in the present description. The ratio of the weight of the crystalline seed to the weight of the compound of formula (I) is 0.1-3%, preferably 0.1-0.2, 0.2-0.5, 0.5-1, 1-1.5, 1, 5-2, 2-2.5 or 2.5-3%.

In some embodiments, a method for preparing crystalline Form A of a compound of formula (I) comprises dissolving a compound of formula (I) in an organic solvent and heating it to 70-100°C;

after dissolving the compound and cooling to 50-65°C, adding a crystalline seed and keeping at a constant temperature;

after the appearance of a solid, slow cooling to 0-25°C and keeping at a constant temperature;

vacuum filtration and drying to obtain crystalline form A, where the crystalline seed is selected from crystalline form A or a solid before drying during the preparation of crystalline form A, and the mass ratio of the crystalline seed and the compound of formula (I) is 0.1-3%, preferably 0.1-0.2, 0.2-0.5, 0.5-1, 1-1.5, 1.5-2, 2-2.5 or 2.5-3%.

In some embodiments, the method of obtaining a crystalline seed includes dissolving the compound of formula (I) in an organic solvent, heating it to 60-100°C and stirring until the compound is dissolved, keeping at a constant temperature, reducing the stirring speed, cooling to 50-65°C, increasing the stirring speed, slow cooling to 0-25° C. to precipitate a solid, keeping at a constant temperature, stirring for 1-24 hours and vacuum filtering to obtain a crystalline seed.

In some embodiments, in methods for preparing crystalline form A and a crystalline seed, the heating temperature is 60-100°C, preferably 70-100°C, preferably 80-100°C, preferably 90-100°C, and the preferred heating temperature is the temperature at which the sample dissolves and becomes transparent.

In some embodiments, in the methods for obtaining crystalline form A and crystalline seed, the temperature is reduced to 0-25°C, preferably to 5-10°C, preferably to 5-20°C, preferably to 10-15°C, preferably to 15- 25°C, and during cooling, cooling may optionally be carried out several times at different temperatures. The cooling rate is preferably 3-15°C/h, preferably 5-10°C/h, preferably 6°C/h, preferably 9°C/h; and cooling methods include, but are not limited to, natural cooling, ice bath cooling, oil bath cooling, refrigeration refrigeration, etc., with natural refrigeration, oil bath refrigeration, and refrigeration refrigeration being preferred in the present invention.

- 3 042455

In some embodiments, regarding mixing in methods for producing crystalline form A and crystalline seed, mixing methods include, but are not limited to, mechanical stirring, magnetic stirring, etc.; and the speed of this mixing is preferably 500-100 rpm, preferably 300 rpm or 150 rpm. and the mixing time is preferably 0.5-10 hours, preferably 0.5-1 hours, preferably 1-6 hours, preferably 1.5-5 hours.

In some embodiments, in methods for preparing crystalline form A and a crystalline seed, a compound of formula (I) is dissolved in an organic solvent selected from one or any combination of two or more of the following solvents:

(1) alcohol solvents selected from fatty alcohol solvents, alicyclic alcohol solvents, and aromatic alcohol solvents, wherein the fatty alcohol solvents are selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, t- butanol, sec-butanol, n-pentanol, n-hexanol, ethylene glycol, propylene glycol or glycerin; alicyclic alcohol solvents are selected from cyclopentanol, cyclopentylmethanol, cyclohexanol, cyclohexylmethanol or cyclohexylethanol; and aromatic alcohol solvents selected from benzyl alcohol, phenylethanol or phenylpropanol;

(2) ketone solvents selected from fatty ketone solvents and cyclic ketone solvents, wherein the fatty ketone solvents are selected from methyl ethyl ketone, methyl isopropyl ketone, acetone, methyl butanone, or methyl isobutyl ketone; and cyclic ketone solvents selected from cyclopropanone, cyclohexanone, isophorone, or N-methylpyrrolidone;

(3) nitrile-based solvents selected from acetonitrile or propionitrile;

(4) ether solvents selected from fatty ether solvents and cyclic ether solvents, wherein the fatty ether solvents are selected from diethyl ether, dipropyl ether, diisopropyl ether, methyl t-butyl ether, ethyl butyl ether, ethyl tert-butyl ether, dibutyl ether or dipentyl ether; and cyclic ether solvents are selected from ethylene oxide, 1,2-propylene oxide, tetrahydrofuran, 2-methylfuran, dioxolane or 1,4-dioxane;

(5) amide solvents selected from formamide, N,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylpropionamide, or N,N-diethylpropionamide; and (6) sulfoxide solvents selected from dimethyl sulfoxide, diethyl sulfoxide, or benzylphenyl sulfoxide.

