Semi-L-tartrate monohydrate inhibiting CDK4 / 6 active compounds

Abstract

The invention relates to a semi-L-tartrate monohydrate (formula II) of an active compound for inhibiting CDK4 / 6, a preparation method of the semi-L-tartrate monohydrate, a medicinal composition of the semi-L-tartrate monohydrate and application of the semi-L-tartrate monohydrate in inhibiting the activity of the CDK4 / 6. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the hemil-L-tartrate monohydrate of (R)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(6-fluoro-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyridin-8-yl)pyrimidin-2-amine, its preparation method, pharmaceutical composition, and its application in inhibiting the activity of CDK4 / 6. Background Art

[0002] Cyclin-dependent kinases (CDKs) are a class of serine / threonine protein kinases that participate in the regulation of processes such as the cell cycle, transcriptional initiation, and certain specific metabolic cascade reactions. Different CDKs form CDK-cyclin complexes with cyclins. If the CDK activity is dysregulated, it will directly or indirectly cause uncontrolled cell proliferation, genomic instability (increased DNA mutations, chromosomal deletions, etc.), and chromosomal instability (changes in chromosome number).

[0003] More than 20 subtypes have been identified in the CDKs family. CDK1, CDK2, CDK4, CDK6, etc. are involved in cell cycle regulation; CDK7, CDK8, CDK9, CDK11, etc. are involved in transcriptional regulation; and other kinases include CDK3, CDK5, etc. Among them, CDK4 / 6 (cyclin-dependent kinases 4 and 6) are key factors regulating the cell cycle. Cell cycle mutations related to cancer mainly occur during the G1 phase and the G1 / S phase transition. CDK4 / 6 binds to Cyclin D to form a kinase-active complex, phosphorylates the product pRb of the tumor suppressor gene Rb, releases the bound transcription factor E2F, initiates the transcription of genes related to the S phase, promotes the cell to pass through the checkpoint, and transfers from the G1 phase to the S phase. The specific activation of CDK4 / 6 is closely related to the proliferation of some tumors. Approximately 80% of human tumors have abnormalities in the cyclin D-CDK4 / 6-INK4-Rb pathway. CDK4 / 6 inhibitors arrest the cell cycle at the G1 phase, thereby playing a role in inhibiting tumor proliferation.

[0004] The research and development of drugs targeting CDK4 / 6 kinases is a significant field. The advantages of its anti-tumor targets are as follows: (1) Most proliferating cells rely on CDK2 or CDK4 / 6 for proliferation, but CDK4 / 6 inhibitors do not exhibit the cytotoxicity of "pan-CDK inhibitors", such as myelosuppression and intestinal reactions; (2) Preclinical experiments have shown that if the cell cyclin D level increases or P16INK4a is inactivated, it can increase the sensitivity of cells to the drug. Since tumor cells have the above phenomena compared to normal cells, to a certain extent, it increases the targeting of the drug.

[0005] PCT International Application PCT / CN2017 / 117950 describes a class of benzimidazole derivatives used as CDK4 / 6 protein kinase inhibitors. Most of these compounds effectively inhibit CDK4 and CDK6. Since there are still unmet needs in the treatment options for kinase-mediated diseases, here we further screen the salt forms and crystal forms of benzimidazole derivatives to meet the medical needs of patients. Summary of the Invention

[0006] To solve the above problems existing in the prior art, the present invention provides a monohydrate of the semi-L-tartrate salt of a compound that inhibits CDK4 / 6 activity. This monohydrate not only has low methanol residue, but also has advantages in druggability such as good crystallinity, good stability, low hygroscopicity, high bioavailability, and good processability for formulation manufacturing. In addition, the present invention also provides a preparation process of this monohydrate, a pharmaceutical composition containing this monohydrate, and their medical uses.

[0007] The technical solutions involved in the present invention are described in detail below:

[0008] An object of the present invention is to provide a compound of formula (II):

[0009]

[0010] Its chemical name is (R)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(6-fluoro-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyridin-8-yl)pyrimidin-2-amine semi-L-tartrate monohydrate. The compound of formula (II), the monohydrate of formula (II), the compound of formula (II), or the above chemical name that appears in this patent application all refer to the same substance. The brackets in formula (II) can be omitted and abbreviated as formula II, and sometimes these terms representing the same substance can be used interchangeably.

[0011] Another object of the present invention is to provide a crystal form of the compound of formula (II).

[0012] In some embodiments, the crystalline form of the compound of formula (II) is crystalline form I, and the X-ray powder diffraction spectrum of the crystalline form I has characteristic peaks with diffraction angles 2θ of 4.3±0.2°, 8.6±0.2°, 10.8±0.2°, 12.9±0.2°, 23.5±0.2° and 26.8±0.2°.

[0013] In some embodiments, the X-ray powder diffraction spectrum of the above-mentioned Form I has characteristic peaks with diffraction angles 2θ of 4.3±0.2°, 8.6±0.2°, 10.8±0.2°, 12.9±0.2°, 18.5±0.2°, 23.5±0.2° and 26.8±0.2°.

[0014] In some embodiments, the above-mentioned Form I has substantially Figure 1 The X-ray powder diffraction spectrum is shown.

[0015] In some embodiments, the differential scanning calorimetry thermogram of the above-mentioned Form I comprises an endothermic peak with an onset temperature of about 231.3°C and / or a peak temperature of about 232.9°C.

[0016] In some embodiments, the above-mentioned Form I has substantially Figure 2 TGA spectrum shown.

[0017] In some embodiments, the above-mentioned Form I has substantially Figure 3 The DSC spectrum shown

[0018] In some embodiments, the above-mentioned Form I has substantially Figure 4 Infrared spectrum shown.

[0019] In some embodiments, the above-mentioned Form I has substantially Figure 5 Raman spectrum shown.