Solvents based on alcohols, ketones, nitriles, ethers, amides and sulfoxides as mentioned above are not limited to the specific examples listed, and any solvents belonging to the above categories can function according to the present invention, i.e. obtaining crystalline Form A of the compound of formula (I).

With respect to organic solvents, any combination of two or more solvents refers to a solvent obtained by mixing organic solvents of the same category or different categories from the above-mentioned organic solvents in a certain ratio. Mixed solvents formed from solvents of the same category include, but are not limited to, the following specific examples: methanol/ethanol, methanol/isopropanol, methanol/ethanol/isopropanol, methanol/t-butanol, methanol/cyclopentanol, methanol/benzyl alcohol , ethanol/isopropanol, ethanol/tert-butanol, diethyl ether/tetrahydrofuran, etc. Mixed solvents formed from different categories of solvents include, but are not limited to, the following mixed solvent systems: alcohols/ketones, alcohols/ethers, alcohols/amides, ketones/amides, etc.

In some embodiments, the organic solvent is an alcohol solvent or a ketone solvent.

In some embodiments, the organic solvent is selected from acetone, isopropanol, butanol, and n-pentanol.

In some embodiments, upon receipt of the crystalline Form A and the crystalline seed, drying methods include, but are not limited to, natural drying at room temperature, drying with an infrared lamp, drying in an oven, drying in an oven, and preferably drying under vacuum; the preferred drying temperature is 30-100°C, preferably 30-80°C, preferably 35-70°C, preferably 40-65°C, preferably 35-55°C; during the drying process, multiple drying at different temperatures may optionally be carried out; and preferred drying times are 5-48, 10-36 and 15-24 hours.

In some embodiments, the present invention also relates to a pharmaceutical composition comprising crystalline form A of the compound of formula (I) according to the present invention and optionally one or more pharmaceutical carriers and/or diluents. In some embodiments, the pharmaceutical composition of the present invention may be in any pharmaceutically acceptable dosage form such as a solution, tablet, capsule, or injection, and such pharmaceutical composition may be administered by injection or oral administration. In some embodiments, the crystalline form A of the compound of formula (I) according to the present invention, or a pharmaceutical composition containing it, is preferably administered orally.

In some embodiments, a pharmaceutical composition comprising a crystalline form (e.g., crystalline form A) of a compound of formula (I) and optionally one or more pharmaceutical carriers and/or diluents as described herein may be in any pharmaceutically acceptable dosage form. It is administered to a patient in need orally, parenterally, rectally, transpulmonary, etc. For oral administration, it may be prepared as conventional solid preparations such as tablets, capsules, pills, granules, etc., or it may also be prepared as oral liquid preparations such as oral solutions, oral suspensions, syrups. etc. In the case of an oral preparation, a suitable excipient, binder, disintegrant, lubricant, etc. may be added. When used for parenteral administration, the composition can be prepared as an injection, including injection solutions, sterile injection powders and concentrated injection solutions. In the case of obtaining an injection preparation, the composition may be prepared in a conventional manner in the existing pharmaceutical field, and in the case of an injection, no additives may be added or suitable additives may be added depending on the nature of the drug. When used for rectal administration, the composition may be in the form of a suppository, etc. When used for administration by the pulmonary route, the composition may be in the form of an inhalant, spray, etc.

In some embodiments, the implementation of the pharmaceutical composition according to the present invention may further contain one or more additional anticancer agents and/or immunosuppressants.

Additional anticancer agents and/or immunosuppressants are selected from one or more of methotrexate, capecitabine, gemcitabine, doxifluridine, pemetrexed disodium, pazopanib, imatinib, erlotinib, lapatinib, gefitinib, vandetanib, herceptin, bevacizumab, rituximab, trastuzumab, paclitaxel, docerel, vinotacel doxorubicin, hydroxycamptothecin, mitomycin, epirubicin, pyrarubicin, bleomycin, letrozole, tamoxifen, fulvestrant, triptorelin, flutamide, leuprorelin, anastrozole, ifosfamide, busulfan, cyclophosphamide, carmustine, nimustine, semustine, nitrogen mustard, melphalan, chlorplatin, cispalybucil , lobaplatin, topotecan, camptothecin, hycamtin, everolimus, sirolimus, temsirolimus, 6-mercaptopurine, 6-thioguanine, azathioprine, mycin D, daunorubicin, amycin, mitoxantrone, blenoxan, plicamycin, and aminoglutethimide.

In some embodiments, the present invention also relates to the use of a crystalline form (e.g., crystalline form A) of a compound of formula (I) according to the present invention or a pharmaceutical composition according to the present invention for the manufacture of a medicament for the treatment and/or prevention of diseases associated with cancer mediated kinase CDK4/6, in a subject.