[0020] The above-mentioned Form I has been characterized by single crystal diffraction analysis. The single crystal diffraction characterization results of Form I show that the single crystal belongs to the monoclinic system, P21 space group, and its unit cell parameters are: The schematic diagram of the asymmetric unit of the single crystal structure obtained by analysis is basically as follows Figure 7 As shown, the unit cell diagram of the single crystal structure model is basically as follows Figure 8 As shown, the hydrogen bond connection diagram between adjacent cations, L-tartrate anions and water molecules in the compound of formula (II) is basically as follows Figure 9 shown.

[0021] The present invention also aims to provide a method for preparing the crystalline form I of the compound of formula (II), which comprises the following steps:

[0022]

[0023] Step 1: Add the compound of formula (I) to a mixed solvent of methanol and water at 60-70 °C to form a clear solution.

[0024] Step 2: Dissolve L-tartaric acid in methanol to obtain a methanol solution of L-tartaric acid. Add the methanol solution of L-tartaric acid to the clear solution obtained in Step 1 at 60-70 °C for reaction to obtain a reaction solution.

[0025] Step 3: Cool the reaction solution obtained in Step 2 to 15-25 °C, stir and filter to obtain a filter cake. The filter cake is obtained as the target substance under a drying process with dry nitrogen and wet nitrogen purging; preferably, dry nitrogen and wet nitrogen are purged in sequence; preferably, dry nitrogen and wet nitrogen are purged in a cycle.

[0026] In some embodiments, in Step 1, the mass ratio of methanol, water to the compound of formula (I) is 12-20:1-1.5:1; preferably 12-15:1.33:1; more preferably 14.77:1.33:1.

[0027] In some embodiments, the methanol solution of L-tartaric acid in Step 2 is added in batches. For example: at 60-70 °C, first add 1 / 15-1 / 10 volume of the methanol solution of L-tartaric acid dropwise to the clear solution in Step 1, stir until a solid precipitates, and then add the remaining methanol solution of L-tartaric acid dropwise to the solution after the solid has precipitated for reaction to obtain a reaction solution. More preferably, at 60-70 °C, first add 1 / 12 volume of the methanol solution of L-tartaric acid dropwise to the clear solution in Step 1, stir until a solid precipitates, and then add the remaining methanol solution of L-tartaric acid dropwise to the solution after the solid has precipitated, and stir for 1-2 h to obtain a reaction solution.

[0028] In some embodiments, in Step 2, the mass ratio of L-tartaric acid, methanol to the compound of formula (I) is 0.15-0.2:3.5-4.5:1; preferably 0.17:3.95:1.

[0029] In some embodiments, in Step 3, the nitrogen purging method is to first use dry nitrogen for purging and then switch to wet nitrogen for purging.

[0030] The object of the present invention also lies in providing a pharmaceutical composition, which contains a therapeutically effective amount of the aforementioned compound of formula (II) or crystalline form I of the compound of formula (II), and a pharmaceutically acceptable excipient and / or carrier.

[0031] In some embodiments, the pharmaceutical composition is further combined with at least one other active ingredient.

[0032] In some embodiments, the pharmaceutical composition is an oral preparation.

[0033] In some embodiments, the oral preparation includes tablets, capsules, cachets, pills, granules, oral liquids, suspensions, dispersions, emulsions, powders, preferably tablets or capsules.

[0034] The present invention also aims to provide the use of the aforementioned compound of formula (II) or crystalline form I of the compound of formula (II) in the preparation of a medicament for treating a disease in a patient, wherein the disease is mediated by CDK4 and / or CDK6.

[0035] In some embodiments, the disease is cancer.

[0036] In some embodiments, the cancer is colon cancer, rectal cancer, mantle cell lymphoma, multiple myeloma, breast cancer, prostate cancer, glioblastoma, squamous cell esophageal cancer, liposarcoma, T-cell lymphoma, melanoma, pancreatic cancer, brain cancer or lung cancer. Preferably, the cancer is breast cancer.

[0037] The present invention also aims to provide a method for treating a disease, including administering to a subject in need thereof a therapeutically effective amount of the aforementioned compound of formula (II) or crystalline form I of the compound of formula (II) or a pharmaceutical composition containing the same, wherein the disease is mediated by CDK4 and / or CDK6.

[0038] In some embodiments, the disease is cancer.

[0039] In some embodiments, the cancer is colon cancer, rectal cancer, mantle cell lymphoma, multiple myeloma, breast cancer, prostate cancer, glioblastoma, squamous cell esophageal cancer, liposarcoma, T-cell lymphoma, melanoma, pancreatic cancer, brain cancer or lung cancer. Preferably, the cancer is breast cancer.

[0040] Terms and Definitions

[0041] All crystalline forms of the present invention are substantially pure.

[0042] The term "substantially pure" used in the present invention means that the content of the crystalline form is not less than 85% by weight, preferably not less than 95%, more preferably not less than 99%, and most preferably not less than 99.5%.

[0043] It should be noted that the numerical values and ranges mentioned in the present invention should not be narrowly construed as the numerical values or ranges themselves. Those skilled in the art should understand that they can fluctuate around specific numerical values according to different specific technical environments without departing from the spirit and principles of the present invention. In the present invention, such foreseeable fluctuation ranges for those skilled in the art are mostly indicated by the terms "about" or "substantially".

[0044] In the present invention, "crystal form" refers to the solid form of a compound, which is different from the amorphous form of the compound. The "amorphous form" refers to a non-crystalline solid form. In the absence of a precise definition, the compound includes an amorphous form, any crystal form, a mixture of any two or more crystal forms, and a mixture of any one or more crystal forms and an amorphous form. The term "crystal form" used refers to a crystal form with the same chemical composition but a different spatial arrangement of molecules, atoms and / or ions that form the crystal. The crystal form includes a single-component crystal form and a multi-component crystal form, including but not limited to a solvent-free form (e.g., anhydrous crystal form), solvates (non-hydrates), hydrates, co-crystals and other molecular complexes and their polymorphs. In some embodiments, the crystal form of a substance may be substantially free of amorphous and / or other crystal forms. In certain embodiments, the crystal form of a substance may contain less than about 50% by weight of one or more amorphous and / or other crystal forms. In some embodiments, the crystal form of a substance may be physically and / or chemically substantially pure.

[0045] Crystalline forms can be detected, identified, classified and characterized using well-known techniques such as, but not limited to, differential scanning calorimetry (DSC), thermogravimetry (TGA), X-ray powder diffractometry (XRPD), single crystal X-ray diffractometry, solid-state nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, Raman spectroscopy and hot-stage optical microscopy.

[0046] In the present invention, "having substantially Figure 1 The term "substantially" used in the context of the "X-ray powder diffraction pattern shown" indicates that the precise positions of the peaks in the accompanying drawings should not be interpreted as absolute values. As those skilled in the art will appreciate, the 2θ values of an X-ray powder diffraction pattern may vary due to different measurement conditions (such as the equipment and instruments used) and different samples (such as samples from different batches), the measurement error of the diffraction angle of an X-ray powder diffraction pattern is 5% or less, and generally, a difference of ±0.2° from a given value is considered appropriate. It should also be understood that the relative intensities of the peaks may fluctuate with experimental conditions and sample preparation, such as the preferred orientation of particles in the sample. The use of automatic or fixed divergence slits will also affect the calculation of relative intensities. The intensities shown in the XRD curves included herein are exemplary only and cannot be used as absolute comparisons, and any crystalline form having a powder diffraction pattern substantially identical to those disclosed herein is within the scope of protection of the present invention.

[0047] Those skilled in the art will appreciate that, in the process of acquiring XRPD spectra, appropriate scientific processing of the relevant data, such as baseline correction, may be performed to reduce errors. Those skilled in the art will also appreciate that, when operating under different laboratory conditions, slight differences may exist in the 2θ angles or resolution of the obtained XRPD spectra.

[0048] Those skilled in the art will understand that due to variations in sample batches, sample purity, sample preparation, and measurement conditions (e.g., heating rate), DSC measured data may vary slightly, and generally, a difference of ±5°C from a given value is acceptable (and still considered characteristic of the specific crystalline form described herein). Therefore, the endotherms cited in this application are not to be taken as absolute values, and such errors should be taken into account when interpreting DSC data.

[0049] In TGA measurements, without being bound by any particular theory, weight loss corresponds to the loss of trace amounts of residual solvent or water. TGA data may vary due to variations in sample batches, sample purity, residual solvent content, sample preparation, and measurement conditions (e.g., heating rate). Therefore, the thermogravimetric curves cited herein are not to be taken as absolute values, and such errors should be taken into account when interpreting TGA data.

[0050] In practice, according to conventional pharmaceutical mixing techniques, the compound of formula (II) of the present invention or its crystalline form, or a mixture thereof, as the active ingredient, is intimately mixed with a pharmaceutical carrier according to conventional pharmaceutical mixing techniques to form a pharmaceutical composition. The pharmaceutical carrier can take a variety of forms, depending on the desired mode of administration, for example, oral or injection (including intravenous injection). Therefore, the pharmaceutical composition of the present invention can be in the form of independent units suitable for oral administration, such as capsules, cachets or tablets containing a predetermined dose of the active ingredient. Furthermore, the pharmaceutical composition of the present invention can be in the form of a powder, granules, solution, aqueous suspension, non-aqueous liquid, oil-in-water emulsion, or water-in-oil emulsion. In addition, in addition to the common dosage forms mentioned above, the compound of formula (II) of the present invention or its crystalline form, or a mixture thereof, can also be administered by controlled release and / or delivery device. The pharmaceutical composition of the present invention can be prepared by any pharmaceutical method. Generally, such a method includes the step of associating the active ingredient with a carrier constituting one or more essential ingredients. Generally, the pharmaceutical composition is prepared by uniformly and uniformly mixing the active ingredient with a liquid carrier or a finely divided solid carrier or a mixture of the two. In addition, the product can be easily prepared into a desired appearance.

[0051] The pharmaceutical carriers used in the present invention can be, for example, solid carriers, liquid carriers, or gaseous carriers. Solid carriers include lactose, gypsum powder, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, and water. Gaseous carriers include carbon dioxide and nitrogen. When preparing oral pharmaceutical preparations, any pharmaceutically convenient medium can be used. Water, ethylene glycol, oils, alcohols, flavor enhancers, preservatives, colorants, etc. can be used to prepare oral liquid preparations such as suspensions, elixirs, and solutions; while carriers, diluents (such as starches, sugars, microcrystalline cellulose, etc.), diluents, granulating agents, lubricants (such as magnesium stearate, micropowdered silica gel), binders (such as povidone, hydroxypropyl cellulose, gelatin), disintegrants (such as sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, etc.), etc. can be used to prepare oral solid preparations such as powders, capsules, and tablets. For ease of administration, tablets and capsules are preferred for oral preparations, where solid pharmaceutical carriers are used. Alternatively, tablets may be coated using standard aqueous or non-aqueous techniques.

[0052] Tablets containing the compounds, crystal forms or pharmaceutical compositions of the present invention can be formed by compression molding or molding, and optionally, tablets can be made together with one or more pharmaceutically acceptable carriers and / or excipients. The active ingredient is in a free-flowing form such as powder or granules, and is mixed with inert diluents, binders, disintegrants, lubricants, glidants and surfactants. In a suitable machine, compressed tablets can be prepared by compression. Among the excipients, the inert diluent can be selected from mannitol, microcrystalline cellulose, starch, lactose, anhydrous calcium hydrogen phosphate, etc.; the binder can be selected from hydroxypropyl methylcellulose, povidone, hydroxypropyl cellulose, etc.; the disintegrant can be selected from sodium carboxymethyl starch crosslinked, sodium carboxymethyl starch, crosslinked povidone, low-substituted hydroxypropyl cellulose, etc.; the lubricant can be selected from stearic acid and its salts; the glidant can be selected from talc, silica, etc.; the surfactant can be selected from poloxamer, sodium lauryl sulfate, Tween, etc. Preferably, each tablet contains about 0.05 mg to 5 g of the active ingredient, and each cachet or capsule contains about 0.05 mg to 5 g of the active ingredient. For example, a formulation intended for oral administration to humans contains about 0.5 mg to about 5 g of the active ingredient, combined with suitable and conveniently metered auxiliary materials, and the auxiliary materials account for about 5% to 95% of the total amount of the pharmaceutical composition. The unit dosage form generally contains about 1 mg to about 2 g of the active ingredient, typically 2.5 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 160 mg, 200 mg, 320 mg, 500 mg, 600 mg, 800 mg, 1000 mg or 2000 mg. However, generally speaking, for treating the conditions or discomforts shown above, it can be understood that the specific dosage level for any particular patient will depend on a variety of factors, including age, weight, general health status, gender, diet, time of administration, route of administration, excretion rate, drug combination situation and the severity of the specific disease being treated.