In some embodiments, the present disclosure also relates to a method of treating and/or preventing diseases associated with CDK4/6 kinase-mediated cancer in a mammal in need thereof, comprising administering to said mammal in need thereof a therapeutically and/or prophylactically effective amount of the crystalline form A compound of formula (I) or a pharmaceutical composition according to the present invention.

In some embodiments, the present invention also relates to crystalline Form A of a compound of formula (I) for use in a medicament for the treatment and/or prevention of diseases associated with cancer mediated by CDK4/6 kinase.

In the present invention, diseases associated with cancer mediated by CDK4/6 kinase are selected from brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head cancer and neck, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, adenocarcinoma of the esophagus, squamous cell carcinoma of the esophagus, prostate cancer, cancer of the female genital tract, carcinoma in situ, lymphoma, neurofibromatosis, thyroid cancer, bone cancer , skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, and sarcoma.

The term approximately, as used herein, for example, to change a certain numerical value or numerical range, refers to the inclusion of a numerical value or numerical range and a range of errors acceptable to a person skilled in the art, in relation to a numerical value or numerical range, such as a range of errors. , co

- 5 042455 factor ±10, ±5, ±4, ±3, ±2, ±1, ±0.5%, etc.

The actual dosage level of each active ingredient in the pharmaceutical composition of the present invention may vary so that the amount of active compound obtained may be effective in achieving the desired therapeutic response for a particular patient, composition and route of administration. The dosage level should be selected based on the activity of the particular compound or its crystalline form, the route of administration, the severity of the condition being treated, and the condition and medical history of the patient being treated. However, it is practice in the art that the dosage of the compound or crystalline form thereof is started at a level lower than that required to obtain the desired therapeutic effect and the dosage is gradually increased until the desired effect is achieved.

When used in the above treatment and/or prevention or other treatments and/or prevention, a therapeutically and/or prophylactically effective amount of the crystalline Form A of the compound of formula (I) according to the present invention may be used alone. Alternatively, crystalline form A of the compound of formula (I) may be administered in a pharmaceutical composition containing crystalline formula A of the compound of formula (I) and one or more pharmaceutically acceptable excipients. The phrase therapeutically and/or prophylactically effective amount of a crystalline form A of a compound of formula (I) according to the present invention refers to that amount of the compound which is sufficient to treat the disorder with a rational effect/risk ratio applicable to any medical treatment and/or prevention. However, it should be understood that the total daily dose of the crystalline form A of the compounds of formula (I) and the pharmaceutical composition, as described in the present description, should be determined by the attending physician in the framework of reliable medical judgment. For each individual patient, the level of a particular therapeutically effective dosage will depend on a number of factors, including the disease being treated and the severity of the disorder; the activity of the particular compound used or its crystalline form; the particular composition used; age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound employed or its crystalline form; duration of treatment; a medicinal product used in combination or simultaneously with the specific compound used or its crystalline form; and similar factors well known in the medical field. For example, as practiced in the art, the dosage of a compound or crystalline form thereof is started at a level lower than that required to obtain the desired therapeutic effect, and the dosage is gradually increased until the desired effect is achieved. In general, the dosage of crystalline form A of the compound of formula (I) according to the present invention for mammals, in particular humans, may be from 0.001 to 1000 mg/kg body weight/day.

The crystalline form A of the compound of formula (I) according to the present invention may be administered alone or in the form of a pharmaceutical composition. The pharmaceutical composition according to the present invention can be obtained in the form of various suitable dosage forms in accordance with the method of administration. The use of one or more physiologically acceptable carriers, including excipients and adjuvants, facilitates the processing of the active compound or its crystalline form to obtain drugs that can be used for pharmaceutical purposes. The appropriate form of the drug depends on the chosen method of administration and can be obtained in accordance with the general knowledge, well known in the art.

The main advantages of the crystalline form, in particular the crystalline form A of the compound of formula (I) according to the present invention, include (1) a production method that is easy to carry out and suitable for industrial production;

(2) good appearance, fluidity, compressibility, convenience for production, detection, preparation, transportation and storage;

(3) high purity, less residual solvent, high solubility, good stability and easy quality control;

(4) good inhibitory activity against CDK4/6 kinase and good exposure and/or bioavailability in vivo; and (5) good in vivo and in vitro efficacy and potential for use in the treatment and/or prevention of diseases associated with cancer mediated by CDK4/6 kinase.