[0053] The present invention provides a pharmaceutical composition suitable for parenteral administration. The active ingredient can be added to water to prepare an aqueous solution or suspension. Appropriate surfactants such as hydroxypropyl cellulose can be included. Dispersed systems can also be prepared in glycerol, liquid polyethylene glycol, and their mixtures in oil. Further, preservatives can also be included in the pharmaceutical composition of the present invention to prevent the growth of harmful microorganisms.

[0054] The present invention provides pharmaceutical compositions suitable for injection, including sterile aqueous solutions or dispersions. Further, the above-mentioned pharmaceutical compositions can be prepared into sterile powder forms for immediate preparation of sterile injections or dispersions. In any case, the final injection form must be sterile and, in order to be easy to inject, must be easy to flow. In addition, the pharmaceutical composition must be stable during preparation and storage. Therefore, preferably, the pharmaceutical composition will be preserved under conditions of antimicrobial such as bacterial and fungal contamination. The carrier can be a solvent or a dispersion medium, for example, water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycol), a vegetable oil and a suitable mixture thereof.

[0055] The pharmaceutical composition provided by the present invention can be in a form suitable for topical use, for example, an aerosol, emulsion, ointment, lotion, dusting powder or other similar dosage forms. Further, the pharmaceutical composition provided by the present invention can be in a form suitable for use with a transdermal drug delivery device. These preparations can be prepared by conventional processing methods using a compound of formula (II) of the present invention, or its crystalline form, or a mixture thereof. As an example, an emulsion or ointment is prepared with an expected consistency by adding about 5wt% to 10wt% of a hydrophilic material and water.

[0056] The pharmaceutical composition provided by the present invention can be in a form suitable for rectal administration using a solid as a carrier. Unit-dose suppositories are the most typical and common dosage form. Suitable excipients include cocoa butter and other materials commonly used in the art. Suppositories can be easily prepared by first mixing the pharmaceutical composition with softened or melted excipients, then cooling and molding.

[0057] In addition to the aforementioned excipient components, the formulation may also include, where appropriate, one or more additional excipient components, such as buffers, flavorings, surfactants, thickeners, and preservatives (including antioxidants). Furthermore, other excipients may include penetration enhancers that adjust the osmotic pressure between the drug and the intended recipient's blood. Pharmaceutical compositions comprising or its crystalline form, or a mixture thereof, may be prepared in the form of a powder or a concentrated solution.

[0058] In the present invention, the term "therapeutically effective amount" refers to an amount of a compound / crystalline form that, when administered to a subject being treated, is sufficient to affect the treatment of a disease, or at least one clinical symptom of a disease or disorder, for the treatment of such disease, disorder or symptom. The "therapeutically effective amount" may vary with the compound, the disease, disorder and / or symptoms of the disease or disorder, the severity of the disease, disorder and / or symptoms of the disease or disorder, the age of the patient being treated, and / or the weight of the patient being treated, etc. In any given case, a suitable amount may be apparent to those skilled in the art or may be determined by routine experimentation. In the case of combination therapy, the "therapeutically effective amount" refers to the total amount of the combination of agents that is effective in treating the disease, disorder or condition.

[0059] The term "disease" refers to any disease, discomfort, illness, symptom or indication.

[0060] Compared with the prior art, the present invention has at least the following advantages:

[0061] In the crystalline form I of the compound of formula (II) in the present invention, the residual methanol is less, and the defect that methanol is difficult to remove due to the formation of methanol participating in the crystal lattice is overcome by introducing water molecules to participate in the crystal lattice.

[0062] The crystalline form I of the compound of formula (II) in the present invention not only has good stability and low hygroscopicity, but also has good bioavailability, can meet the basic requirements of drugability, and meet the needs of commercial manufacturing of bulk drugs and preparations.

[0063] In the preparation process of the present invention, by introducing a certain amount of water, not only the amount of methanol used is reduced, but also it is beneficial to improve the solubility of the crystalline form I of the compound of formula (II), effectively avoiding the phenomenon of sudden precipitation of the product, preventing the inclusion of impurities in the product and the problem that the solvent residue is difficult to remove during drying due to sample caking, which is beneficial to the improvement of product quality.

[0064] In the preparation process of the present invention, the methanol solution of L-tartaric acid is added to the supernatant of step one of the foregoing preparation process in two batches, further preventing the phenomenon of sudden precipitation during the salt formation process and improving the crystallinity and quality of the product.

[0065] In the process of achieving low residual methanol solvent of the crystalline form I of the compound of formula (II), the present invention not only adopts a specific process of using dry and wet nitrogen purging in combination, but also successfully realizes that this process can be applied to large-scale production (from several kilograms to dozens of kilograms of active pharmaceutical ingredients (API)). BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 : XRPD spectrum of the crystalline form I of the compound of formula (II);

[0067] Figure 2 : TGA spectrum of Polymorph I of the compound of formula (II);

[0068] Figure 3 : DSC spectrum of Polymorph I of the compound of formula (II);

[0069] Figure 4 : Fourier transform infrared spectrum of Polymorph I of the compound of formula (II);

[0070] Figure 5 : Raman spectrum of Polymorph I of the compound of formula (II);

[0071] Figure 6 : Overlay of XRPD of single crystal sample of Polymorph I of the compound of formula (II) and XRPD of Polymorph I (process sample) of the compound of formula (II);

[0072] Figure 7 : Schematic diagram of the asymmetric unit of the single crystal structure model of Polymorph I of the compound of formula (II);

[0073] Figure 8 : Schematic diagram of the unit cell of the single crystal structure model of Polymorph I of the compound of formula (II);

[0074] Figure 9 : Schematic diagram of hydrogen bond connection in the single crystal structure model of Polymorph I of the compound of formula (II). Detailed implementation mode

[0075] Unless otherwise specified, the information of the detection instruments and the parameters of the detection methods used in the present invention are as follows:

[0076] Table 1

[0077]

[0078] Table 2

[0079] Device Name Thermogravimetric Analyzer (TGA) Instrument Model TA Discovery TGA 550 Temperature range Room temperature (<30)~400℃ Heating rate 10℃ / min

[0080] Table 3

[0081] Device Name Differential Scanning Calorimetry (DSC) Instrument Model TA Discovery DSC 2500 Temperature range 30～250℃ Heating rate 10℃ / min Purge gas flow rate 50mL / min

[0082] Table 4

[0083] Device Name Infrared spectroscopy Instrument Model Brook Alpha Test conditions Potassium bromide tableting method Resolution <![CDATA[1cm -1 ]]> Sample scan times 4 Background scan times 4 Scan range <![CDATA[4000~400cm -1 ]]>

[0084] Table 5

[0085]

[0086] Moisture determination: The moisture of the test sample was determined using a Karl Fischer moisture meter, specifically as follows:

[0087] Instrument equipment

[0088] METTLER TOLEDO volumetric Karl Fischer titrator V30, V30S, or equivalent equipment; one in a hundred thousandth analytical balance; headspace vial, 20 mL; Milli-Q pure water preparation instrument or equivalent instrument.

[0089] Reagents

[0090] Karl Fischer reagent solution A, HYDRANAL TM / Composite5, Honeywell, Cat#34805-1L-CN or equivalent reagent; methanol, chromatographically pure; two-component volumetric solvent, HYDRANAL T M / Solvent, Honeywell, Cat#34800-1L, or equivalent reagent; dichloromethane, chromatographically pure.

[0091] Instrument parameters

[0092]

[0093]

[0094] Determination of L-tartaric acid content: The content of L-tartaric acid in the test sample is determined by high performance liquid chromatography as follows:

[0095] Instrument and equipment

[0096] Agilent 1260 high performance liquid chromatography system or equivalent instrument; one in a hundred thousandth analytical balance; Milli-Q pure water preparation instrument or equivalent instrument.

[0097] Reagents (equivalent or higher grade)

[0098] Purified water from Milli-Q; acetonitrile (ACN), chromatographically pure; phosphoric acid, analytically pure; L-tartaric acid, reference substance.

[0099] Chromatographic conditions

[0100]

[0101]

[0102] The present invention is further illustrated by the following examples, but the examples do not constitute any limitation to the scope of the present invention claimed. In the specific embodiments of the present invention, unless otherwise specified, the technologies or methods are conventional technologies or methods in the art, etc.

[0103] Preparation of the precursor of the hemihydrate of L-tartrate of (R)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(6-fluoro-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyridin-8-yl)pyrimidin-2-amine (precursor of the compound of formula (II)):

[0104] Take 5009 mg of the compound of formula (I) in a 250 mL crystallization kettle, add 100 mL of methanol / water (14:1, v:v) solvent, heat up to 60 °C and stir until dissolved clearly, and keep warm for 50 min. Take 0.728 g of L-tartaric acid in a beaker, add 25 mL of methanol to prepare an L-tartaric acid methanol solution. First, take 2 mL of the L-tartaric acid methanol solution and add it to the aforementioned solution, and stir until a solid precipitates. Then continue to slowly add the remaining L-tartaric acid methanol solution thereto, stir for 1 - 2 h, cool down to 20 °C and stir for 1 - 2 h, filter, and vacuum dry at 45 °C for 20 - 30 h to obtain a sample, which is defined as the precursor of the compound of formula (II).

[0105] Characterize the obtained precursor of the compound of formula (II), and it is found that the water content in this precursor is 2.2%, and the methanol content is 1.6%, indicating that this precursor is a co-solvent hydrate containing methanol - water. Since methanol participates in the formation of the crystal lattice in this precursor, it is difficult to remove the residual methanol contained therein by conventional vacuum drying methods, far exceeding the requirement that the residual methanol limit specified in ICH Q3C (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use: Impurities: Residual Solvents) should be ≤ 3000 ppm.

[0106] Preparation of the hemihydrate of L-tartrate of (R)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(6-fluoro-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyridin-8-yl)pyrimidin-2-amine (formula II):

[0107]

[0108] Add methanol (224.5 kg) and purified water (20.2 kg) to Reactor 1, start stirring, adjust the temperature to 15 - 30 °C, and add the compound of formula (I) (15.2 kg); subsequently, adjust the temperature of Reactor 1 to 60 - 70 °C, and under the condition of maintaining the temperature at 60 - 70 °C, stir until the system is clarified; preheat the jacket of Reactor 2 to 40 - 60 °C in advance, and keep the temperature of the jacket of Reactor 2 controlled at 40 - 60 °C, and filter the system of Reactor 1 through a filter and transfer it hot to Reactor 2.

[0109] Adjust the temperature of Reactor 2 to 60-70°C and control the temperature at 60-70°C. Slowly dropwise add a methanol solution of L-tartaric acid (prepared in advance by dissolving L-tartaric acid (2.58 kg) in methanol (60.2 kg)) to Reactor 2 in an amount equal to 1 / 12 of the prepared methanol solution of L-tartaric acid. Stir for 1-2 hours after the addition is complete until solids precipitate. Slowly dropwise add the remaining methanol solution of L-tartaric acid to Reactor 2. Continue stirring for 1-2 hours after the addition is complete. Adjust the temperature of Reactor 2 to 15-25°C and stir for 1-2 hours.