Brief description of graphic materials

The drawings described in the present description are used to further explain the present invention and form part of the present application, and illustrative examples of the present invention and its description are used to explain the present invention and are not restrictive in relation to the present invention. On the graphics of Fig. 1 is an X-ray powder diffraction (XRPD) pattern of a crystalline

- 6 042455 Form A of the compound of formula (I), where the ordinate indicates the diffraction intensity, and the abscissa indicates the diffraction angle (29);

fig. 2 is a TGA-DSC analysis chart of crystalline form A of the compound of formula (I), where the ordinate on the right is the mass (%), on the y-axis on the left is the heat flux (W/g), and on the abscissa is the temperature T (° WITH);

fig. 3 is the H-NMR spectrum of the crystalline form A of the compound of formula (I).

Detailed description of embodiments

The main content of the present description is further illustrated below in conjunction with specific examples of the present invention, and it should be understood that the following examples are only intended to illustrate the present invention and not to limit the scope of protection of the present invention. In the following examples, where no specific conditions are indicated, they were carried out in accordance with the usual conditions or manufacturer's recommendations. If manufacturers are not indicated for the drugs or reagents used, they are all common products that are commercially available.

While many of the materials and modes of operation used in the following examples are well known in the art, the present invention has been described in as much detail as possible. It will be clear to a person skilled in the art that, unless otherwise indicated, the materials and modes of operation used in the following examples are well known in the art.

The compound of formula (I) used in the following examples or experimental examples was obtained in accordance with the preparation method of example 1 of the specification of patent application PCT/CN 2014/095615.

Receipt examples.

Methods for preparing crystalline form A of a compound of formula (I).

Method of obtaining I.

5.0 g of the compound of formula (I) was taken, 75 ml of acetone was added and the compound of formula (I) was dissolved with magnetic stirring, a white solid immediately precipitated, and after 5 hours of stirring it was filtered off under vacuum to obtain a white powder, which was dried in vacuo at 35° C. for 16 hours to give a solid identified as crystalline Form A by XRPD.

Production method II.

500 mg of the compound of formula (I) was taken, 15 ml of isopropanol was added and the compound of formula (I) was dissolved with stirring in an oil bath at a temperature of 80°C until the solution became clear (within 4.0 h), cooled naturally to room temperature (32°C) until a white solid precipitated, then stirred at room temperature for 12 h, cooled to 15°C, stirred for 4.0 h and filtered under vacuum to obtain a solid, which was dried in vacuum at 45°C for 16 h, and identified as crystalline form A using XRPD and analysis of ¹ H-NMR.

Production method III.

5.0 g of the compound of formula (I) was taken and placed in a 100 ml three-necked flask, 75 ml of sec-butanol was added, the oil bath temperature was maintained at 95° C., and dissolution was carried out with mechanical stirring for 0.5 h until until the solution becomes clear. The oil bath was then cooled to 70° C., approximately 25 mg of seed crystal (crystal form A obtained by Preparation Method I or II) was added, a white solid began to slowly precipitate, the oil bath was further cooled to 60° C., maintained at constant temperature for 0.5 h, cooled to 55°C, maintained at constant temperature for 0.5 h, and then cooled to approximately 20°C, and then stirred for 1.5 h, filtered under vacuum and washed methyl tert-butyl ether (2x10 ml) to give a solid which was dried in vacuo at 65°C for 24 h. form A.

Method of obtaining IV.

1) Obtaining a crystalline seed. Took 40 g of the compounds of formula (I) and 300 g of n-pentanol and heated to 75°C; after about 0.5 h at a speed of rotation of the mixing blade R=300 rpm, the raw material began to dissolve; and, after the feed was completely dissolved and the solution became clear, the feed was held at 75°C for 0.5 h. .5 hours After reaching 55°C, the stirring speed was increased to R=300 rpm, the system was further cooled to 5°C at a rate of 9°C/h, and it slowly became cloudy and a solid precipitated. After reaching 5° C., the system was kept at a constant temperature for 2 hours and filtered under vacuum to obtain a solid, i.e. crystal seed.

- 7 042455

2) Process for preparing crystalline form A 50 g of a compound of formula (I) was dispersed in 300 g of n-pentanol, heated to 85°C, completely dissolved until the solution became clear, and quickly cooled to 58°C (85 -58°C/h). 0.1 g of the seed crystal mentioned in 1 above was added), the temperature was kept constant for 60 minutes. The system at this time slowly became turbid until a white solid appeared. The system was slowly cooled to 5°C at a rate of 6°C/h, then kept overnight at a constant temperature, and then filtered under vacuum. The resulting solid was placed in a vacuum dryer and dried at 100° C. for 6 hours. The resulting dry solid was dried under vacuum at 65° C. for ²⁴ hours. XRPD showed that the solid was crystalline Form A.

Testing of the products obtained by preparation methods I to IV showed that the purity of crystalline form A was above 99.7%, the single maximum impurity content was less than 0.08%, and practically no solvent remained.

XRPD test.