[0110] The reactor 2 system was transferred to a filter dryer for pressure filtration until no liquid flowed out. After purging with dry nitrogen for at least 2 hours, the filter dryer was vacuum dried for approximately 20 to 30 hours at a temperature of T≤45°C. Subsequently, dry nitrogen was bubbled into a water storage tank containing an appropriate amount of water. Wet nitrogen containing water was then passed through the upper pipe of the water storage tank into the filter dryer, and drying was continued for approximately 30 hours under the condition of wet nitrogen purge. The filter dryer jacket was cooled, and the solid was removed to obtain the target substance, which was defined as Form I of the compound of formula (II). The methanol content in this form was measured to be 1732 ppm, which meets the requirement of the methanol residual limit of ≤3000 ppm specified in ICH Q3C (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use: Guidelines for Impurities: Residual Solvents).

[0111] In order to effectively reduce the problem of excessive residual methanol in the precursor of the compound of formula (II) in the aforementioned Example 1 due to the participation of methanol in the crystal lattice, Example 2 of the present invention first uses dry nitrogen to purge to remove free methanol, and then switches to wet nitrogen purge to replace the methanol in the precursor crystal lattice with water molecules. This method effectively solves the problem that the residual methanol solvent in the precursor of the compound of formula (II) cannot meet the limit requirements. It is found that such a technical effect can only be achieved through the monohydrate crystal form, which also provides a theoretical basis for the drying process using wet nitrogen purge, successfully achieving the ability to very efficiently remove the residual methanol solvent in the precursor of the compound of formula (II).

[0112] To confirm that the crystalline form I of the compound of formula (II) achieves these assumptions of low methanol solvent residue, the present invention conducted the following tests and characterizations:

[0113] The XRPD spectrum of the crystalline form I of the compound of formula (II) is as follows: Figure 1 The analytical data are shown in Table 6.

[0114] Table 6 XRPD spectrum analysis data

[0115]

[0116]

[0117] The TGA spectrum of the crystalline form I of the compound of formula (II) is as follows: Figure 2 As shown in the figure, the TGA results show that the crystal loses about 2.9% of its weight before the temperature rises from room temperature to 150°C, indicating that the sample contains a certain amount of crystal water. Figure 3 As shown in the figure, the DSC results show that the target substance has a sharp melting endothermic peak (peak temperature) at 232.9℃ and two broad endothermic signals at 68.9℃ and 134.3℃. Its Fourier transform infrared spectrum is shown in the figure Figure 4 As shown, its Raman spectrum is as follows Figure 5 shown.

[0118] The crystal form I of the compound of formula (II) was tested using a Karl Fischer moisture meter, and the moisture content of the crystal form was found to be 3.0%, which is consistent with the theoretical content of its monohydrate of 2.94%.

[0119] The L-tartrate content of Form I of the compound of formula (II) was tested using high performance liquid chromatography, and the L-tartaric acid content in the crystalline form was found to be 12.25%, demonstrating that the molar ratio of the salt formation was: free base: L-tartaric acid = 2:1.

[0120] Example 3 Single crystal growth and structure analysis

[0121] In order to further clarify the structure of the crystalline form I of the compound of formula (II) prepared in Example 2, single crystal cultivation and structural analysis of the substance were performed in this example. The single crystal cultivation method is:

[0122] About 30 mg of the crystalline form I of the compound of formula (II) prepared in Example 2 was weighed and dissolved in 1.5 mL of a mixed solution of MeOH / H2O (2:1, v / v). After dissolving at 60°C, the temperature was slowly lowered to 20°C to precipitate crystals. After wiping the solvent on the surface of the sample, the sample was placed under forced air drying conditions at 40°C for single crystal-single crystal conversion. The obtained sample was subjected to single crystal testing and structural analysis.

[0123] The single crystal diffraction characterization results show that the single crystal belongs to the monoclinic system, P21 space group, and its unit cell parameters are: Table 7 lists other crystallographic data and structure refinement parameters of the single crystal.

[0124] Single crystal testing instrument: Single crystal diffraction data were collected using a Rigaku XtaLAB Synergy R single crystal diffractometer (Cu target light source, ) was performed at 120K.

[0125] Table 7 Crystallographic data and structure refinement parameters of single crystal

[0126]

[0127]

[0128] The overlay graph of the measured XRPD of the obtained single-crystal sample and the XRPD of polymorph I of the compound of formula (II) obtained by the preparation method of Example 2 is as Figure 6 shown, and the results show that the measured XRPD of the single-crystal sample is consistent with the XRPD of polymorph I of the compound of formula (II). It indicates that the crystal form of the sample obtained by single-crystal cultivation is the same as that of polymorph I of the compound of formula (II) prepared in Example 2.

[0129] Single-crystal structure analysis shows that the asymmetric unit of the crystal structure consists of two crystallographically independent cations (+1 valence) of the compound of formula (I), one L-tartrate anion (-2 valence), and two crystallized water molecules. This indicates that polymorph I of the compound of formula (II) contains 1 / 2 L-tartaric acid relative to the compound of formula (I) and is a monohydrate. The schematic diagram of the asymmetric unit of the obtained single-crystal structure is as Figure 7 shown.

[0130] Figure 8 is the schematic diagram of the unit cell of the single-crystal structure model. The hydrogen bond connections between adjacent cations of the compound of formula (I), L-tartrate anions, and water molecules are shown as Figure 9 shown. Table 8 lists the detailed information of the corresponding hydrogen bonds.

[0131] Table 8 Hydrogen Bond Table in the Single-Crystal Structure Model

[0132]

[0133] Analyzed using the general crystallographic software PLATON (version: 140621), (hydrogen bond analysis method: d(D…A) < R(D)+R(A)+0.5Ang., d(H…A) < R(H)+R(A)-0.12Ang., D-H…A>100.0Deg). Symmetry operation codes: #1 (-1+x, 1+y, -1+z); #2 (1+x, -1+y, 1+z); #3 (2-x, 1 / 2+y, 1-z)

[0134] Example 4 Stability Determination

[0135] The crystalline form I of the compound of formula (II) prepared in Example 2 was placed under accelerated conditions (40±2°C / 75±5% RH) for 6 months, and samples were taken at 0 month, 1 month, 2 months, 3 months and 6 months for stability determination. It was also placed under long-term conditions (30±2°C / 65±5% RH) for 12 months, and samples were taken at 0 month, 3 months, 6 months, 9 months and 12 months for stability determination. The determination results are shown in Tables 9 and 10.

[0136] Table 9 Summary of accelerated stability test data

[0137]

[0138]

[0139] Table 10 Summary of long-term stability test data

[0140]

[0141] As shown in Tables 9 and 10, Form I of the compound of formula (II) was placed under accelerated conditions (40±2°C / 75±5% RH) for 6 months and under long-term conditions (30±2°C / 65±5% RH) for 12 months. All test results met the limit requirements and showed good stability.

[0142] Example 5: Moisture absorption test

[0143] The preparation method of Example 2 was repeated three times to obtain three batches of Form I of the compound of formula (II). Hygroscopicity testing was carried out in accordance with the Guidelines for Hygroscopicity Testing of Drugs (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 9103). The three batches were tested for hygroscopicity under storage conditions of 25°C ± 1°C and 80% ± 2% relative humidity (saturated ammonium chloride or saturated ammonium sulfate solution). The results are as follows:

[0144] batch Moisture-induced weight gain 1 1.25％ 2 1.32％ 3 1.36％

[0145] The results showed that the product was slightly hygroscopic, indicating that the crystalline form I of the compound of formula (II) had weak hygroscopicity and was suitable for the preparation of solid preparations.

[0146] Example 6 Pharmacokinetic Test

[0147] Twenty-four beagle dogs were divided into 4 groups with 6 dogs in each group, half male and half female. The animals in the first group were administered a 5% glucose solution of polymorph I of the compound of formula (II) at a single intravenous injection dose of 3 mg / kg. The animals in the second, third, and fourth groups were administered aqueous solutions of polymorph I of the compound of formula (II) at single oral doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg, respectively. Oral administration was by gavage after an overnight fast. Plasma samples were collected before dosing and at 5 min (only the intravenous dosing group), 15 min (only the intravenous dosing group), 1 h, 4 h, 6 h (only the gavage dosing group), 8 h, 12 h, and 24 h after dosing. The concentration of the target substance in plasma was detected using a validated LC-MS / MS method and the pharmacokinetic parameters were calculated using WinNonlin. The experimental data are shown in Table 11.

[0148] After intravenous injection of the target substance solution at 3 mg / kg to beagle dogs, C0 was 508 ng / mL, t 1 / 2 was 12 h, AUC 0-24h was 3,220 h·ng / mL, CL was 15.7 mL / min / kg, and Vd ss was 13.2 L / kg. This indicates that the target substance has a moderate clearance rate in dogs, a relatively high apparent volume of distribution, and is relatively easily distributed in tissues.

[0149] After oral administration of the target substance solution at 3, 10, and 30 mg / kg to beagle dogs, t 1 / 2 were 11 h, 18 h, NA respectively, T max were 6.3 h, 9.7 h, 11 h respectively, C max were 83.1 ng / mL, 172 ng / mL, 205 ng / mL respectively, AUC 0-24h were 1,710 h·ng / mL, 5,680 h·ng / mL, 10,000 h·ng / mL respectively, and the absolute bioavailability was 53.7%, 53.6%, 38.3% respectively. The above results indicate that the target substance has a slow absorption rate, a long half-life, a good absorption degree in dogs, and conforms to linear pharmacokinetic characteristics within the dose range of 3 - 30 mg / kg.

[0150] Table 11 Main pharmacokinetic parameters of beagle dogs after single administration of polymorph I

[0151]

[0152] Thus, it can be seen that polymorph I of the compound of formula (II) in the present invention has excellent physical and chemical stability, low hygroscopicity, excellent pharmacokinetic properties, and a suitable crystallization process. In particular, the excellent effect of low residual methanol content in the crystal form has been successfully achieved, which is beneficial to drug development.

[0153] Embodiment

[0154] The following clauses describe specific embodiments of the present invention.

[0155] 1. A compound of formula (II):

[0156]

[0157] which is the hemihydrate of L-tartaric acid of (R)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(6-fluoro-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyridin-8-yl)pyrimidin-2-amine.

[0158] 2. Polymorph I of a compound of formula (II), characterized in that the compound of formula (II) is as described in Embodiment 1.

[0159] 3. The polymorph I according to Embodiment 2, characterized in that the X-ray powder diffraction pattern of the polymorph has characteristic peaks at diffraction angles 2θ of 4.3 ± 0.2°, 8.6 ± 0.2°, 10.8 ± 0.2°, 12.9 ± 0.2°, 23.5 ± 0.2° and 26.8 ± 0.2°.

[0160] 4. The polymorph I according to Embodiment 2 or 3, characterized in that the X-ray powder diffraction pattern of the polymorph has characteristic peaks at diffraction angles 2θ of 4.3 ± 0.2°, 8.6 ± 0.2°, 10.8 ± 0.2°, 12.9 ± 0.2°, 18.5 ± 0.2°, 23.5 ± 0.2° and 26.8 ± 0.2°.

[0161] 5. The polymorph I according to any one of Embodiments 2-4, characterized in that the polymorph has an X-ray powder diffraction pattern substantially as Figure 1 shown.

[0162] 6. The polymorph I according to any one of Embodiments 2-5, characterized in that the differential scanning calorimetry thermogram of the polymorph includes an endothermic peak with an onset temperature of about 231.3 °C and / or a peak temperature of about 232.9 °C.

[0163] 7. The polymorph I according to any one of Embodiments 2-6, characterized in that the polymorph has a TGA pattern substantially as Figure 2 shown.

[0164] 8. The polymorph I according to any one of Embodiments 2-7, characterized in that the polymorph has a DSC pattern substantially as Figure 3 shown.

[0165] 9. The crystalline form I according to any one of Embodiments 2-8, characterized in that the crystalline form has substantially as Figure 4 shown in the infrared spectrogram.

[0166] 10. The crystalline form I according to any one of Embodiments 2-9, characterized in that the crystalline form has substantially as Figure 5 shown in the Raman spectrogram.