Instrument used: Bruker D2 X-ray powder diffractometer.

X-ray reflection parameters: Cu, Ka; entry gap: 0.6 mm; divergence gap: 1 mm; scan mode: continuous; scan range: 3.0-45.0°; sampling step: 0.02°; scan time per step: 19.8 s; detector angle: 2.0°.

The crystalline form A of the compound of formula (I) is shown by powder x-ray diffraction pattern in FIG. 1, where the crystal form has peaks at the following 2θ diffraction angles: 6.6±0.2, 8.7±0.2, 10.0±0.2, 10.9±0.2, 13.2±0 .2, 15.7±0.2, 16.4±0.2, 16.7±0.2, 17.4±0.2, 19.3±0.2, 20.1±0.2 , 20.6±0.2, 22.2±0.2, 23.3±0.2, 24.0±0.2, 25.9±0.2, 28.1±0.2 and 30 .4±0.2°.

Differential scanning calorimetry.

Solid state thermal properties of crystalline form A of the compound of formula (I) are studied using differential scanning calorimetry (DSC).

Instrument used: Q2000 differential scanning calorimeter purchased from TA.

Measurement conditions: nitrogen flush at 50 ml/min, data acquisition at a heating rate of 10°C/min in the range of 25 to 220°C and plotted with endothermic peaks down.

Measurement results: crystalline form A of the compound of formula (I) shows an endothermic peak in the range of 195 to 215°C, and the differential scanning calorimetry plot is shown in FIG. 2.

Thermogravimetric analysis.

Instrument used: Q50 thermogravimetric analyzer purchased from TA.

Test conditions: nitrogen purge at 60 ml/min and data collection at a heating rate of 10°C/min over a range of room temperature to 350°C.

Measurement results: crystalline form A of the compound of formula (I) has no apparent weight loss in the range from 0 to 250°C, and its TG curve is shown in FIG. 2.

Analysis by nuclear magnetic resonance ( ¹ H-NMR).

Tool: Bruker Advance III 400; solvent: deuterated DMSO.

Measurement results: H-NMR of the crystalline form A of the compound of formula (I) is as shown in FIG. 3.

Experimental examples for testing properties.

Experimental example 1. Investigation of the properties of the crystalline form A.

1) Stability test.

Test product: crystalline form A of the compound of formula (I) obtained in accordance with preparation methods I, II, III or IV mentioned above.

Experimental procedure: the test product was placed in high humidity conditions (25°C/92.5% relative humidity or 40°C/75% relative humidity) or 60°C for 10 days and samples were taken at 5- and 10- th days respectively; and exposed to light (4500±500 lux) for 10 days, sampled on day 10, tested for co-substances and by XRPD and compared with the results of the same tests for the sample on day 0.

Associated substances: measured according to Chinese Pharmacopoeia, 2015 Edition, Volume II, Appendix VD. High performance liquid chromatography.

XRPD measurement: measured according to Chinese Pharmacopoeia 2015 Edition Volume IV, 0451 X-ray diffraction method.

The results of the experiment on the stability of the crystalline Form And the compounds of formula (I) are shown in table. 1.

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Table 1

The results of the study of the stability of the crystalline form A of the compound of formula (I)

Tested product Test conditions Sampling time View Associated substance (%) XRPD Crystal form A of the compound of formula (I) 0 day 0 day off-white powder To oh and Crystal Form A 25°C / 92.5% RH Open system Day 5 off-white powder to 0.09 Crystal Form A Day 10 off-white powder to 0.09 Crystal Form A 60°C Open system Day 5 off-white powder To oh and Crystal Form A Day 10 off-white powder To 0.14 Crystal Form A 60°С Closed system Day 5 off-white powder To 0.09 Crystal Form A Day 10 off-white powder To oh and Crystal Form A 40°C / 75% relative humidity Open system Day 5 off-white powder To 0.10 Crystal Form A Day 10 off-white powder To 0.20 Crystal Form A

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40°C / 75% relative humidity Closed system Day 5 off-white powder To 0.10 Crystal Form A Day 10 off-white powder To 0.20 Crystal Form A Light Day 10 off-white powder To 0.09 Crystal Form A

2) Hygroscopicity test of the compound of formula (I) and crystalline form A.

Tested product.

The compound of formula (I), obtained in accordance with the method of obtaining example 1 from the description of patent application PCT/CN 2014/095615.

Crystal form A of the compound of formula (I) obtained in accordance with preparation methods I, II, III or IV mentioned above.

Measurement Method: The measurements were carried out in accordance with the Chinese Pharmacopoeia 2015 Edition, Volume IV, General Principles, 9103 Guidelines for Hygroscopicity Testing of Medicinal Products.

See table. 2, which shows the results of the experiment on hygroscopicity.

table 2

Hygroscopicity Test Results

Tested product Mass gain due to moisture absorption (%) Results in respect hygroscopicity Compound of formula (I) Crystal form A of compound of formula (I) 6.3 0.003 Moisture absorption No or little or no moisture absorption

3) Particle size measurement test of crystalline form A.

Test product: crystalline Form A of the compound of formula (I) obtained according to preparation method I, II, III or IV mentioned above.

Reagents: Tween 80 and ultrapure water.

Devices and equipment: laser particle size analyzer, sample disperser.

Measurement method.

An appropriate amount of test product was taken, 0.1 ml of a 1% Tween 80 solution was added to reduce surface tension, and water was used as a dispersant to obtain a uniform dispersed suspension. It was placed in a sample disperser, the stirring speed was set to 2000 rpm, sonication was performed for 2 minutes at an ultrasound frequency of 7 kHz, and measurements were taken.

Experiment results.

The particle size distribution of the crystalline form A of the compound of formula (I) is as follows: 10% of the sample is 2.501 µm or less, 50% is 10.432 µm or less, and 90% is 59.852 µm or less.

Experimental Example 2 Stability study of the compound of formula (I) in amorphous form.

Test product: Compound of formula (I) (i.e., amorphous form) obtained according to the preparation method of Example 1 of the specification of patent application PCT/CN 2014/095615.

Experimental technique.

The test product was placed under conditions of 25° C./92.5% relative humidity or under conditions of 60° C. for 10 days and samples were taken on days 5 and 10, respectively; the test product was exposed to light (4500±500 lux) or 40°C/75% relative humidity for 10 days, samples were taken for

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Day 10, tested for co-substances and XRPD, and compared to day 0 sample.

Associated substances: measured according to Chinese Pharmacopoeia, 2015 Edition, Volume II, Appendix VD. High performance liquid chromatography.

XRPD Measurement: Chinese Pharmacopoeia 2015 Edition Volume IV, 0451 X-ray diffraction method.

The results of the experiment on the stability of the amorphous form of the compounds of formula (I) are shown in table. 3.

Table 3

The results of the study of the stability of the amorphous form

Tested product Test conditions Sampling time View Concomitant substance (%) XRPD Compound of formula (I) 0 day 0 day off-white powder 0.14 amorphous form 25°C / 92.5% RH Open system Day 5 off white powder 0.14 amorphous form Day 10 off white powder 0.19 amorphous form 60°C open system Day 5 Light yellow powder 0.67 amorphous form Day 10 Light yellow powder 1D5 amorphous form 40°C / 75% RH Open system Day 10 off-white powder 0.19 amorphous form Light Day 10 Light yellow powder 10.54 amorphous form

Conclusions after the experiment.

After being at 60°C or under lighting conditions for 10 days, the properties such as appearance, co-substances and XRPD of the crystalline form A did not change overtly, while the amorphous form contained up to 10.54% co-substances under lighting conditions. .

The results show that the crystalline form A of the present invention has good stability characteristics and low hygroscopicity, which is convenient in drug manufacturing, drug manufacturing, transportation and storage, and is more conducive to ensuring drug stability and safety. In addition, compared to the amorphous form, the crystalline form A has good exposure and/or bioavailability in vivo, as well as good in vivo and in vitro efficacy.

Experimental Example 3 Compressibility experiment of amorphous form and crystalline form A of the compound of formula (I).

Test products: crystalline form A of the compound of formula (I), obtained in accordance with the method of obtaining I, II, III or IV, mentioned above; and amorphous form compound form

- 11 042455 ly (I), obtained in accordance with the method of obtaining example 1 from the description of patent application PCT/CN 2014/095615.

Experimental technique.

Appropriate amounts of the crystalline form A and the amorphous form of the compound of formula (I), respectively, were taken as raw materials and tableted separately, the weight of the tablets was recorded, the thickness of the tablets (tabletting strength) was adjusted, and the hardness of the tablets was measured. Change in tablet hardness with change in tablet thickness was investigated and compressibility was compared.

See table. 4, which shows the results of the experiment.

Table 4

The results of the compressibility experiment

Tablet thickness/mm 2.4 2.0 1.8 1.3 0.9 Hardness of the crystalline form 0 1.76 5.40 3.50 2.97 A/kg Amorphous hardness/kg 0.72 0.59 2.75 / /

Notes.

(1) 7 means that a tablet of this thickness could not be obtained.

(2) The smaller the tablet thickness, the greater the tableting force.