[0167] 11. The crystalline form I according to any one of Embodiments 2-10, characterized in that the crystalline form I is a single crystal, which belongs to the monoclinic system, P21 space group, and the unit cell parameters are:

[0168] 12. A method for preparing the crystalline form I according to any one of Embodiments 2-11, characterized in that the method comprises the following steps:

[0169]

[0170] Step 1: Add the compound of formula (I) to a mixed solvent of methanol and water at 60-70 °C to form a clear solution;

[0171] Step 2: Dissolve L-tartaric acid in methanol to obtain a methanol solution of L-tartaric acid, and add the methanol solution of L-tartaric acid to the clear solution obtained in Step 1 at 60-70 °C for reaction to obtain a reaction solution;

[0172] Step 3: Cool the reaction solution obtained in Step 2 to 15-25 °C, stir and filter to obtain a filter cake, and the filter cake is obtained as the target substance under the drying process of purging with dry nitrogen and wet nitrogen.

[0173] 13. According to the preparation method described in Embodiment 12, characterized in that the addition of the methanol solution of L-tartaric acid in Step 2 specifically comprises the following steps: First, add 1 / 15-1 / 10 volume of the methanol solution of L-tartaric acid dropwise to the clear solution obtained in Step 1 at 60-70 °C, stir until solid precipitates, and then add the remaining methanol solution of L-tartaric acid dropwise to the solution after solid precipitation for reaction to obtain a reaction solution.

[0174] 14. According to the preparation method described in Embodiment 12 or 13, characterized in that in Step 1, the mass ratio of methanol, water to the compound of formula (I) is 12-20:1-1.5:1.

[0175] 15. According to the preparation method described in any one of Embodiments 12-14, characterized in that in Step 1, the mass ratio of methanol, water to the compound of formula (I) is 12-15:1.33:1.

[0176] 16. The preparation method according to any one of embodiments 12-15, characterized in that in step 2, the mass ratio of L-tartaric acid, methanol and the compound of formula (I) is 0.15-0.2:3.5-4.5:1.

[0177] 17. A pharmaceutical composition, characterized in that it contains a therapeutically effective amount of the compound of embodiment 1 or the crystalline form I of any one of embodiments 2-11, and a pharmaceutically acceptable excipient and / or carrier.

[0178] 18. Use of the compound of embodiment 1, the crystalline form I of any one of embodiments 2-11, or the pharmaceutical composition of embodiment 17 in the preparation of a medicament for treating a disease in a patient, wherein the disease is mediated by CDK4 and / or CDK6.

[0179] 19. The use according to embodiment 18, wherein the disease is cancer.

[0180] 20. The use according to embodiment 19, characterized in that the cancer is colon cancer, rectal cancer, mantle cell lymphoma, multiple myeloma, breast cancer, prostate cancer, glioblastoma, squamous cell esophageal cancer, liposarcoma, T-cell lymphoma, melanoma, pancreatic cancer, brain cancer or lung cancer.

[0181] 21. The use according to embodiment 20, characterized in that the cancer is breast cancer.

Claims

1. A compound of formula (II): It is the hemil-L-tartrate monohydrate of (R)-N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(6-fluoro-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyridin-8-yl)pyrimidin-2-amine.

2. Polymorph I of a compound of formula (II), characterized in that, The compound of formula (II) is as described in claim 1.

3. The crystalline form I according to claim 2, wherein The X-ray powder diffraction pattern of the crystal form has characteristic peaks at diffraction angles 2θ of 4.3±0.2°, 8.6±0.2°, 10.8±0.2°, 12.9±0.2°, 23.5±0.2° and 26.8±0.2°.

4. The crystalline form I according to claim 2 or 3, characterized in that, The X-ray powder diffraction pattern of the crystal form has characteristic peaks at diffraction angles 2θ of 4.3±0.2°, 8.6±0.2°, 10.8±0.2°, 12.9±0.2°, 18.5±0.2°, 23.5±0.2° and 26.8±0.2°.

5. The crystalline form I according to any one of claims 2-4, characterized in that, The crystal form has an X-ray powder diffraction pattern substantially as shown in Figure 1.

6. A preparation method of polymorphic form I as described in any one of claims 2-5, characterized in that, The method comprises the following steps: Step 1: Add the compound of formula (I) to a mixed solvent of methanol and water at 60-70 °C to form a clear solution; Step 2: Dissolve L-tartaric acid in methanol to obtain a methanol solution of L-tartaric acid, and add the methanol solution of L-tartaric acid to the clear solution obtained in Step 1 at 60-70 °C for reaction to obtain a reaction solution; Step 3: Cool the reaction solution obtained in Step 2 to 15-25 °C, stir and filter to obtain a filter cake, and obtain the target substance under a drying process with dry nitrogen and wet nitrogen purging.

7. The preparation method according to claim 6, characterized in that, The addition of the methanol solution of L-tartaric acid in Step 2 specifically comprises the following steps: First, add 1 / 15-1 / 10 volume of the methanol solution of L-tartaric acid dropwise to the clear solution obtained in Step 1 at 60-70 °C, stir until a solid precipitates, and then add the remaining methanol solution of L-tartaric acid dropwise to the solution after the solid has precipitated for reaction to obtain a reaction solution.

8. A pharmaceutical composition, characterized in that, Containing a therapeutically effective amount of the compound according to claim 1 or the crystal form I according to any one of claims 2-5, and a pharmaceutically acceptable excipient and / or carrier.

9. Use of the compound according to claim 1, crystalline form I according to any one of claims 2 - 5, or the pharmaceutical composition according to claim 8 in the manufacture of a medicament for treating a disease in a patient, characterized in that, The disease is mediated by CDK4 and / or CDK6.

10. The use according to claim 9, characterized in that, The disease is colon cancer, rectal cancer, mantle cell lymphoma, multiple myeloma, breast cancer, prostate cancer, glioblastoma, squamous cell esophageal cancer, liposarcoma, T cell lymphoma, melanoma, pancreatic cancer, brain cancer or lung cancer.

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