From the results of the experiments given in table. 4, it can be seen that, at the same tablet thickness, the hardness of the crystalline form A exceeds that of the amorphous form. In addition, as the tablet thickness decreases (tabletting strength increases), the tablet hardness of the crystalline form A can reach about 5.4 kg, while the maximum hardness of the amorphous form is about 2.75 kg; furthermore, in the tableting process, the amorphous form is prone to sticking to the walls, causing the surface of the tablet to become rough or defective, and also prone to chipping, indicating that the crystalline form A has better compressibility than the amorphous form and is more favorable for drug formulation development.

Experimental Example 4 Release Experiment of Amorphous Form and Crystalline Form A of the Compound of Formula (I).

Test products: crystalline form A of the compound of formula (I), obtained in accordance with the method of obtaining I, II, III or IV, mentioned above; and an amorphous form of the compound of formula (I), obtained in accordance with the method of obtaining example 1 from the description of patent application PCT/CN 2014/095615.

Experimental technique.

40 mg of the crystalline form A and 40 mg of the amorphous form were taken and respectively added to the same types and amounts of adjuvants and compressed into tablets. Disintegration and dissolution were investigated under the following conditions.

Dissolution method: Paddle method (Chinese Pharmacopoeia 2015 Edition Volume IV, 0931 Methods for Measuring Dissolution and Release Method II); medium: aqueous 0.2% SDS solution; rotation speed: 50 rpm.

See table. 5 and 6, which show the results of the experiment.

Table 5

Decay time

Tested product Crystal form A amorphous form Disintegration time/min 4 > 30 min Absorbed water turns into a colloid and slowly dissolves

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Claims (15)

Table 6 Dissolution Crystal form Total dissolution (%) (1) alcohol solvents selected from fatty alcohol solvents, alicyclic alcohol solvents, and aromatic alcohol solvents, wherein the fatty alcohol solvents are selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, t- butanol, sec-butanol, n-pentanol, n-hexanol, ethylene glycol, propylene glycol or glycerin; alicyclic alcohol solvents are selected from cyclopentanol, cyclopentylmethanol, cyclohexanol, cyclohexylmethanol or cyclohexylethanol; and aromatic alcohol solvents selected from benzyl alcohol, phenylethanol or phenylpropanol; 1. Crystal form A of the compound of formula (I), characterized by an X-ray powder diffraction pattern containing characteristic peaks at 6.6±0.2, 10.0±0.2, 13.2±0.2, 17.4±0, 2, 20.1±0.2 and 20.6±0.2° expressed by 2Θ angles using Cu-Ka radiation, F Formula (I) (2) ketone solvents selected from fatty ketone solvents and cyclic ketone solvents, wherein the fatty ketone solvents are selected from methyl ethyl ketone, methyl isopropyl ketone, acetone, methyl butanone, or methyl isobutyl ketone; and cyclic ketone solvents selected from cyclopropanone, cyclohexanone, isophorone, or N-methylpyrrolidone; 2. Crystalline form A according to claim 1, characterized by an X-ray powder diffraction pattern containing characteristic peaks at 6.6±0.2, 8.7±0.2, 10.0±0.2, 10.9±0.2 , 13.2±0.2, 15.7±0.2, 16.4±0.2, 17.4±0.2, 20.1±0.2, 20.6±0.2 and 30 .4±0.2° expressed as 2θ angles using Cu-Ka radiation. (3) nitrile-based solvents selected from acetonitrile or propionitrile; 3. Crystal form A according to claim 1 or 2, characterized by an X-ray powder diffraction pattern containing characteristic peaks at 6.6±0.2, 8.7±0.2, 10.0±0.2, 10.9±0 .2, 13.2±0.2, 15.7±0.2, 16.4±0.2, 16.7±0.2, 17.4±0.2, 19.3±0.2 , 20.1±0.2, 20.6±0.2, 22.2±0.2, 23.3±0.2, 24.0±0.2, 25.9±0.2, 28 ,1 ±0.2 and 30.4±0.2° expressed as 2Θ angles using Cu-Ka radiation. (4) ether solvents selected from fatty ether solvents and cyclic ether solvents, wherein the fatty ether solvents are selected from diethyl ether, dipropyl ether, diisopropyl ether, methyl t-butyl ether, ethyl butyl ether, ethyl tert-butyl ether, dibutyl ether or dipentyl ether; and cyclic ether solvents are selected from ethylene oxide, 1,2-propylene oxide, tetrahydrofuran, 2-methylfuran, dioxolane or 1,4-dioxane; 4. Crystalline Form A according to any one of claims 1 to 3, characterized by an X-ray powder diffraction pattern obtained using Cu-Ka radiation, as shown in FIG. 1. (5) amide solvents selected from formamide, N,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylpropionamide, or N,N-diethylpropionamide; and (6) sulfoxide solvents selected from dimethyl sulfoxide, diethyl sulfoxide, or benzylphenyl sulfoxide. 5. Crystalline form A according to any one of claims 1-4, characterized by a differential scanning calorimetry (DSC) diagram containing an endothermic peak at approximately 195-215°C. 5 minutes 10 min 15 minutes 30 min 45 min 60 min Crystal Form A 69.2 90.7 93.8 96.6 97.5 97.9 amorphous form 1.0 2.7 4.6 9.2 13.2 18.5 The results of the experiment are given in table. 5 and 6 show that the crystalline form A decomposes and dissolves faster and completely dissolves within 60 minutes, while the decomposition and dissolution rate of the amorphous form is significantly reduced compared to the crystalline form A, indicating that the crystalline form A more suitable for drug development. Finally, it should be noted that the above examples are used only to illustrate and not to limit the technical solutions according to the present invention. Although the present invention has been described in detail with reference to the preferred examples, it should be clear to a person skilled in the art that modifications to certain embodiments of the present invention or equivalent substitutions of some of its technical features can be made without deviating from the essence of the technical solutions according to the present invention, and all they are within the scope of the technical solutions claimed in the present invention. CLAIM 6. Crystalline form A according to any one of claims 1 to 5, characterized by a differential scanning calorimetry (DSC) diagram containing an endothermic peak at 205±3°C. 7. Crystal form A according to any one of claims 1 to 6, characterized by a differential scanning calorimetry (DSC) diagram as shown in FIG. 2. 8. A method for obtaining crystalline form A of the compound of formula (I) according to any one of claims 1-7, including dissolving the compound of formula (I) in an organic solvent, heating it to 60-100 ° C and stirring until the compound is dissolved, further stirring and cooling to 0-25°C to precipitate a solid, keeping at a constant temperature, stirring for 1-24 hours, vacuum filtration and drying to obtain crystalline Form A. 9. The method of obtaining a crystalline form And the compounds of formula (I) according to any one of paragraphs.1-7, including the dissolution of the compounds of formula (I) in an organic solvent and heating it to 70-100°C; after dissolving the compound, cooling to 50-75°C, adding a crystalline seed and keeping at a constant temperature to precipitate a solid; And 10. The method for obtaining crystalline form A according to any one of claims 8, 9, where the organic solvent is selected from one or from any combination of two or more of the following solvents: 11. The method for obtaining crystalline Form A according to claim 8 or 9, wherein the organic solvent is selected from acetone, isopropanol, butanol and n-pentanol. 12. Pharmaceutical composition containing crystalline form A of the compound of formula (I) according to any one of claims 1 to 7 and optionally one or more pharmaceutical carriers and/or diluents. 13. Pharmaceutical composition according to claim 12, further comprising one or more additional anticancer agents and/or immunosuppressants selected from one or more of methotrexate, capecitabine, gemcitabine, doxifluridine, pemetrexed disodium, pazopanib, imatinib, erlotinib, lapatinib, gefitinib, vandetanib , herceptin, bevacizumab, rituximab, trastuzumab, paclitaxel, vinorelbine, docetaxel, doxorubicin, hydroxycamptothecin, mitomycin, epirubicin, pyrarubicin, bleomycin, letrozole, tamoxifen, fulvestrant, triptorelin, flutamide, leuprorelin, anastrozole, ifosfanamide, nifosfamide, cyclosulfamide , semustine, nitrogen mustard, melphalan, chlorambucil, carboplatin, cisplatin, oxaliplatin, lobaplatin, topotecan, camptothecin, hycamtin, everolimus, sirolimus, temsirolimus, 6-mercaptopurine, 6-thioguanine, azathioprine, mycin D, daunorubicin, mitokamisan , plicamycin and aminoglutethimide. - 13 042455 slow cooling to 0-25°C, keeping at a constant temperature, collecting the precipitated solid and drying to obtain crystalline form A, where the crystalline seed is a crystalline form A of the compound of formula (I) or a solid precipitated during preparation crystalline form A of the compound of formula (I), and the amount of added crystalline seed is 0.1-3% relative to the mass of the compound of formula (I). 14. A method of treating and/or preventing diseases associated with CDK4/6 kinase-mediated cancer in a mammal in need, comprising administering to said mammal in need a therapeutically and/or prophylactically effective amount of a crystalline form A of a compound of formula (I) according to any from claims 1 to 7 or a pharmaceutical composition according to claims 12 or 13. 15. The method of treating and/or preventing diseases according to claim 14, wherein the diseases associated with cancer mediated by CDK4/6 kinase are selected from brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, stomach cancer, ovarian cancer, cancer peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female genital tract cancer, carcinomas in situ, lymphoma, neurofibromatosis, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, and sarcoma. -